Sample records for adenoviral vector carrying

  1. Targeted adenoviral vectors

    NASA Astrophysics Data System (ADS)

    Douglas, Joanne T.

    The practical implementation of gene therapy in the clinical setting mandates gene delivery vehicles, or vectors, capable of efficient gene delivery selectively to the target disease cells. The utility of adenoviral vectors for gene therapy is restricted by their dependence on the native adenoviral primary cellular receptor for cell entry. Therefore, a number of strategies have been developed to allow CAR-independent infection of specific cell types, including the use of bispecific conjugates and genetic modifications to the adenoviral capsid proteins, in particular the fibre protein. These targeted adenoviral vectors have demonstrated efficient gene transfer in vitro , correlating with a therapeutic benefit in preclinical animal models. Such vectors are predicted to possess enhanced efficacy in human clinical studies, although anatomical barriers to their use must be circumvented.

  2. High-capacity 'gutless' adenoviral vectors.

    PubMed

    Kochanek, S; Schiedner, G; Volpers, C

    2001-10-01

    Adenoviral vectors are promising gene transfer vehicles for different gene therapy applications. High-capacity adenoviral (HC-Ad) vectors address some of the problems that have been observed with replication-defective, E1-deleted first-generation adenoviral vectors: toxicity and immunogenicity due to viral gene expression and 7 to 8 kb capacity limit for the transport of therapeutic DNA. This review summarizes HC-Ad vector-related publications from the past 18 months that are mainly concerned with vector design/production and in vivo applications in different murine models.

  3. Germline incorporation of a replication-defective adenoviral vector in mice does not alter immune responses to adenoviral vectors.

    PubMed

    Camargo, F D; Huey-Louie, D A; Finn, A V; Sassani, A B; Cozen, A E; Moriwaki, H; Schneider, D B; Agah, R; Dichek, D A

    2000-11-01

    The utility of adenoviral vectors is limited by immune responses to adenoviral antigens. We sought to develop immune-competent mice in which the immune response to adenoviral antigens was selectively absent. To do so, we generated mice that were transgenic for a replication-defective vector. Adenoviral antigens might be seen as self-antigens by these mice, and the mice could exhibit immunologic tolerance after postnatal exposure to adenoviral vectors. In addition, characterization of these mice could reveal potential consequences of germline transmission of an adenoviral vector, as might occur in a gene therapy trial. Injection of a "null" (not containing a transgene) E1, E3-deleted vector genome into mouse zygotes yielded five founders that were capable of transmitting the vector genome. Among offspring of these mice, transgenic pups were significantly underrepresented: 108 of 255 pups (42%) were transgenic (P<0.02 versus expected frequency of 50%). Postnatal transgenic mice, however, had no apparent abnormalities. Persistence of an adenoviral vector after intravenous injection was equivalent in livers of transgenic mice and their nontransgenic littermates. Transgenic and nontransgenic mice also had equivalent humoral and cellular immune responses to adenoviral vector injection. Mice that are transgenic for an E1, E3-deleted adenoviral genome can be easily generated; however, they are not tolerant of adenovirus. Moreover, germline transmission of an adenoviral vector genome does not prevent generation of a robust immune response after exposure to adenoviral antigens.

  4. Bone formation in vivo induced by Cbfa1-carrying adenoviral vectors released from a biodegradable porous β-tricalcium phosphate (β-TCP) material.

    PubMed

    Uemura, Toshimasa; Kojima, Hiroko

    2011-06-01

    Overexpression of Cbfa1 (a transcription factor indispensable for osteoblastic differentiation) is expected to induce the formation of bone directly and indirectly in vivo by accelerating osteoblastic differentiation. Adenoviral vectors carrying the cDNA of Cbfa1/til-1(Adv-Cbf1) were allowed to be adsorbed onto porous blocks of β-tricalcium phosphate (β-TCP), a biodegradable ceramic, which were then implanted subcutaneously and orthotopically into bone defects. The adenoviral vectors were released sustainingly by biodegradation, providing long-term expression of the genes. Results of the subcutaneous implantation of Adv-Cbfa1-adsorbed β-TCP/osteoprogenitor cells suggest that a larger amount of bone formed in the pores of the implant than in the control material. Regarding orthotopic implantation into bone defects, the released Adv-Cbfa1 accelerated regeneration in the cortical bone, whereas it induced bone resorption in the marrow cavity. A safer gene transfer using a smaller amount of the vector was achieved using biodegradable porous β-TCP as a carrier.

  5. Bone formation in vivo induced by Cbfa1-carrying adenoviral vectors released from a biodegradable porous β-tricalcium phosphate (β-TCP) material

    NASA Astrophysics Data System (ADS)

    Uemura, Toshimasa; Kojima, Hiroko

    2011-06-01

    Overexpression of Cbfa1 (a transcription factor indispensable for osteoblastic differentiation) is expected to induce the formation of bone directly and indirectly in vivo by accelerating osteoblastic differentiation. Adenoviral vectors carrying the cDNA of Cbfa1/til-1(Adv-Cbf1) were allowed to be adsorbed onto porous blocks of β-tricalcium phosphate (β-TCP), a biodegradable ceramic, which were then implanted subcutaneously and orthotopically into bone defects. The adenoviral vectors were released sustainingly by biodegradation, providing long-term expression of the genes. Results of the subcutaneous implantation of Adv-Cbfa1-adsorbed β-TCP/osteoprogenitor cells suggest that a larger amount of bone formed in the pores of the implant than in the control material. Regarding orthotopic implantation into bone defects, the released Adv-Cbfa1 accelerated regeneration in the cortical bone, whereas it induced bone resorption in the marrow cavity. A safer gene transfer using a smaller amount of the vector was achieved using biodegradable porous β-TCP as a carrier.

  6. Modification of liposomal concentration in liposome/adenoviral complexes allows significant protection of adenoviral vectors from neutralising antibody, in vitro.

    PubMed

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Dingwall, Daniel J; Kalle, Wouter H J

    2005-06-01

    Adenoviral vectors have been commonly used in gene therapy protocols, however the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced which limits further administration. This study examines the efficacy of complexing liposomes to adenovirus for the protection of the adenovirus from neutralising antibodies in an in vitro setting. Dimethyldioctadecylammonium bromide (DDAB)-dioleoyl-l-phosphatidylethanolamine (DOPE) liposomes were bound at varying concentrations to adenovirus to form AL complexes and tested these complexes' ability to prevent adenoviral neutralisation. It is shown that by increasing the concentration of liposomes in the adenoviral-liposome (AL) complexes we can increase the level of immuno-shielding afforded the adenovirus. It is also shown that the increase in liposomal concentration may lead to drawbacks such as increased cytotoxicity and reductions in expression levels.

  7. Helper-Dependent Adenoviral Vectors.

    PubMed

    Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

    2011-10-29

    Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology.

  8. Helper-Dependent Adenoviral Vectors

    PubMed Central

    Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

    2012-01-01

    Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology. PMID:24533227

  9. Chromosomal integration of adenoviral vector DNA in vivo.

    PubMed

    Stephen, Sam Laurel; Montini, Eugenio; Sivanandam, Vijayshankar Ganesh; Al-Dhalimy, Muhseen; Kestler, Hans A; Finegold, Milton; Grompe, Markus; Kochanek, Stefan

    2010-10-01

    So far there has been no report of any clinical or preclinical evidence for chromosomal vector integration following adenovirus (Ad) vector-mediated gene transfer in vivo. We used liver gene transfer with high-capacity Ad vectors in the FAH(Deltaexon5) mouse model to analyze homologous and heterologous recombination events between vector and chromosomal DNA. Intravenous injection of Ad vectors either expressing a fumarylacetoacetate hydrolase (FAH) cDNA or carrying part of the FAH genomic locus resulted in liver nodules of FAH-expressing hepatocytes, demonstrating chromosomal vector integration. Analysis of junctions between vector and chromosomal DNA following heterologous recombination indicated integration of the vector genome through its termini. Heterologous recombination occurred with a median frequency of 6.72 x 10(-5) per transduced hepatocyte, while homologous recombination occurred more rarely with a median frequency of 3.88 x 10(-7). This study has established quantitative and qualitative data on recombination of adenoviral vector DNA with genomic DNA in vivo, contributing to a risk-benefit assessment of the biosafety of Ad vector-mediated gene transfer.

  10. The evolution of adenoviral vectors through genetic and chemical surface modifications.

    PubMed

    Capasso, Cristian; Garofalo, Mariangela; Hirvinen, Mari; Cerullo, Vincenzo

    2014-02-17

    A long time has passed since the first clinical trial with adenoviral (Ad) vectors. Despite being very promising, Ad vectors soon revealed their limitations in human clinical trials. The pre-existing immunity, the marked liver tropism and the high toxicity of first generation Ad (FG-Ad) vectors have been the main challenges for the development of new approaches. Significant effort toward the development of genetically and chemically modified adenoviral vectors has enabled researchers to create more sophisticated vectors for gene therapy, with an improved safety profile and a higher transduction ability of different tissues. In this review, we will describe the latest findings in the high-speed, evolving field of genetic and chemical modifications of adenoviral vectors, a field in which different disciplines, such as biomaterial research, virology and immunology, co-operate synergistically to create better gene therapy tools for modern challenges.

  11. Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination.

    PubMed

    Campos, Samuel K; Barry, Michael A

    2004-11-01

    There are extensive efforts to develop cell-targeting adenoviral vectors for gene therapy wherein endogenous cell-binding ligands are ablated and exogenous ligands are introduced by genetic means. Although current approaches can genetically manipulate the capsid genes of adenoviral vectors, these approaches can be time-consuming and require multiple steps to produce a modified viral genome. We present here the use of the bacteriophage lambda Red recombination system as a valuable tool for the easy and rapid construction of capsid-modified adenoviral genomes.

  12. Gene Transfer into Rat Brain Using Adenoviral Vectors

    PubMed Central

    Puntel, Mariana; Kroeger, Kurt M.; Sanderson, Nicholas S.R.; Thomas, Clare E.; Castro, Maria G.; Lowenstein, Pedro R.

    2010-01-01

    Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc. PMID:20066657

  13. Regulated Expression of Adenoviral Vectors-Based Gene Therapies

    PubMed Central

    Curtin, James F.; Candolfi, Marianela; Puntel, Mariana; Xiong, Weidong; Muhammad, A. K. M.; Kroeger, Kurt; Mondkar, Sonali; Liu, Chunyan; Bondale, Niyati; Lowenstein, Pedro R.; Castro, Maria G.

    2008-01-01

    Summary Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the “OFF” state, and expression should quickly reach therapeutic levels in the “ON” state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA2S-M2 inducer and tTSKid silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo. PMID:18470649

  14. Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

    PubMed Central

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

  15. Genetically engineering adenoviral vectors for gene therapy.

    PubMed

    Coughlan, Lynda

    2014-01-01

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

  16. Microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro.

    PubMed

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Kalle, Wouter H J

    2004-11-01

    Adenoviral vectors have been commonly used in gene therapy protocols but the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced, which limits further administration. This study examines the effectiveness of a novel combination of microspheres and liposomes for the shielding of adenovirus from neutralising antibodies in an in-vitro setting. We show that liposomes are effective in the protection of adenovirus from neutralising antibody and that the conjugation of these complexes to microspheres augments the level of protection. This study further reveals that previously neutralised adenovirus may still be transported into the cell via liposome-cell interactions and is still capable of expressing its genes, making this vector an effective tool for circumvention of the humoral immune response. We also looked at possible side effects of using the complexes, namely increases in cytotoxicity and reductions in transfection efficiency. Our results showed that varying the liposome:adenovirus ratio can reduce the cytotoxicity of the vector as well as increase the transfection efficiency. In addition, in cell lines that are adenoviral competent, transfection efficiencies on par with uncomplexed adenoviral vectors were achievable with the combination vector.

  17. Adenoviral vector tethering to metal surfaces via hydrolysable cross-linkers for the modulation of vector release and transduction

    PubMed Central

    Fishbein, Ilia; Forbes, Scott P.; Chorny, Michael; Connolly, Jeanne M.; Adamo, Richard F.; Corrales, Ricardo; Alferiev, Ivan S.; Levy, Robert J.

    2013-01-01

    The use of arterial stents and other medical implants as a delivery platform for surface immobilized gene vectors allows for safe and efficient localized expression of therapeutic transgenes. In this study we investigate the use of hydrolysable cross-linkers with distinct kinetics of hydrolysis for delivery of gene vectors from polyallylamine bisphosphonate-modified metal surfaces. Three cross-linkers with the estimated t1/2 of ester bonds hydrolysis of 5, 12 and 50 days demonstrated a cumulative 20%, 39% and 45% vector release, respectively, after 30 days exposure to physiological buffer at 37°C. Transgene expression in endothelial and smooth muscles cells transduced with substrate immobilized adenovirus resulted in significantly different expression profiles for each individual cross-linker. Furthermore, immobilization of adenoviral vectors effectively extended their transduction effectiveness beyond the initial phase of release. Transgene expression driven by adenovirus-tethered stents in rat carotid arteries demonstrated that a faster rate of cross-linker hydrolysis resulted in higher expression levels at day 1, which declined by day 8 after stent implantation, while inversely, slower hydrolysis was associated with increased arterial expression at day 8 in comparison with day 1. In conclusion, adjustable release of transduction-competent adenoviral vectors from metallic surfaces can be achieved, both in vitro and in vivo, through surface immobilization of adenoviral vectors using hydrolysable cross-linkers with structure-specific release kinetics. PMID:23777912

  18. Adenoviral Vector Immunity: Its Implications and circumvention strategies

    PubMed Central

    Ahi, Yadvinder S.; Bangari, Dinesh S.; Mittal, Suresh K.

    2014-01-01

    Adenoviral (Ad) vectors have emerged as a promising gene delivery platform for a variety of therapeutic and vaccine purposes during last two decades. However, the presence of preexisting Ad immunity and the rapid development of Ad vector immunity still pose significant challenges to the clinical use of these vectors. Innate inflammatory response following Ad vector administration may lead to systemic toxicity, drastically limit vector transduction efficiency and significantly abbreviate the duration of transgene expression. Currently, a number of approaches are being extensively pursued to overcome these drawbacks by strategies that target either the host or the Ad vector. In addition, significant progress has been made in the development of novel Ad vectors based on less prevalent human Ad serotypes and nonhuman Ad. This review provides an update on our current understanding of immune responses to Ad vectors and delineates various approaches for eluding Ad vector immunity. Approaches targeting the host and those targeting the vector are discussed in light of their promises and limitations. PMID:21453277

  19. Avidin-Based Targeting and Purification of a Protein IX-Modified, Metabolically Biotinylated Adenoviral Vector

    PubMed Central

    Campos, Samuel K.; Parrott, M. Brandon; Barry, Michael A.

    2014-01-01

    While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (Kd = 10−15 M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (Kd = 10−7 M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors. PMID:15194061

  20. Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE.

    PubMed

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Abu-Asab, Mones S; Tsokos, Maria; Morris, John C; Kalle, Wouter H J

    2007-04-01

    We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

  1. Helper-dependent adenoviral vectors for liver-directed gene therapy

    PubMed Central

    Brunetti-Pierri, Nicola; Ng, Philip

    2011-01-01

    Helper-dependent adenoviral (HDAd) vectors devoid of all viral-coding sequences are promising non-integrating vectors for liver-directed gene therapy because they have a large cloning capacity, can efficiently transduce a wide variety of cell types from various species independent of the cell cycle and can result in long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd for liver-directed gene therapy is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration resulting in acute toxicity, the severity of which is dependent on vector dose. Intense efforts have been focused on elucidating the factors involved in this acute response and various strategies have been investigated to improve the therapeutic index of HDAd vectors. These strategies have yielded encouraging results with the potential for clinical translation. PMID:21470977

  2. Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography.

    PubMed

    Eglon, Marc N; Duffy, Aoife M; O'Brien, Timothy; Strappe, Padraig M

    2009-11-01

    Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. HEK-293 cells were cultured in ten-layer CellStacks() and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP). Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader. Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF-AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX-GF) resulted in a vector yield of 2.3 x 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 10(7) pfu/cm(2) for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation.

  3. Development of an adenoviral vector with robust expression driven by p53

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bajgelman, Marcio C.; Biotechnology Program, Biomedical Sciences Institute, University of Sao Paulo; Millennium Institute-Gene Therapy Network, Ministry of Science and Technology

    2008-02-05

    Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG servedmore » as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.« less

  4. Chimpanzee adenoviral vectors as vaccines for outbreak pathogens

    PubMed Central

    2017-01-01

    ABSTRACT The 2014–15 Ebola outbreak in West Africa highlighted the potential for large disease outbreaks caused by emerging pathogens and has generated considerable focus on preparedness for future epidemics. Here we discuss drivers, strategies and practical considerations for developing vaccines against outbreak pathogens. Chimpanzee adenoviral (ChAd) vectors have been developed as vaccine candidates for multiple infectious diseases and prostate cancer. ChAd vectors are safe and induce antigen-specific cellular and humoral immunity in all age groups, as well as circumventing the problem of pre-existing immunity encountered with human Ad vectors. For these reasons, such viral vectors provide an attractive platform for stockpiling vaccines for emergency deployment in response to a threatened outbreak of an emerging pathogen. Work is already underway to develop vaccines against a number of other outbreak pathogens and we will also review progress on these approaches here, particularly for Lassa fever, Nipah and MERS. PMID:29083948

  5. Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.

    PubMed

    Kibbe, M R; Murdock, A; Wickham, T; Lizonova, A; Kovesdi, I; Nie, S; Shears, L; Billiar, T R; Tzeng, E

    2000-02-01

    Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ineffective viral-target cell interactions. Viral attachment, which largely determines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while internalization follows binding of the adenoviral RGD motif to alpha(v)-integrin receptors. Modifications of the fiber coat protein sequence have been successful for targeting the adenovirus to more prevalent receptors in the vasculature, including heparan sulfate-containing receptors and alpha(v)-integrin receptors. Modified adenoviral vectors targeted to receptors more prevalent in the vasculature result in an increased transfer efficiency of the virus in vitro and in vivo even in the presence of clinically relevant doses of heparin. We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple positively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-binding sequence in the fiber coat protein that allows binding to alpha(v)-integrin receptors. The gene transfer efficiency of these modified viruses was compared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin during most vascular surgical procedures and the concern that heparin might interfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the effect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosidase expression compared with the unmodified adenovirus (mean +/- SEM, 1766.3 +/- 89.1 and 44

  6. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons

    PubMed Central

    Morral, Núria; O’Neal, Wanda; Rice, Karen; Leland, Michele; Kaplan, Johanne; Piedra, Pedro A.; Zhou, Heshan; Parks, Robin J.; Velji, Rizwan; Aguilar-Córdova, Estuardo; Wadsworth, Samuel; Graham, Frank L.; Kochanek, Stefan; Carey, K. Dee; Beaudet, Arthur L.

    1999-01-01

    The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes. PMID:10536005

  7. Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

    PubMed Central

    Campos, Samuel K.; Barry, Michael A.

    2008-01-01

    Gene delivery vectors based on Adenoviral (Ad) vectors have enormous potential for the treatment of both hereditary and acquired disease. Detailed structural analysis of the Ad virion, combined with functional studies has broadened our knowledge of the structure/function relationships between Ad vectors and host cells/tissues and substantial achievement has been made towards a thorough understanding of the biology of Ad vectors. The widespread use of Ad vectors for clinical gene therapy is compromised by their inherent immunogenicity. The generation of safer and more effective Ad vectors, targeted to the site of disease, has therefore become a great ambition in the field of Ad vector development. This review provides a synopsis of the structure/function relationships between Ad vectors and host systems and summarizes the many innovative approaches towards achieving Ad vector targeting. PMID:17584037

  8. Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

    PubMed Central

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-01-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

  9. Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines.

    PubMed

    Duisit, G; Salvetti, A; Moullier, P; Cosset, F L

    1999-01-20

    We have generated three different E1-deleted replication-defective adenoviral vectors expressing either Moloney murine leukemia virus (Mo-MuLV) Gag-Pol core particle proteins, gibbon ape leukemia virus (GALV) envelope glycoproteins, or an MuLV-derived retroviral vector genome encoding mCD2 antigen, a murine cell surface marker easily detectable by flow cytometry. Each of the three vectors was first characterized individually by infection of cells providing the complementary retroviral function(s) and able to induce the production of retroviral vectors with an efficiency similar to or higher than that of FLY stable retroviral packaging cells [Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R.A., and Collins, M.K.L., (1995). J. Virol. 69, 7430-7436]. In small-scale pilot experiments, TE671 cells simultaneously coinfected with the three adenoviral vectors efficiently released helper-free retroviral vectors in their supernatant, with titers greater than 10(6) infectious particles per milliliter by end-point titrations. Our results also indicated that in contrast to retroviral vector-packageable RNAs, the adenovirus-mediated overexpression of both Gag-Pol and Env packaging functions had limited impact on retroviral titers. The primary mechanism suspected is the premature intracellular cleavage of the Pr65gag precursor that we found in gag-pol-expressing cells, which in turn may impair the normal incorporation of high loads of functional Env. Last, the characterization of the adenoviral/retroviral chimeric vectors allowed the screening of various primate cells for retroviral production and we found that three hepatocyte-derived cell lines were highly efficient in the assembly and release of infectious retroviral particles.

  10. Vaccination with an adenoviral vector that encodes and displays a retroviral antigen induces improved neutralizing antibody and CD4+ T-cell responses and confers enhanced protection.

    PubMed

    Bayer, Wibke; Tenbusch, Matthias; Lietz, Ruth; Johrden, Lena; Schimmer, Simone; Uberla, Klaus; Dittmer, Ulf; Wildner, Oliver

    2010-02-01

    We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env- and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4(+) T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.

  11. Combination recombinant simian or chimpanzee adenoviral vectors for vaccine development.

    PubMed

    Cheng, Cheng; Wang, Lingshu; Ko, Sung-Youl; Kong, Wing-Pui; Schmidt, Stephen D; Gall, Jason G D; Colloca, Stefano; Seder, Robert A; Mascola, John R; Nabel, Gary J

    2015-12-16

    Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development. Published by Elsevier Ltd.

  12. Pancreatic Transduction by Helper-Dependent Adenoviral Vectors via Intraductal Delivery

    PubMed Central

    Morró, Meritxell; Teichenne, Joan; Jimenez, Veronica; Kratzer, Ramona; Marletta, Serena; Maggioni, Luca; Mallol, Cristina; Ruberte, Jesus; Kochanek, Stefan; Bosch, Fatima

    2014-01-01

    Abstract Pancreatic gene transfer could be useful to treat several diseases, such as diabetes mellitus, cystic fibrosis, chronic pancreatitis, or pancreatic cancer. Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, we have generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days postinjection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days postinjection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery. These data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue. PMID:25046147

  13. In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.

    PubMed

    Mathison, Megumi; Singh, Vivek P; Chiuchiolo, Maria J; Sanagasetti, Deepthi; Mao, Yun; Patel, Vivekkumar B; Yang, Jianchang; Kaminsky, Stephen M; Crystal, Ronald G; Rosengart, Todd K

    2017-02-01

    The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05). Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans. Copyright © 2016 The American

  14. Development of nonhuman adenoviruses as vaccine vectors

    PubMed Central

    Bangari, Dinesh S.; Mittal, Suresh K.

    2006-01-01

    Human adenoviral (HAd) vectors have demonstrated great potential as vaccine vectors. Preclinical and clinical studies have demonstrated the feasibility of vector design, robust antigen expression and protective immunity using this system. However, clinical use of adenoviral vectors for vaccine purposes is anticipated to be limited by vector immunity that is either preexisting or develops rapidly following the first inoculation with adenoviral vectors. Vector immunity inactivates the vector particles and rapidly removes the transduced cells, thereby limiting the duration of transgene expression. Due to strong vector immunity, subsequent use of the same vector is usually less efficient. In order to circumvent this limitation, nonhuman adenoviral vectors have been proposed as alternative vectors. In addition to eluding HAd immunity, these vectors possess most of the attractive features of HAd vectors. Several replication-competent or replication-defective nonhuman adenoviral vectors have been developed and investigated for their potential as vaccine delivery vectors. Here, we review recent advances in the design and characterization of various nonhuman adenoviral vectors, and discuss their potential applications for human and animal vaccination. PMID:16297508

  15. Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

    PubMed

    Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L

    2016-07-01

    The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases.

  16. MicroRNA Silencing Improves the Tumor Specificity of Adenoviral Transgene Expression

    PubMed Central

    Card, Paul B.; Hogg, Richard T.; del Alcazar, Carlos Gil

    2012-01-01

    Adenoviral technology has been thoroughly evaluated for delivering genetic material to tumor tissue and the surrounding microenvironment. Almost any gene can be cloned into an adenovirus (Ad) vector, which when combined with strong, constitutively active promoters permit up to a million-fold amplification of the transgene in a single adenoviral particle, thus facilitating their use in cancer therapy and imaging. However, widespread infection of the liver and other non-targeted tissues by Ad vectors is a substantial problem that often results in significant liver inflammation and hepatotoxicity at doses required to achieve efficient tumor transduction. miR-122 is a highly expressed liver-specific microRNA that provides a unique opportunity for down-regulating adenoviral transgene expression in liver tissue. The binding of endogenous miRNAs to complementary miRNA targeting elements (miRTs) incorporated into the 3′ untranslated region of adenoviral transgenes interferes with message stability and/or protein translation, and miRT elements against miR-122 (miRT-122) can selectively reduce adenoviral transgene expression in the liver. Previous studies using miR-122-based regulation, with and without other types of transcriptional targeting, have yielded promising preliminary results. However, investigations to date evaluating miRT-122 elements for improving tumor specificity have used either non-tumor bearing animals or direct intratumoral injection as the mode of delivery. In the present study, we confirmed the ability of miRT-122 sequences to selectively down-regulate adenoviral luciferase expression in the liver in vitro and in vivo, and show that this strategy can improve tumor specific transgene expression in a HT1080 human fibrosarcoma model. Rapid growth and the inefficient flow of blood through tumor neovasculature often results in profound hypoxia, which provides additional opportunities for targeting solid tumors and their microenvironment using vectors

  17. A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma.

    PubMed

    Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Morshed, Ramin A; Miska, Jason; Ahmed, Atique U; Balyasnikova, Irina V; Han, Yu; Zhang, Lingjiao; Curiel, David T; Lesniak, Maciej S

    2015-09-01

    The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy.

  18. Transducing Airway Basal Cells with a Helper-Dependent Adenoviral Vector for Lung Gene Therapy.

    PubMed

    Cao, Huibi; Ouyang, Hong; Grasemann, Hartmut; Bartlett, Claire; Du, Kai; Duan, Rongqi; Shi, Fushan; Estrada, Marvin; Seigel, Kyle E; Coates, Allan L; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Moraes, Theo J; Hu, Jim

    2018-06-01

    A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells, of which basal cells are the major type in human airways. In this study, helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery, and direct bronchoscopic instillation, respectively. Vector transduction was assessed by immunostaining of lung tissue sections, which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition, efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore, we successfully delivered the human CFTR gene to airway basal cells from CF patients, and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases.

  19. Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.

    PubMed

    Armendáriz-Borunda, Juan; Bastidas-Ramírez, Blanca Estela; Sandoval-Rodríguez, Ana; González-Cuevas, Jaime; Gómez-Meda, Belinda; García-Bañuelos, Jesús

    2011-11-01

    Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

    PubMed Central

    2011-01-01

    Background Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. Methods In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. Results The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. Conclusion The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin. PMID:21255430

  1. [Experimental study of human umbilical cord blood derived stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1 and GFP].

    PubMed

    Zhang, Xi; Si, Ying-Jian; Chen, Xing-Hua; Liu, Yao; Gao, Li; Gao, Lei; Peng, Xian-Gui; Wang, Qing-Yu

    2008-06-01

    This study was aimed to investigate the effect of vcam-1 gene-modified human umbilical cord blood derived stromal cells (CBDSCs) on hematopoietic regulation so as to establish the experimental foundation for further study. The target gene vcam-1 was cloned into the shuttle plasmid with the report gene GFP. The recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l, and the recombinant adenoviral vector ad-vcam-1-gfp was confirmed after transfection with CBDSCs. The results indicated that two fragments of about 9 kb and 2 kb were obtained after digestion of recombinant plasmid pAdTrack-vcam-1 with NotIand XhoI, and single fragment of 600 bp was obtained after amplification with PCR; two fragments of about 31 kb and 4 kb were obtained after digestion of recombinant plasmid pad-vcam-1-gfp with PacI, which suggested a successful homologous recombination. The expression of vcam-1 gene in ad-vcam-1-gfp transfected CBDSCs could be detected by immunocytochemistry, RT-PCR and fluorescent microscopy. It is concluded that the recombinant adenoviral vector ad-vcam-1-gfp has been constructed successfully, and the expression of vcam-1 is up-regulated in CBDSCs transfected by gene ad-vcam-1-gfp.

  2. Radiolabeled Adenoviral Sub-unit Proteins for Molecular Imaging and Therapeutic Applications in Oncology

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Srivastava, S.; Meinken, G.; Springer, K. Awasthi, V.

    2004-10-06

    The objective of this project was to develop and optimize new ligand systems, based on adenoviral vectors (intact adenovirus, adeno-viral fiber protein, and the knob protein), for delivering suitable radionuclides into tumor cells for molecular imaging and combined gene/radionuclide therapy of cancer.

  3. Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors.

    PubMed

    Penny, Gervette M; Cochran, Rebecca B; Pihlajoki, Marjut; Kyrönlahti, Antti; Schrade, Anja; Häkkinen, Merja; Toppari, Jorma; Heikinheimo, Markku; Wilson, David B

    2017-10-01

    Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse Gata4 and Gata6 , two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult Gata4 flox/flox ; Gata6 flox/flox mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes ( Hsd3b1 , Cyp17a1 and Hsd17b3 ) was reduced, whereas expression of another Leydig cell marker, Insl3 , was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function in vivo Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.Free Finnish abstract: A Finnish translation of this abstract is freely available at http://www.reproduction-online.org/content/154/4/455/suppl/DC2. © 2017 Society for Reproduction and Fertility.

  4. Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector.

    PubMed

    Sasaki, Makoto; Mathis, J Michael; Jennings, Merilyn H; Jordan, Paul; Wang, Yuping; Ando, Tomoaki; Joh, Takashi; Alexander, J Steven

    2005-10-31

    Genetic deficiency in the expression of interleukin-10 (IL-10) is associated with the onset and progression of experimental inflammatory bowel disease (IBD). The clinical significance of IL-10 expression is supported by studies showing that immune-augmentation of IL-10 prevents inflammation and mucosal damage in animal models of colitis and in human colitis. Interleukin-10 (IL-10), an endogenous anti-inflammatory and immunomodulating cytokine, has been shown to prevent some inflammation and injury in animal and clinical studies, but the efficacy of IL-10 treatment remains unsatisfactory. We found that intra-peritoneal administration of adenoviral IL-10 to mice significantly reversed colitis induced by administration of 3% DSS (dextran sulfate), a common model of colitis. Adenoviral IL-10 (Ad-IL10) transfected mice developed high levels of IL-10 (394 +/- 136 pg/ml) within the peritoneal cavity where the adenovirus was expressed. Importantly, when given on day 4 (after the induction of colitis w/DSS), Ad-IL10 significantly reduced disease activity and weight loss and completely prevented histopathologic injury to the colon at day 10. Mechanistically, compared to Ad-null and DSS treated mice, Ad-IL10 and DSS-treated mice were able to suppress the expression of MAdCAM-1, an endothelial adhesion molecule associated with IBD. Our results suggest that Ad-IL10 (adenoviral IL-10) gene therapy of the intestine or peritoneum may be useful in the clinical treatment of IBD, since we demonstrated that this vector can reverse the course of an existing gut inflammation and markers of inflammation.

  5. Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector

    PubMed Central

    Sasaki, Makoto; Mathis, J Michael; Jennings, Merilyn H; Jordan, Paul; Wang, Yuping; Ando, Tomoaki; Joh, Takashi; Alexander, J Steven

    2005-01-01

    Genetic deficiency in the expression of interleukin-10 (IL-10) is associated with the onset and progression of experimental inflammatory bowel disease (IBD). The clinical significance of IL-10 expression is supported by studies showing that immune-augmentation of IL-10 prevents inflammation and mucosal damage in animal models of colitis and in human colitis. Interleukin-10 (IL-10), an endogenous anti-inflammatory and immunomodulating cytokine, has been shown to prevent some inflammation and injury in animal and clinical studies, but the efficacy of IL-10 treatment remains unsatisfactory. We found that intra-peritoneal administration of adenoviral IL-10 to mice significantly reversed colitis induced by administration of 3% DSS (dextran sulfate), a common model of colitis. Adenoviral IL-10 (Ad-IL10) transfected mice developed high levels of IL-10 (394 +/- 136 pg/ml) within the peritoneal cavity where the adenovirus was expressed. Importantly, when given on day 4 (after the induction of colitis w/DSS), Ad-IL10 significantly reduced disease activity and weight loss and completely prevented histopathologic injury to the colon at day 10. Mechanistically, compared to Ad-null and DSS treated mice, Ad-IL10 and DSS-treated mice were able to suppress the expression of MAdCAM-1, an endothelial adhesion molecule associated with IBD. Our results suggest that Ad-IL10 (adenoviral IL-10) gene therapy of the intestine or peritoneum may be useful in the clinical treatment of IBD, since we demonstrated that this vector can reverse the course of an existing gut inflammation and markers of inflammation. PMID:16259632

  6. Upgrading HepG2 cells with adenoviral vectors that encode drug-metabolizing enzymes: application for drug hepatotoxicity testing.

    PubMed

    Gómez-Lechón, M José; Tolosa, Laia; Donato, M Teresa

    2017-02-01

    Drug attrition rates due to hepatotoxicity are an important safety issue considered in drug development. The HepG2 hepatoma cell line is currently being used for drug-induced hepatotoxicity evaluations, but its expression of drug-metabolizing enzymes is poor compared with hepatocytes. Different approaches have been proposed to upgrade HepG2 cells for more reliable drug-induced liver injury predictions. Areas covered: We describe the advantages and limitations of HepG2 cells transduced with adenoviral vectors that encode drug-metabolizing enzymes for safety risk assessments of bioactivable compounds. Adenoviral transduction facilitates efficient and controlled delivery of multiple drug-metabolizing activities to HepG2 cells at comparable levels to primary human hepatocytes by generating an 'artificial hepatocyte'. Furthermore, adenoviral transduction enables the design of tailored cells expressing particular metabolic capacities. Expert opinion: Upgraded HepG2 cells that recreate known inter-individual variations in hepatic CYP and conjugating activities due to both genetic (e.g., polymorphisms) or environmental (e.g., induction, inhibition) factors seems a suitable model to identify bioactivable drug and conduct hepatotoxicity risk assessments. This strategy should enable the generation of customized cells by reproducing human pheno- and genotypic CYP variability to represent a valuable human hepatic cell model to develop new safer drugs and to improve existing predictive toxicity assays.

  7. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

    PubMed

    Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

    2017-12-07

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

  8. HIV-1 adenoviral vector vaccines expressing multi-trimeric BAFF and 4-1BBL enhance T cell mediated anti-viral immunity.

    PubMed

    Kanagavelu, Saravana; Termini, James M; Gupta, Sachin; Raffa, Francesca N; Fuller, Katherine A; Rivas, Yaelis; Philip, Sakhi; Kornbluth, Richard S; Stone, Geoffrey W

    2014-01-01

    Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia

  9. Adenoviral Gene Delivery to Primary Human Cutaneous Cells and Burn Wounds

    PubMed Central

    Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

    2006-01-01

    The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2 × 108 pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P > 0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P = 0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting

  10. Adenoviral gene delivery to primary human cutaneous cells and burn wounds.

    PubMed

    Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

    2006-01-01

    The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has

  11. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

    PubMed

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-04-01

    Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

  12. Guinea pig adenovirus infection does not inhibit cochlear transfection with human adenoviral vectors in a model of hearing loss.

    PubMed

    Hankenson, F Claire; Wathen, Asheley B; Eaton, Kathryn A; Miyazawa, Toru; Swiderski, Donald L; Raphael, Yehoash

    2010-04-01

    Routine surveillance of guinea pigs maintained within a barrier facility detected guinea pig adenovirus (GPAdV) in sentinel animals. These guinea pigs served as models of induced hearing loss followed by regeneration of cochlear sensory (hair) cells through transdifferentiation of nonsensory cells by using human adenoviral (hAV) gene therapy. To determine whether natural GPAdV infection affected the ability of hAV vectors to transfect inner ear cells, adult male pigmented guinea pigs (n = 7) were enrolled in this study because of their prolonged exposure to GPAdV-seropositive conspecifics. Animals were deafened chemically (n = 2), received an hAV vector carrying the gene for green fluorescent protein (hAV-GFP) surgically without prior deafening (n = 2), or were deafened chemically with subsequent surgical inoculation of hAV-GFP (n = 3). Cochleae were evaluated by using fluorescence microscopy, and GFP expression in supporting cells indicated that the hAV-GFP vector was able to transfect inner ears in GPAdV-seropositive guinea pigs that had been chemically deafened. Animals had histologic evidence of interstitial pneumonia, attributable to prior infection with GPAdV. These findings confirmed that the described guinea pigs were less robust animal models with diminished utility for the overall studies. Serology tests confirmed that 5 of 7 animals (71%) were positive for antibodies against GPAdV at necropsy, approximately 7 mo after initial detection of sentinel infection. Control animals (n = 5) were confirmed to be seronegative for GPAdV with clinically normal pulmonary tissue. This study is the first to demonstrate that natural GPAdV infection does not negatively affect transfection with hAV vectors into guinea pig inner ear cells, despite the presence of other health complications attributed to the viral infection.

  13. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma

    PubMed Central

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P.; Al-Hendy, Ayman

    2016-01-01

    Background: Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. Study design: An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Materials and methods: Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. Results: In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Conclusion: Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. PMID:26884457

  14. Inhibition of Choroidal Neovascularization by Intravenous Injection of Adenoviral Vectors Expressing Secretable Endostatin

    PubMed Central

    Mori, Keisuke; Ando, Akira; Gehlbach, Peter; Nesbitt, David; Takahashi, Kyoichi; Goldsteen, Donna; Penn, Michael; Chen, Cheauyan T.; Mori, Keiko; Melia, Michele; Phipps, Sandrina; Moffat, Diana; Brazzell, Kim; Liau, Gene; Dixon, Katharine H.; Campochiaro, Peter A.

    2001-01-01

    Endostatin is a cleavage product of collagen XVIII that inhibits tumor angiogenesis and growth. Interferon α2a blocks tumor angiogenesis and causes regression of hemangiomas, but has no effect on choroidal neovascularization (CNV). Therefore, inhibitors of tumor angiogenesis do not necessarily inhibit ocular neovascularization. In this study, we used an intravenous injection of adenoviral vectors containing a sig-mEndo transgene consisting of murine immunoglobulin κ-chain leader sequence coupled to sequence coding for murine endostatin to investigate the effect of high serum levels of endostatin on CNV in mice. Mice injected with a construct in which sig-mEndo expression was driven by the Rous sarcoma virus promoter had moderately high serum levels of endostatin and significantly smaller CNV lesions at sites of laser-induced rupture of Bruch’s membrane than mice injected with null vector. Mice injected with a construct in which sig-mEndo was driven by the simian cytomegalovirus promoter had ∼10-fold higher endostatin serum levels and had nearly complete prevention of CNV. There was a strong inverse correlation between endostatin serum level and area of CNV. This study provides proof of principle that gene therapy to increase levels of endostatin can prevent the development of CNV and may provide a new treatment for the leading cause of severe loss of vision in patients with age-related macular degeneration. PMID:11438478

  15. In vivo marking of spontaneous or vaccine-induced fibrosarcomas in the domestic house cat, using an adenoviral vector containing a bifunctional fusion protein, GAL-TEK.

    PubMed

    Marini, F C; Cannon, J P; Belmont, J W; Shillitoe, E J; Lapeyre, J N

    1995-09-01

    We evaluated the ability of a replication-deficient, recombinant adenoviral vector to transfer the bifunctional gene GAL-TEK, which expresses a marking/therapeutic gene product, to naturally occurring cat fibrosarcomas in situ. GAL-TEK contains an in-frame fusion of the bacterial LacZ gene for histochemical marking of tumors with beta-galactosidase (beta-Gal) and the HSV tk gene for enzyme-prodrug activation of the prodrug ganciclovir (GCV) to induce selective tumor cell killing. GAL-TEK bifunctional marking and cell killing activities were tested in vitro after adenoviral vector infection of HT1080 human fibrosarcoma cells. The tk activity of GAL-TEK is shown to be almost as potent as HSV tk to catalyze conversion of GCV to GCV nucleotides and promote selective cell killing. Using 8 cats with recurring 2.5-cm2 fibrosarcomas that either arose spontaneously or were induced by vaccine, we determined experimentally the administration routes and times required for optimum GAL-TEK gene transfer by beta-Gal histological staining and reverse transcriptase polymerase chain reaction to the multiple compartments of the growing fibrosarcomas consonant with minimizing collateral infection of neighboring tissues and other unwanted side effects.

  16. Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1

    PubMed Central

    Castello, Raffaele; Borzone, Roberta; D’Aria, Stefania; Annunziata, Patrizia; Piccolo, Pasquale; Brunetti-Pierri, Nicola

    2015-01-01

    Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT) which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate which ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared to saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with Ethylene Glycol (EG), a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy. PMID:26609667

  17. Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction.

    PubMed

    Broaddus, W C; Liu, Y; Steele, L L; Gillies, G T; Lin, P S; Loudon, W G; Valerie, K; Schmidt-Ullrich, R K; Fillmore, H L

    1999-12-01

    The goal of this study was to determine whether adenoviral vector-mediated expression of human wildtype p53 can enhance the radiosensitivity of malignant glioma cells that express native wild-type p53. The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth. Recent studies suggest that reconstitution of wild-type p53 can render cells more susceptible to radiation-mediated death, primarily by p53-mediated apoptosis. Rat RT2 glioma cells were analyzed for native p53 status by reverse transcriptase-polymerase chain reaction and sequence analysis and for p53 expression by Western blot analysis. Clonogenic survival and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were used to characterize RT2 cell radiosensitivity and apoptosis, respectively, with and without prior transduction with p53-containing and control adenoviral vectors. Animal survival length was monitored after intracerebral implantation with transduced and nontransduced RT2 cells, with and without cranial radiation. The RT2 cells were demonstrated to express native rat wild-type p53 and to markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation resulted in marked decreases in RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. Animals receiving cranial radiation after intracerebral implantation with RT2 cells previously transduced with p53 survived significantly longer than control animals (p<0.01). The ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 offers hope for this approach as a therapeutic strategy

  18. Efficacy and site-specificity of adenoviral vector integration mediated by the phage φC31 integrase.

    PubMed

    Robert, Marc-André; Zeng, Yue; Raymond, Benoît; Desfossé, Laurie; Mairey, Emilie; Tremblay, Jacques P; Massie, Bernard; Gilbert, Rénald

    2012-12-01

    Adenoviral vectors deleted of all their viral genes (helper-dependent [HD]) are efficient gene-transfer vehicles. Because transgene expression is rapidly lost in actively dividing cells, we investigated the feasibility of using phage φC31 integrase (φC31-Int) to integrate an HD carrying an attB site and the puromycin resistance gene into human cells (HeLa) and murine myoblasts (C2C12) by co-infection with a second HD-expressing φC31-Int. Because the HD genome is linear, we also investigated whether its circularization, through expression of Cre using a third HD, affects integration. Efficacy and specificity were determined by scoring the number of puromycin-resistant colonies and by sequencing integration sites. Unexpectedly, circularization of HD was unnecessary and it even reduced the integration efficacy. The maximum integration efficacy achieved was 0.5% in HeLa cells and 0.1% in C2C12 myoblasts. Up to 76% of the integration events occurred at pseudo attP sites and previously characterized hotspots were found. A small (two- to three-fold) increase in the number of γ-H2AX positive foci, accompanied by no noticeable change in γ-H2AX expression, indicated the low genotoxicity of φC31-Int. In conclusion, integration of HD mediated by φC31-Int is an attractive alternative to engineer cells, because it permits site-specific integration of large DNA fragments with low genotoxicity.

  19. Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques.

    PubMed

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides

  20. Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV-1-Specific T-Cell Responses in Macaques

    PubMed Central

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN (‘A’), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 (‘P’), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques

  1. Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via "Antigen Capsid-Incorporation" strategy.

    PubMed

    Gu, Linlin; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Farrow, Anitra L; Derdeyn, Cynthia A; Matthews, Qiana L

    2016-01-01

    Adenoviral (Ad) vectors in combination with the "Antigen Capsid-Incorporation" strategy have been applied in developing HIV-1 vaccines, due to the vectors׳ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the "Antigen Capsid-Incorporation" strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2. Copyright © 2015. Published by Elsevier Inc.

  2. A Potent, Imaging Adenoviral Vector Driven by the Cancer-selective Mucin-1 Promoter That Targets Breast Cancer Metastasis

    PubMed Central

    Huyn, Steven T.; Burton, Jeremy B.; Sato, Makoto; Carey, Michael; Gambhir, Sanjiv S.; Wu, Lily

    2009-01-01

    Purpose With breast cancer, early detection and proper staging are critical, and will often influence both the treatment regimen and the therapeutic outcome for those affected with this disease. Improvements in these areas will play a profound role in reducing mortality from breast cancer. Experimental Design In this work we developed a breast cancer – targeted serotype 5 adenoviral vector, utilizing the tumor-specific mucin-1 promoter in combination with the two-step transcriptional amplification system, a system used to augment the activity of weak tissue – specific promoters. Results We showed the strong specificity of this tumor-selective adenovirus to express the luciferase optical imaging gene, leading to diagnostic signals that enabled detection of sentinel lymph node metastasis of breast cancer. Furthermore, we were able to target hepatic metastases following systemic administration of this mucin-1 selective virus. Conclusions Collectively, we showed that the amplified mucin-1 promoter – driven vector is able to deliver to and selectively express a desirable transgene in metastatic lesions of breast tumors. This work has strong clinical relevance to current diagnostic staging approaches, and could add to targeted therapeutic strategies to advance the fight against breast cancer. PMID:19366829

  3. Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging

    PubMed Central

    Sato, M; Figueiredo, ML; Burton, JB; Johnson, M; Chen, M; Powell, R; Gambhir, SS; Carey, M; Wu, L

    2009-01-01

    Effective treatment for recurrent, disseminated prostate cancer is notably limited. We have developed adenoviral vectors with a prostate-specific two-step transcriptional amplification (TSTA) system that would express therapeutic genes at a robust level to target metastatic disease. The TSTA system employs the prostate-specific antigen (PSA) promoter/enhancer to drive a potent synthetic activator, which in turn activates the expression of the therapeutic gene. In this study, we explored different configurations of this bipartite system and discovered that physical separation of the two TSTA components into E1 and E3 regions of adenovirus was able to enhance androgen regulation and cell-discriminatory expression. The TSTA vectors that express imaging reporter genes were assessed by noninvasive imaging technologies in animal models. The improved selectivity of the E1E3 configured vector was reflected in silenced ectopic expression in the lung. Significantly, the enhanced specificity of the E1E3 vector enabled the detection of lung metastasis of prostate cancer. An E1E3 TSTA vector that expresses the herpes simplex virus thymidine kinase gene can effectively direct positron emission tomography (PET) imaging of the tumor. The prostate-targeted gene delivery vectors with robust and cell-specific expression capability will advance the development of safe and effective imaging guided therapy for recurrent metastatic stages of prostate cancer. PMID:18305574

  4. Improvement in adenoviral gene transfer efficiency after preincubation at +37 degrees C in vitro and in vivo.

    PubMed

    Kossila, Maija; Jauhiainen, Suvi; Laukkanen, Mikko O; Lehtolainen, Pauliina; Jääskeläinen, Maiju; Turunen, Päivi; Loimas, Sami; Wahlfors, Jarmo; Ylä-Herttuala, Seppo

    2002-01-01

    Adenovirus is a widely used vector in gene transfer experiments because it produces high transduction efficiency in vitro and in vivo by means of the coxsackie-adenovirus receptor (CAR) and major histocompatibility complex (MHC) class I alpha-2 domain. Adenoviral gene transfer efficiency has been reported to correlate with cellular CAR expression. We report here a simple method to increase adenoviral gene transfer efficiency in cells that do not express high levels of CAR: preincubation of adenovirus for 30-40 minutes at +37 degrees C significantly increased the transduction efficiency in vitro in CHO and BALB/3T3 cells, in which CAR is expressed at very low levels. Increased transduction efficiency of preincubated adenovirus was also detected in vivo in rat brain tissue. In addition, we found that adenoviruses were rapidly inactivated in human serum in a complement-independent manner, whereas fetal bovine serum (FBS) had hardly any effects on the viral infectivity. We conclude that preincubation of adenoviral vectors at +37 degrees C may substantially increase gene transfer efficiency in applications in which target cells do not express high levels of CAR.

  5. Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes.

    PubMed

    Pohle, D; Kasch, R; Herlyn, P; Bader, R; Mittlmeier, T; Pützer, B M; Müller-Hilke, B

    2012-09-01

    The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins. Copyright © 2012 Wiley Periodicals, Inc.

  6. Comparative Analysis of the Magnitude, Quality, Phenotype and Protective Capacity of SIV Gag-Specific CD8+ T Cells Following Human-, Simian- and Chimpanzee-Derived Recombinant Adenoviral Vector Immunisation

    PubMed Central

    Quinn, Kylie M.; Costa, Andreia Da; Yamamoto, Ayako; Berry, Dana; Lindsay, Ross W.B.; Darrah, Patricia A.; Wang, Lingshu; Cheng, Cheng; Kong, Wing-Pui; Gall, Jason G.D.; Nicosia, Alfredo; Folgori, Antonella; Colloca, Stefano; Cortese, Riccardo; Gostick, Emma; Price, David A.; Gomez, Carmen E.; Esteban, Mariano; Wyatt, Linda S.; Moss, Bernard; Morgan, Cecilia; Roederer, Mario; Bailer, Robert T.; Nabel, Gary J.; Koup, Richard A.; Seder, Robert A.

    2013-01-01

    Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8+ T cell-mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. Here we show low seroreactivity in humans against simian- (sAd11, sAd16), or chimpanzee-derived (chAd3, chAd63) compared to human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype and protective capacity of CD8+ T cell responses in mice vaccinated with rAds encoding SIV Gag. Using a dose range (1 × 107 to 109 PU), we defined a hierarchy among rAd vectors based on the magnitude and protective capacity of CD8+ T cell responses, from most to least as: rAd5 and chAd3, rAd28 and sAd11, chAd63, sAd16, and rAd35. Selection of rAd vector or dose could modulate the proportion and/or frequency of IFNγ+TNFα+IL-2+ and KLRG1+CD127- CD8+ T cells, but strikingly ~30–80% of memory CD8+ T cells co-expressed CD127 and KLRG1. To further optimise CD8+ T cell responses, we assessed rAds as part of prime-boost regimens. Mice primed with rAds and boosted with NYVAC generated Gag-specific responses that approached ~60% of total CD8+ T cells at peak. Alternatively, priming with DNA or rAd28 and boosting with rAd5 or chAd3 induced robust and equivalent CD8+ T cell responses compared to prime or boost alone. Collectively, these data provide the immunologic basis for using specific rAd vectors alone or as part of prime-boost regimens to induce CD8+ T cells for rapid effector function or robust long-term memory, respectively. PMID:23390298

  7. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Naumov, Inna; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; Kazanov, Dina

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1more » and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.« less

  8. Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors

    PubMed Central

    Chorny, Michael; Fishbein, Ilia; Tengood, Jillian E.; Adamo, Richard F.; Alferiev, Ivan S.; Levy, Robert J.

    2013-01-01

    Gene therapeutic strategies have shown promise in treating vascular disease. However, their translation into clinical use requires pharmaceutical carriers enabling effective, site-specific delivery as well as providing sustained transgene expression in blood vessels. While replication-deficient adenovirus (Ad) offers several important advantages as a vector for vascular gene therapy, its clinical applicability is limited by rapid inactivation, suboptimal transduction efficiency in vascular cells, and serious systemic adverse effects. We hypothesized that novel zinc oleate-based magnetic nanoparticles (MNPs) loaded with Ad would enable effective arterial cell transduction by shifting vector processing to an alternative pathway, protect Ad from inactivation by neutralizing factors, and allow site-specific gene transfer to arteries treated with stent angioplasty using a 2-source magnetic guidance strategy. Ad-loaded MNPs effectively transduced cultured endothelial and smooth muscle cells under magnetic conditions compared to controls and retained capacity for gene transfer after exposure to neutralizing antibodies and lithium iodide, a lytic agent causing disruption of free Ad. Localized arterial gene expression significantly stronger than in control animal groups was demonstrated after magnetically guided MNP delivery in a rat stenting model 2 and 9 d post-treatment, confirming feasibility of using Ad-loaded MNPs to achieve site-specific transduction in stented blood vessels. In conclusion, Ad-loaded MNPs formed by controlled precipitation of zinc oleate represent a novel delivery system, well-suited for efficient, magnetically targeted vascular gene transfer.—Chorny, M., Fishbein, I., Tengood, J. E., Adamo, R. F., Alferiev, I. S., Levy, R. J. Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors. PMID:23407712

  9. Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors.

    PubMed

    Kolb, Andreas F; Knowles, Christopher; Pultinevicius, Patrikas; Harbottle, Jennifer A; Petrie, Linda; Robinson, Claire; Sorrell, David A

    2017-01-01

    Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

  10. Use of adenoviral vectors as veterinary vaccines.

    PubMed

    Ferreira, T B; Alves, P M; Aunins, J G; Carrondo, M J T

    2005-10-01

    Vaccines are the most effective and inexpensive prophylactic tool in veterinary medicine. Ideally, vaccines should induce a lifelong protective immunity against the target pathogen while not causing clinical or pathological signs of diseases in the vaccinated animals. However, such ideal vaccines are rare in the veterinary field. Many vaccines are either of limited effectiveness or have harmful side effects. In addition, there are still severe diseases with no effective vaccines. A very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and even more so for poultry vaccination, where vaccines must sell for a few cents a dose. Traditional approaches include inactivated vaccines, attenuated live vaccines and subunit vaccines. Recently, genetic engineering has been applied to design new, improved vaccines. Adenovirus vectors are highly efficient for gene transfer in a broad spectrum of cell types and species. Moreover, adenoviruses often induce humoral, mucosal and cellular immune responses to antigens encoded by the inserted foreign genes. Thus, adenoviruses have become a vector of choice for delivery and expression of foreign proteins for vaccination. Consequently, the market requirements for adenovirus vaccines are increasing, creating a need for production methodologies of concentrated vectors with warranted purity and efficacy. This review summarizes recent developments and approaches of adenovirus production and purification as the application of these vectors, including successes and failures in clinical applications to date.

  11. Cloning and characterization of an adenoviral vector for highly efficient and doxycycline – suppressible expression of bioactive human single – chain interleukin 12 in colon cancer

    PubMed Central

    Wulff, Holger; Krieger, Thorsten; Krüger, Karen; Stahmer, Ingrid; Thaiss, Friedrich; Schäfer, Hansjörg; Block, Andreas

    2007-01-01

    Background Interleukin-12 (IL-12) is well characterized to induce cellular antitumoral immunity by activation of NK-cells and T-lymphocytes. However, systemic administration of recombinant human IL-12 resulted in severe toxicity without perceptible therapeutic benefit. Even though intratumoral expression of IL-12 leads to tumor regression and long-term survival in a variety of animal models, clinical trials have not yet shown a significant therapeutic benefit. One major obstacle in the treatment with IL-12 is to overcome the relatively low expression of the therapeutic gene without compromising the safety of such an approach. Our objective was to generate an adenoviral vector system enabling the regulated expression of very high levels of bioactive, human IL-12. Results High gene expression was obtained utilizing the VP16 herpes simplex transactivator. Strong regulation of gene expression was realized by fusion of the VP16 to a tetracycline repressor with binding of the fusion protein to a flanking tetracycline operator and further enhanced by auto-regulated expression of its fusion gene within a bicistronic promoter construct. Infection of human colon cancer cells (HT29) at a multiplicity of infection (m.o.i.) of 10 resulted in the production of up to 8000 ng/106 cells in 48 h, thus exceeding any published vector system so far. Doxycycline concentrations as low as 30 ng/ml resulted in up to 5000-fold suppression, enabling significant reduction of gene expression in a possible clinical setting. Bioactivity of the human single-chain IL-12 was similar to purified human heterodimeric IL-12. Frozen sections of human colon cancer showed high expression of the coxsackie adenovirus receptor with significant production of human single chain IL-12 in colon cancer biopsies after infection with 3*107 p.f.u. Ad.3r-scIL12. Doxycycline mediated suppression of gene expression was up to 9000-fold in the infected colon cancer tissue. Conclusion VP16 transactivator-mediated and

  12. Adenoviral vector-mediated GM-CSF gene transfer improves anti-mycobacterial immunity in mice - role of regulatory T cells.

    PubMed

    Singpiel, Alena; Kramer, Julia; Maus, Regina; Stolper, Jennifer; Bittersohl, Lara Friederike; Gauldie, Jack; Kolb, Martin; Welte, Tobias; Sparwasser, Tim; Maus, Ulrich A

    2018-03-01

    Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in differentiation, survival and activation of myeloid and non-myeloid cells with important implications for lung antibacterial immunity. Here we examined the effect of pulmonary adenoviral vector-mediated delivery of GM-CSF (AdGM-CSF) on anti-mycobacterial immunity in M. bovis BCG infected mice. Exposure of M. bovis BCG infected mice to AdGM-CSF either applied on 6h, or 6h and 7days post-infection substantially increased alveolar recruitment of iNOS and IL-12 expressing macrophages, and significantly increased accumulation of IFNγ pos T cells and particularly regulatory T cells (Tregs). This was accompanied by significantly reduced mycobacterial loads in the lungs of mice. Importantly, diphtheria toxin-induced depletion of Tregs did not influence mycobacterial loads, but accentuated immunopathology in AdGM-CSF-exposed mice infected with M. bovis BCG. Together, the data demonstrate that AdGM-CSF therapy improves lung protective immunity against M. bovis BCG infection in mice independent of co-recruited Tregs, which however critically contribute to limit lung immunopathology in BCG-infected mice. These data may be relevant to the development of immunomodulatory strategies to limit immunopathology-based lung injury in tuberculosis in humans. Copyright © 2017 Elsevier GmbH. All rights reserved.

  13. Gene therapy in the inner ear using adenovirus vectors.

    PubMed

    Husseman, Jacob; Raphael, Yehoash

    2009-01-01

    Therapies for the protection and regeneration of auditory hair cells are of great interest given the significant monetary and lifestyle impact of hearing loss. The past decade has seen tremendous advances in the use of adenoviral vectors to achieve these aims. Preliminary data demonstrated the functional capacity of this technique as adenoviral-induced expression of neurotrophic and growth factors protected hair cells and spiral ganglion neurons from ototoxic insults. Subsequent efforts confirmed the feasibility of adenoviral transfection of cells in the auditory neuroepithelium via cochleostomy into the scala media. Most recently, efforts have focused on regeneration of depleted hair cells. Mammalian hearing loss is generally considered a permanent insult as the auditory epithelium lacks a basal layer capable of producing new hair cells. Recently, the transcription factor Atoh1 has been found to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo have shown the ability to regenerate auditory and vestibular hair cells by causing transdifferentiation of neighboring epithelial-supporting cells. Functional recovery of both the auditory and vestibular systems has been documented following adenoviral induced Atoh1 overexpression. Copyright (c) 2009 S. Karger AG, Basel.

  14. Standard Free Droplet Digital Polymerase Chain Reaction as a New Tool for the Quality Control of High-Capacity Adenoviral Vectors in Small-Scale Preparations

    PubMed Central

    Boehme, Philip; Stellberger, Thorsten; Solanki, Manish; Zhang, Wenli; Schulz, Eric; Bergmann, Thorsten; Liu, Jing; Doerner, Johannes; Baiker, Armin E.

    2015-01-01

    Abstract High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy. PMID:25640117

  15. Oral vaccination with an adenovirus-vectored vaccine protects against botulism

    PubMed Central

    Chen, Shan; Xu, Qingfu; Zeng, Mingtao

    2013-01-01

    We have previously shown that an adenovirus vectored vaccine delivered intramuscularly or intranasally was effective in protection against botulism in a mouse model. The adenoviral vector encodes a human codon-optimized heavy chain C-fragment (HC50) of botulinum neurotoxin type C (BoNT/C). Here, we evaluate the same vaccine candidate as an oral vaccine against BoNT/C in a mouse model. To elicit protective immunity, the mice were orally vaccinated with a single dose of 1×104 to 1×107 plaque forming units (pfu) of the adenoviral vector. The immune sera, collected six weeks after oral vaccination with 2×107 pfu adenovirus, has shown an ability to neutralize the biological activity of BoNT/C in vitro. Additionally, animals receiving a single dose of 2×106 pfu adenovirus or greater were completely protected against challenge with 100×MLD50 of BoNT/C. The data demonstrated the feasibility to develop an adenovirus-based oral vaccine against botulism. PMID:23295065

  16. Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

    PubMed Central

    Li, Wenyan; Shen, Jun

    2016-01-01

    Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction. PMID:28116172

  17. Anti-adenoviral effect of anti-HIV agents in vitro in serotypes inducing keratoconjunctivitis.

    PubMed

    Uchio, Eiichi; Fuchigami, Aki; Kadonosono, Kazuaki; Hayashi, Akio; Ishiko, Hiroaki; Aoki, Koki; Ohno, Shigeaki

    2007-09-01

    Around one million people are affected by adenoviral keratoconjunctivitis a year in Japan, and it is recognized as one of the major pathogens of ophthalmological nosocomial infection worldwide. Although cidofovir can be used systemically for immunocompromised patients with disseminated adenoviral infection, no specific anti-adenoviral agent has been established for the treatment of adenoviral infection. We evaluated the anti-adenoviral effect of anti-HIV (human immunodeficiency virus) agents in this study. Five anti-HIV agents (zalcitabine, stavudine, nevirapine, indinavir and amprenavir) were subjected to in vitro evaluation. A549 cells were used for viral cell culture, and adenovirus serotypes 3, 4, 8, 19 and 37 were used. After calculating CC(50) (50% cytotoxic concentration) of each agent by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we cultured adenovirus with the agents for seven days and quantitatively measured extracted adenoviral DNA by real-time PCR. Among the anti-HIV drugs, zalcitabine and stavudine, both nucleoside reverse transcriptase inhibitors, showed significant anti-adenoviral activity. In contrast, nevirapine, a non-nucleoside reverse transcriptase inhibitor, and indinavir and amprenavir, which are both protease inhibitors, were ineffective against adenovirus. These results indicate that zalcitabine and stavudine are possible candidates for the local and systemic treatment of adenoviral infection, and the anti-adenoviral effect might depend on the pharmacological properties of anti-HIV agents. The chemical properties on the clinical safety for systemic and local application need to be determined in order to for these drugs to be accepted for the treatment of adenovirus in clinical settings.

  18. Isolation and characterization of anti-adenoviral secondary metabolites from marine actinobacteria.

    PubMed

    Strand, Mårten; Carlsson, Marcus; Uvell, Hanna; Islam, Koushikul; Edlund, Karin; Cullman, Inger; Altermark, Björn; Mei, Ya-Fang; Elofsson, Mikael; Willassen, Nils-Peder; Wadell, Göran; Almqvist, Fredrik

    2014-01-28

    Adenovirus infections in immunocompromised patients are associated with high mortality rates. Currently, there are no effective anti-adenoviral therapies available. It is well known that actinobacteria can produce secondary metabolites that are attractive in drug discovery due to their structural diversity and their evolved interaction with biomolecules. Here, we have established an extract library derived from actinobacteria isolated from Vestfjorden, Norway, and performed a screening campaign to discover anti-adenoviral compounds. One extract with anti-adenoviral activity was found to contain a diastereomeric 1:1 mixture of the butenolide secondary alcohols 1a and 1b. By further cultivation and analysis, we could isolate 1a and 1b in different diastereomeric ratio. In addition, three more anti-adenoviral butenolides 2, 3 and 4 with differences in their side-chains were isolated. In this study, the anti-adenoviral activity of these compounds was characterized and substantial differences in the cytotoxic potential between the butenolide analogs were observed. The most potent butenolide analog 3 displayed an EC50 value of 91 μM and no prominent cytotoxicity at 2 mM. Furthermore, we propose a biosynthetic pathway for these compounds based on their relative time of appearance and structure.

  19. Randomized, Controlled, Phase 2 Trial of Povidone-Iodine/Dexamethasone Ophthalmic Suspension for Treatment of Adenoviral Conjunctivitis.

    PubMed

    Pepose, Jay S; Ahuja, Arjun; Liu, Wenlei; Narvekar, Abhijit; Haque, Reza

    2018-05-19

    To evaluate the efficacy/safety of an ophthalmic suspension of povidone-iodine (PVP-I) 0.6% and dexamethasone 0.1% in patients with acute adenoviral conjunctivitis. Multicenter, randomized, vehicle-controlled, double-masked trial. Adults with a positive Rapid Pathogen Screening Adeno-Detector Plus TM test were randomized 1:1:1 to PVP-I 0.6%/dexamethasone 0.1%, PVP-I 0.6%, or vehicle, bilaterally 4 times daily for 5 days (days 1-5). Patients were evaluated on days 3, 6, and 12 (+1-day window). Efficacy measures included clinical resolution and adenoviral eradication. Overall, 144 patients were included in the efficacy analysis (PVP-I/dexamethasone, n = 48; PVP-I, n = 50; vehicle, n = 46). The proportion of patients with clinical resolution (primary study eye with last observation carried forward [LOCF]) at the day 6 visit was higher with PVP-I/dexamethasone (31.3%) than vehicle (10.9%; P = .0158) and PVP-I (18.0%; P = nonsignificant). The proportion with adenoviral eradication (primary study eye with LOCF) was higher with PVP-I/dexamethasone than vehicle at the day 3 (35.4% vs 8.7%; P = .0019) and day 6 (79.2% vs 56.5%; P = .0186) visits and versus PVP-I (day 3 visit, 32.0%; day 6 visit, 62.0%; each P = nonsignificant). Treatment-emergent adverse events (AEs) occurred in 69.0% (vehicle), 62.7% (PVP-I), and 53.4% (PVP-I/dexamethasone) of patients in the safety dataset. Discontinuation owing to AEs occurred in 37 patients (vehicle, n = 16; PVP-I, n = 12; PVP-I/dexamethasone, n = 9). PVP-I/dexamethasone appeared safe and well tolerated, and significantly improved clinical resolution and adenoviral eradication in patients with acute adenoviral conjunctivitis. Copyright © 2018. Published by Elsevier Inc.

  20. Electric cell-substrate impedance sensing (ECIS) based real-time measurement of titer dependent cytotoxicity induced by adenoviral vectors in an IPI-2I cell culture model.

    PubMed

    Müller, Jakob; Thirion, Christian; Pfaffl, Michael W

    2011-01-15

    Recombinant viral vectors are widespread tools for transfer of genetic material in various modern biotechnological applications like for example RNA interference (RNAi). However, an accurate and reproducible titer assignment represents the basic step for most downstream applications regarding a precise multiplicity of infection (MOI) adjustment. As necessary scaffold for the studies described in this work we introduce a quantitative real-time PCR (qPCR) based approach for viral particle measurement. Still an implicated problem concerning physiological effects is that the appliance of viral vectors is often attended by toxic effects on the individual target. To determine the critical viral dose leading to cell death we developed an electric cell-substrate impedance sensing (ECIS) based assay. With ECIS technology the impedance change of a current flow through the cell culture medium in an array plate is measured in a non-invasive manner, visualizing effects like cell attachment, cell-cell contacts or proliferation. Here we describe the potential of this online measurement technique in an in vitro model using the porcine ileal epithelial cell line IPI-2I in combination with an adenoviral transfection vector (Ad5-derivate). This approach shows a clear dose-depending toxic effect, as the amount of applied virus highly correlates (p<0.001) with the level of cell death. Thus this assay offers the possibility to discriminate the minimal non-toxic dose of the individual transfection method. In addition this work suggests that the ECIS-device bears the feasibility to transfer this assay to multiple other cytotoxicological questions. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. Getting genetic access to natural adenovirus genomes to explore vector diversity.

    PubMed

    Zhang, Wenli; Ehrhardt, Anja

    2017-10-01

    Recombinant vectors based on the human adenovirus type 5 (HAdV5) have been developed and extensively used in preclinical and clinical studies for over 30 years. However, certain restrictions of HAdV5-based vectors have limited their clinical applications because they are rather inefficient in specifically transducing cells of therapeutic interest that lack the coxsackievirus and adenovirus receptor (CAR). Moreover, enhanced vector-associated toxicity and widespread preexisting immunity have been shown to significantly hamper the effectiveness of HAdV-5-mediated gene transfer. However, evolution of adenoviruses in the natural host is driving the generation of novel types with altered virulence, enhanced transmission, and altered tissue tropism. As a consequence, an increasing number of alternative adenovirus types were identified, which may represent a valuable resource for the development of novel vector types. Thus, researchers are focusing on the other naturally occurring adenovirus types, which are structurally similar but functionally different from HAdV5. To this end, several strategies have been devised for getting genetic access to adenovirus genomes, resulting in a new panel of adenoviral vectors. Importantly, these vectors were shown to have a host range different from HAdV5 and to escape the anti-HAdV5 immune response, thus underlining the great potential of this approach. In summary, this review provides a state-of-the-art overview of one essential step in adenoviral vector development.

  2. [The preparation of recombinant adenovirus Ad-Rad50-GFP and detection of the optimal multiplicity of infection in CNE1 transfected hv Ad-Rad50-GFP].

    PubMed

    Yan, Ruicheng; Huang, Jiancong; Zhu, Ling; Chang, Lihong; Li, Jingjia; Wu, Xifu; Ye, Jin; Zhang, Gehua

    2015-12-01

    The optimal multiplicity of infection (MOI) of the recombinant adenovirus Ad-Rad50-GFP carrying a mutant Rad50 gene expression region on the cell growth of nasopharyngeal carcinoma and the viral amplification efficiency of CNE1 cell infected by this adenovirus were studied. The biological titer of Ad-Rad50-GFP was measured by end point dilution method. The impact of recombinant adenoviral vector transfection on the growth of CNE1 cells was observed by cell growth curve. Transfection efficacy of recombinant adenoviral vector was observed and calculated through fluorescence microscope. The expression f mutant Rad50 in the Ad-Rad50-GFP transfected CNE1 cells with optimal MOI was detected by Western Blot after transfection. The biological titer of Ad-Rad50-GFP was 1.26 x 10¹¹ pfu/ml. CNE1 cell growth was not influenced significantly as they were transfected by recombinant adenoviral vector with MOI less than 50. Transfection efficacy of recombinant adenoviral vector was most salient at 24 hours after transfection, with the high expression of mutant Rad50, and the efficiency still remained about 70% after 72 hours. Recombinant adenoviral vector Ad-Rad50-GFP could transfect CNE1 cells as well as result in the expression of mutant Rad50 in CNE1 cells effectively. MOI = 50 was the optimal multiplicity of infection of CNE1 cells transfected by recombinant adenoviral vector Ad-Rad50-GFP.

  3. Adenoviral receptor expression of normal bladder and transitional cell carcinoma of the bladder.

    PubMed

    Buscarini, Maurizio; Quek, Marcus L; Gilliam-Hegarich, Susan; Kasahara, Nori; Bochner, Bernard

    2007-01-01

    The insertion of absent or underexpressed genes into cancer cells to alter their malignant phenotype is an important potential application of available gene therapy technology. One of the more common viral vector systems that has been extensively studied for this purpose are the replication-deficient adenoviruses (Ad). Adenoviral infection of cells is mediated through a complex pathway, initiated following viral-cell attachment. Adenoviral-cell attachment occurs following interactions with a 46-kDa transmembrane protein with high affinity for both the Coxsackie and adenovirus, designated the CAR (Coxsackie and adenoviral receptor). Additional important cell-viral interactions that occur involve the alpha(v)-based integrins, specifically alpha(v)beta3 and alpha(v)beta5. The purpose of the present study was to determine the extent of expression and localization of the known Ad receptor proteins (CAR, alpha(v)beta3, and alpha(v)beta5) in normal and cancerous human bladders. Frozen tissue samples of normal bladder and invasive transitional cell cancers of the bladder were evaluated. Tissue blocks containing muscle-invasive transitional cell carcinoma (TCC) were obtained following radical cystectomy, which were performed at our institution. Thirty-two invasive transitional cell bladder tumors were evaluated, each with a matched sample of histologically normal-appearing bladder used as a control. Four additional samples of normal bladder were obtained from patients with no evidence of disease of the bladder and served as further controls. Three additional cases of invasive bladder cancer with no matching normal tissue were also evaluated. Identification of the CAR receptor was performed using the anti-CAR mouse monoclonal antibody designated RmBC. The integrins alpha(v)beta3 and alpha(v)beta5 were identified using the mouse monoclonal antibodies designated LM609 and P1F6 respectively. All slides were evaluated by two of the authors (M.B., B.B.) without knowledge of the

  4. Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene.

    PubMed

    Halbert, Christine L; Allen, James M; Miller, A Dusty

    2002-07-01

    The small packaging capacity of adeno-associated virus (AAV) vectors limits the utility of this promising vector system for transfer of large genes. We explored the possibility that larger genes could be reconstituted following homologous recombination between AAV vectors carrying overlapping gene fragments. An alkaline phosphatase (AP) gene was split between two such AAV vectors (rec vectors) and packaged using AAV2 or AAV6 capsid proteins. Rec vectors having either capsid protein recombined to express AP in cultured cells at about 1-2% of the rate observed for an intact vector. Surprisingly, the AAV6 rec vectors transduced lung cells in mice almost as efficiently as did an intact vector, with 10% of airway epithelial cells, the target for treatment of cystic fibrosis (CF), being positive. Thus AAV rec vectors may be useful for diseases such as CF that require transfer of large genes.

  5. [Construction and identification of recombinant human platelet-derived growth factor-B adenoviral vector and transfection into periodontal ligament stem cells].

    PubMed

    Shang, Shu-huan; Zhang, Yu-feng; Shi, Bin; Cheng, Xiang-rong

    2008-10-01

    To construct a recombinant human platelet-derived growth factor-B (PDGF-B) adenoviral vector and to transfect it into human periodontal ligament stem cells (PDLSC). The recombinant plasmid pAd-PDGF-B was constructed by homologous recombination and confirmed by restriction endonucleases digestion. Recombinant adenovirus was packaged in HEK293 cells. PDLSC were transfected with recombinant adenovirus and PDGF-B expression was confirmed. Expression of collagen type I gene was determined by quantitative analysis of the products of RT-PCR. The cell proliferation was determined with MTT colorimetric assay. The recombinant plasmid pAd-PDGF-B was confirmed by restriction endonucleases digestion. EGFP expression was observed on the third day after transfecting, and the expression of PDGF-B was detected. Immunohistochemical methods revealed that PDGF-B was expressed in PDLSC. Levels of expression of collagen type I gene were increased significantly by transfer of the exogenous PDGF-B gene to PDLSC. At the same time, findings indicated that Ad-PDGF-B stimulated PDLSC proliferation. MTT assay indicated the absorbance of PDLSC by stimulating with Ad-PDGF-B was (0.68 +/- 0.02), P < 0.01. Using the AdEasy system, the human PDGF-B recombinant adenovirus can be rapidly obtained. These results indicate that recombinant adenoviruses encoding PDGF-B transgenes could modulate proliferative activity of PDLSC, enhance the high expression of collagen type I and lay the foundation for periodontal tissue regeneration and dental implant gene therapy.

  6. Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71.

    PubMed

    Zhang, Chao; Yang, Yong; Chi, Yudan; Yin, Jieyun; Yan, Lijun; Ku, Zhiqiang; Liu, Qingwei; Huang, Zhong; Zhou, Dongming

    2015-09-22

    Hand, foot and mouth disease (HFMD) is a major public health concern in Asia; more efficient vaccines against HFMD are urgently required. Adenoviral (Ad) capsids have been used widely for the presentation of foreign antigens to induce specific immune responses in the host. Here, we describe a novel bivalent vaccine for HFMD based on the hexon-modified, E1-deleted chimpanzee adenovirus serotype 68 (AdC68). The novel vaccine candidate was generated by incorporating the neutralising epitope of Coxsackievirus A16 (CA16), PEP71, into hypervariable region 1 (HVR1), and a shortened neutralising epitope of Enterovirus 71 (EV71), sSP70, into HVR2 of the AdC68 hexon. In order to enhance the immunogenicity of EV71, VP1 of EV71 was cloned into the E1-region of the AdC68 vectors. The results demonstrated that these two epitopes were well presented on the virion surface and had high affinity towards specific antibodies, and VP1 of EV71 was also significantly expressed. In pre-clinical mouse models, the hexon-modified AdC68 elicited neutralising antibodies against both CA16 and EV71, which conferred protection to suckling mice against a lethal challenge of CA16 and EV71. In summary, this study demonstrates that the hexon-modified AdC68 may represent a promising bivalent vaccine carrier against EV71 and CA16 and an epitope-display platform for other pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Recombinant low-seroprevalent adenoviral vectors Ad26 and Ad35 expressing the respiratory syncytial virus (RSV) fusion protein induce protective immunity against RSV infection in cotton rats.

    PubMed

    Widjojoatmodjo, Myra N; Bogaert, Lies; Meek, Bob; Zahn, Roland; Vellinga, Jort; Custers, Jerome; Serroyen, Jan; Radošević, Katarina; Schuitemaker, Hanneke

    2015-10-05

    RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Intra-testicular injection of adenoviral constructs results in Sertoli cell-specific gene expression and disruption of the seminiferous epithelium

    PubMed Central

    Hooley, R P; Paterson, M; Brown, P; Kerr, K; Saunders, P T K

    2009-01-01

    Spermatogenesis is a complex process that cannot be modelled in vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-down in vivo. This paper describes a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC function in vivo and future work will therefore focus on the use of lentiviral delivery systems. PMID:18955374

  9. Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

    PubMed Central

    Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.

    2018-01-01

    ABSTRACT Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the

  10. [Effects of adenoviral vector containing human angiotensin II type 1 receptor antisense cDNA on biological action of human pulmonary artery smooth muscle cells].

    PubMed

    Tu, Ming-li; Wang, Han-qin; Lei, Huai-ding; Luo, Guo-shi; Liu, Xian-jun; Liu, Wei-shun; Xiong, Chang; Liu, Yu-quan; Ren, Si-qun

    2005-04-01

    To investigate the effect of human angiotensin II (AngII) type 1 receptor (AT(1)R) antisense cDNA (ahAT(1)) on migration, proliferation, and apoptosis of cultured human pulmonary artery smooth muscle cells (PASMC). Two recombinant adenoviral vectors, AdCMVahAT(1) containing full length antisense cDNA targeting to human AT(1)R mRNA, and AdCMVLacZ containing LacZ, were constructed by orientation clone technology and homologous recombination. The PASMC was divided into 3 groups (DMEM, AdCMVLacZ, AdCMVahAT(1)) and different interventions were given to different groups. AT(1)R expression was detected by RT-PCR and immunohistochemistry method; migration of PASMC was measured by Boyden's Chamer method. Other PASMC was divided into 4 groups (DMEM, AngII, AdCMVLacZ + AngII and AdCMVahAT(1) + AngII), and only the last 2 groups were respectively transfected with AdCMVLacZ and AdCMVahAT(1) before administration of AngII. From 6 h to 96 h after stimulation by AngII (10(-7) mol/L), proliferation index (PI) and apoptosis of PASMC were determined by flow cytometry. At the 48 h the level of AT(1)R mRNA was significantly less in PASMC transfected AdCMVahAT(1) than that in group DMEM and in group AdCMVLacZ. The protein level showed a same difference (P < 0.01). At 24 h the migration distance of PASMC also was significantly less in group AdCMVahAT(1) than that in group DMEM and Group AdCMVLacZ (P < 0.01). Stimulated by AngII for 48 h, in group AngII the PI of PASMC markedly increased (P < 0.01 vs group DMEM). But in Group AdCMVahAT(1) + AngII PI of PASMC clearly decreased (P < 0.01 vs group AngII and group DMEM respectively). There was no statistic difference of PI between group AdCMVLacZ + AngII and group AngII. Moreover, apoptosis peak emerged only in group AdCMVahAT(1) + AngII. The rate of apoptosis in those PASMC used AdCMVahAT(1) and AngII was 24.70 +/- 4.04 (P < 0.01 vs the other 3 groups respectively). These results indicate that AngII stimulates proliferation via AT(1

  11. Homologous and heterologous recombination between adenovirus vector DNA and chromosomal DNA.

    PubMed

    Stephen, Sam Laurel; Sivanandam, Vijayshankar Ganesh; Kochanek, Stefan

    2008-11-01

    Adenovirus vector DNA is perceived to remain as episome following gene transfer. We quantitatively and qualitatively analysed recombination between high capacity adenoviral vector (HC-AdV) and chromosomal DNA following gene transfer in vitro. We studied homologous and heterologous recombination with a single HC-AdV carrying (i) a large genomic HPRT fragment with the HPRT CHICAGO mutation causing translational stop upon homologous recombination with the HPRT locus and (ii) a selection marker to allow for clonal selection in the event of heterologous recombination. We analysed the sequences at the junctions between vector and chromosomal DNA. In primary cells and in cell lines, the frequency of homologous recombination ranged from 2 x 10(-5) to 1.6 x 10(-6). Heterologous recombination occurred at rates between 5.5 x 10(-3) and 1.1 x 10(-4). HC-AdV DNA integrated via the termini mostly as intact molecules. Analysis of the junction sequences indicated vector integration in a relatively random manner without an obvious preference for particular chromosomal regions, but with a preference for integration into genes. Integration into protooncogenes or tumor suppressor genes was not observed. Patchy homologies between vector termini and chromosomal DNA were found at the site of integration. Although the majority of integrations had occurred without causing mutations in the chromosomal DNA, cases of nucleotide substitutions and insertions were observed. In several cases, deletions of even relative large chromosomal regions were likely. These results extend previous information on the integration patterns of adenovirus vector DNA and contribute to a risk-benefit assessment of adenovirus-mediated gene transfer.

  12. Neonatal adenoviral infection: a seventeen year experience and review of the literature.

    PubMed

    Ronchi, Andrea; Doern, Christopher; Brock, Evangeline; Pugni, Lorenza; Sánchez, Pablo J

    2014-03-01

    To describe the clinical manifestations and short-term outcomes of adenoviral infections in neonates and review all published cases to better determine impact and treatment outcomes. Retrospective cohort study of all neonates hospitalized at Children's Medical Center (CMC) and Parkland Memorial Hospital (PMH), Dallas, TX with laboratory-confirmed adenoviral infection from January 1,1995-December 31, 2012. Neonates were identified by review of the CMC Virology Laboratory's prospective database of all positive adenovirus tests performed in the inpatient and ambulatory settings, and at PMH, of a prospective neonatal database that included all neonatal intensive care unit admissions. Patients also were identified by discharge International Classification of Disease, 9th edition codes for adenoviral infection. The medical records were reviewed, and a review of the English literature was performed. During 17 years, 26 neonates had adenoviral infection (25, CMC; 1, PMH). The principle reasons for hospitalization were respiratory signs (88%) and temperature instability (65%). Five (19%) had disseminated disease and 4 (80%) of these infants died. Ribavirin or cidofovir treatment, as well as immune globulin intravenous, did not improve outcomes except in 1 neonate. Literature review (n = 72) combined with our data found that disseminated infection was associated with death (68% vs 21% with localized infection, P < .001). In addition, neonates <14 days of age were more likely to have disseminated disease (44% vs 12%, P = .004) and death (48% vs 8%; P < .001). Adenoviral infection in hospitalized neonates was associated with severe morbidity and mortality, especially when infection was disseminated and involved the respiratory tract. Development of new therapeutic strategies is needed. Copyright © 2014 Mosby, Inc. All rights reserved.

  13. The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

    PubMed

    Lu, Zhi Hong; Kaliberov, Sergey; Zhang, Jingzhu; Muz, Barbara; Azab, Abdel K; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Curiel, David T; Arbeit, Jeffrey M

    2014-08-01

    Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

  14. Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats.

    PubMed

    Arnhold, Stefan; Heiduschka, Peter; Klein, Helmut; Absenger, Yvonne; Basnaoglu, Serkan; Kreppel, Florian; Henke-Fahle, Sylvia; Kochanek, Stefan; Bartz-Schmidt, Karl-Ulrich; Addicks, Klaus; Schraermeyer, Ulrich

    2006-09-01

    To determine the potential of adenovirally transduced bone marrow stromal cells (BMSCs) to differentiate into retinal pigment epithelial-like cells and to evaluabe possible rescue effects after transplantation into the retinas of Royal College of Surgeons (RCS) rats. Through a high-capacity adenoviral vector expressing either green fluorescent protein (GFP) or pigment epithelial-derived factor (PEDF), rat MSCs were transduced in vitro before subretinal transplantation into Wistar rats or, alternatively, RCS rats. Two months after cell injection, the rats were killed and the eyes enucleated. The eyes were then investigated light microscopically or processed for electron microscopic investigations. Cell differentiation and integration were analyzed immunocytochemically using antibodies against cytokeratin and the tight junction protein ZO-1. Electroretinography was performed 16 days after injection of cells, to check whether a functional rescue could be detected. In vitro experiments in cocultured human MSCs and human RPE cells showed that MSCs adopted RPE-like characteristics. In grafting experiments, some rat MSCs integrate into the host RPE cell layer of Wistar and RCS rats, indicated by their hexagonal morphology. Subretinally transplanted cells express the epithelial marker cytokeratin and establish tight junctions with the host RPE cells. Furthermore, rescue effects can be demonstrated after grafting of vector-transduced and nontransduced MSCs in semithin sections of dystrophic retinas. Ultrastructurally, MSCs can be detected on top of host RPE and in close contact with photoreceptor outer segments phagocytosing rod outer segments. Taken together, these results raise the possibility that MSCs have the potency to replace diseased RPE cells and deliver therapeutic proteins into the subretinal space to protect photoreceptor cells from degeneration.

  15. Mucosal immunization with recombinant adenoviral vectors expressing murine gammaherpesvirus-68 genes M2 and M3 can reduce latent viral load.

    PubMed

    Hoegh-Petersen, Mette; Thomsen, Allan R; Christensen, Jan P; Holst, Peter J

    2009-11-12

    Gammaherpesviruses establish life-long latent infections in their hosts. If the host becomes immunosuppressed, these viruses may reactivate and cause severe disease, and even in immunocompetent individuals the gammaherpesviruses are presumed to have an oncogenic potential. Murine gammaherpesvirus-68 (MHV-68) is a member of the Gammaherpesvirinae subfamily and represents a useful murine model for this category of infections, in which new vaccination strategies may initially be evaluated. Two attenuated variants of MHV-68 have successfully been used as vaccines, but the oncogenic potential of the gammaherpesvirinae speaks against using a similar approach in humans. DNA immunization with plasmids encoding the MHV-68 genes M2 or M3 caused a reduction in either acute or early latent viral load, respectively, but neither immunization had an effect at times later than 14 days post-infection. Adenovirus-based vaccines are substantially more immunogenic than DNA vaccines and can be applied to induce mucosal immunity. Here we show that a significant reduction of the late viral load in the spleens, at 60 days post-infection, was achieved when immunizing mice both intranasally and subcutaneously with adenoviral vectors encoding both M2 and M3. Additionally we show that M3 immunization prevented the usual development of virus-induced splenomegaly at 2-3 weeks post-infection. This is the first time that immunization with a non-replicating vaccine has lead to a significantly reduced viral load at time points beyond 14 days post-infection, and thus demonstrates that a non-replicating vaccine may successfully be employed to reduce the viral burden during chronic gammaherpesvirus infection.

  16. p53 adenoviral vector (Ad-CMV-p53) induced prostatic growth inhibition of primary cultures of human prostate and an experimental rat model.

    PubMed

    Shirakawa, T; Gotoh, A; Gardner, T A; Kao, C; Zhang, Z J; Matsubara, S; Wada, Y; Hinata, N; Fujisawa, M; Hanioka, K; Matsuo, M; Kamidono, S

    2000-01-01

    Benign prostatic hyperplasia (BPH) is the most common proliferative disease affecting men. Numerous minimally invasive technologies are being developed or are currently in use to obviate the need for transurethral surgery. The goal of the present study was to develop a novel molecular based approach for the treatment of BPH using recombinant p53 adenoviral vector. The over-expression of wt-p53 can cause cell apoptosis or cell growth arrest, thus preventing the uncontrolled cell proliferation underlying BPH pathophysiology. Ad-CMV-p53, a replication-deficient recombinant adenovirus containing cytomegalovirus promoter driving p53 gene, was used. Human prostate stromal (PS) cells were evaluated for apoptosis (TUNEL assay), mRNA levels of key cell cycle regulators influencing apoptosis (p-53, Bax and Bcl-2) using quantitative RT-PCR and cytotoxicity after Ad-CMV-p53. Ad-CMV-p53 was unilaterally injected into rat ventral prostates and growth inhibition was measured by prostate weight 3 weeks after injection. In vitro exposure to Ad-CMV-p53 significantly inhibited the proliferation of PS cells, induced mRNA over-expression of both wt-p53 and Bax, and increased the proportion of apoptotic cells. A 30% decrease in average prostate weight was demonstrated in rodents after Ad-CMV-p53 injection. The results suggest that further investigation of molecular gene therapy with recombinant wt-p53 adenovirus for the treatment of BPH is warranted.

  17. Treatment of Adenoviral Acute Respiratory Distress Syndrome Using Cidofovir With Extracorporeal Membrane Oxygenation.

    PubMed

    Lee, Minhyeok; Kim, Seulgi; Kwon, Oh Jung; Kim, Ji Hye; Jeong, Inbeom; Son, Ji Woong; Na, Moon Jun; Yoon, Yoo Sang; Park, Hyun Woong; Kwon, Sun Jung

    2017-03-01

    Adenovirus infections are associated with respiratory (especially upper respiratory) infection and gastrointestinal disease and occur primarily in infants and children. Although rare in adults, severe lower respiratory adenovirus infections including pneumonia are reported in specific populations, such as military recruits and immunocompromised patients. Antiviral treatment is challenging due to limited clinical experience and lack of well-controlled randomized trials. Several previously reported cases of adenoviral pneumonia showed promising efficacy of cidofovir. However, few reports discussed the efficacy of cidofovir in acute respiratory distress syndrome (ARDS). We experienced 3 cases of adenoviral pneumonia associated with ARDS and treated with cidofovir and respiratory support, including extracorporeal membrane oxygenation (ECMO). All 3 patients showed a positive clinical response to cidofovir and survival at 28 days. Cidofovir with early ECMO therapy may be a therapeutic option in adenoviral ARDS. A literature review identified 15 cases of adenovirus pneumonia associated with ARDS.

  18. Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine

    PubMed Central

    Lee, Cody S.; Bishop, Elliot S.; Zhang, Ruyi; Yu, Xinyi; Farina, Evan M.; Yan, Shujuan; Zhao, Chen; Zheng, Zongyue; Shu, Yi; Wu, Xingye; Lei, Jiayan; Li, Yasha; Zhang, Wenwen; Yang, Chao; Wu, Ke; Wu, Ying; Ho, Sherwin; Athiviraham, Aravind; Lee, Michael J.; Wolf, Jennifer Moriatis; Reid, Russell R.; He, Tong-Chuan

    2017-01-01

    With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology, it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies. Despite numerous setbacks, efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases. It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies. There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development. In fact, among over 2,000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors, including adenoviral biology, approaches to engineering adenoviral vectors, and their applications in clinical and pre-clinical studies with an emphasis in the areas of cancer treatment, vaccination and regenerative medicine. Current challenges and future directions regarding the use of adenoviral vectors are also discussed. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine. PMID:28944281

  19. Adenoviral Mediated Gene Transfer of IGF-1 Enhances Wound Healing and Induces Angiogenesis

    PubMed Central

    Balaji, S.; LeSaint, M.; Bhattacharya, S. S.; Moles, C.; Dhamija, Y.; Kidd, M.; Le, L.D.; King, A.; Shaaban, A.; Crombleholme, T. M.; Bollyky, P.; Keswani, S. G.

    2014-01-01

    Background Chronic wounds are characterized by a wound healing and neovascularization deficit. Strategies to increase neovascularization can significantly improve chronic wound healing. Insulin like growth factor (IGF-1) is reported to be a keratinocyte mitogen and is believed to induce angiogenesis via a vascular endothelial growth factor (VEGF) dependent pathway. Using a novel ex vivo human dermal wound model and a diabetic impaired wound healing murine model, we hypothesized that adenoviral over expression of IGF-1 (Ad-IGF-1) will enhance wound healing and induce angiogenesis through a VEGF dependent pathway. Methods Ex vivo: 6 mm full thickness punch biopsies were obtained from normal human skin, and 3 mm full thickness wounds were created at the center. Skin explants were maintained at air liquid interface. Db/db murine model: 8 mm full thickness dorsal wounds in diabetic (db/db) mice were created. Treatment groups in both human ex vivo and in vivo db/db wound models include 1×108 PFU of Ad-IGF-1 or Ad-LacZ, and PBS (n=4–5/group). Cytotoxicity (LDH) was quantified at days 3, 5 and 7 for the human ex vivo wound model. Epithelial gap closure (H&E; Trichrome), VEGF expression (ELISA) and capillary density (CD 31+ CAPS/HPF) were analyzed at day 7. Results In the human ex vivo organ culture, the adenoviral vectors did not demonstrate any significant difference in cytotoxicity compared to PBS. Ad-IGF-1 over expression significantly increases basal keratinocyte migration, with no significant effect on epithelial gap closure. There was a significant increase in capillary density in the Ad-IGF-1 wounds. However, there was no effect on VEGF levels in Ad-IGF-1 samples compared to controls. In db/db wounds, Ad-IGF-1 over expression significantly improves epithelial gap closure and granulation tissue with a dense cellular infiltrate compared to controls. Ad-IGF-1 also increases capillary density, again with no significant difference in VEGF levels in the wounds compared

  20. Adenoviral Vectors Armed with Cell Fusion-Inducing Proteins as Anti-Cancer Agents

    PubMed Central

    Del Papa, Joshua; Parks, Robin J.

    2017-01-01

    Cancer is a devastating disease that affects millions of patients every year, and causes an enormous economic burden on the health care system and emotional burden on affected families. The first line of defense against solid tumors is usually extraction of the tumor, when possible, by surgical methods. In cases where solid tumors can not be safely removed, chemotherapy is often the first line of treatment. As metastatic cancers often become vigorously resistant to treatments, the development of novel, more potent and selective anti-cancer strategies is of great importance. Adenovirus (Ad) is the most commonly used virus in cancer clinical trials, however, regardless of the nature of the Ad-based therapeutic, complete responses to treatment remain rare. A number of pre-clinical studies have shown that, for all vector systems, viral spread throughout the tumor mass can be a major limiting factor for complete tumor elimination. By expressing exogenous cell-fusion proteins, many groups have shown improved spread of Ad-based vectors. This review summarizes the research done to examine the potency of Ad vectors expressing fusogenic proteins as anti-cancer therapeutics. PMID:28106842

  1. Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment.

    PubMed

    Kraljević Pavelić, Sandra; Marjanović, Marko; Poznić, Miroslav; Kralj, Marijeta

    2009-12-01

    p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents--cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed. p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21(WAF1/CIP1) and cyclin B1 proteins was examined using immunocytochemistry and western blot methods. In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis. This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and

  2. Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene.

    PubMed

    Hashimoto, Haruo; Mizushima, Tomoko; Chijiwa, Tsuyoshi; Nakamura, Masato; Suemizu, Hiroshi

    2017-06-15

    The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Recent Advances in Preclinical Developments Using Adenovirus Hybrid Vectors.

    PubMed

    Ehrke-Schulz, Eric; Zhang, Wenli; Gao, Jian; Ehrhardt, Anja

    2017-10-01

    Adenovirus (Ad)-based vectors are efficient gene-transfer vehicles to deliver foreign DNA into living organisms, offering large cargo capacity and low immunogenicity and genotoxicity. As Ad shows low integration rates of their genomes into host chromosomes, vector-derived gene expression decreases due to continuous cell cycling in regenerating tissues and dividing cell populations. To overcome this hurdle, adenoviral delivery can be combined with mechanisms leading to maintenance of therapeutic DNA and long-term effects of the desired treatment. Several hybrid Ad vectors (AdV) exploiting various strategies for long-term treatment have been developed and characterized. This review summarizes recent developments of preclinical approaches using hybrid AdVs utilizing either the Sleeping Beauty transposase system for somatic integration into host chromosomes or designer nucleases, including transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease for permanent gene editing. Further options on how to optimize these vectors further are discussed, which may lead to future clinical applications of these versatile gene-therapy tools.

  4. Development of Novel Adenoviral Vectors to Overcome Challenges Observed With HAdV-5–based Constructs

    PubMed Central

    Alonso-Padilla, Julio; Papp, Tibor; Kaján, Győző L; Benkő, Mária; Havenga, Menzo; Lemckert, Angelique; Harrach, Balázs; Baker, Andrew H

    2016-01-01

    Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in preclinical models and clinical trials over the past two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread preexisting immunity have been shown to significantly impede the effectiveness of HAdV-5–mediated gene transfer. It is therefore that the in-depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes. PMID:26478249

  5. Adenoviral infection in a collection of juvenile inland bearded dragons (Pogona vitticeps).

    PubMed

    Doneley, R J T; Buckle, K N; Hulse, L

    2014-01-01

    Juvenile inland bearded dragons (Pogona vitticeps) from a breeding collection in south-east Queensland were presented at age 6-10 weeks with neurological signs, poor growth and occasional deaths. Histopathological examination revealed that six of eight lizards had multifocal non-suppurative hepatitis associated with 5-10 μm diameter, smudgy, basophilic, hyaline intranuclear inclusion bodies that marginated the nuclear chromatin. These histological lesions were considered consistent with adenoviral hepatitis. Infection with adenovirus was confirmed positive in one of the eight dragons by PCR for adenoviral DNA. DNA was extracted from formalin-fixed paraffin-embedded pooled tissues of the juvenile inland bearded dragons and tested using a nested-PCR protocol with primers specific for identification of adenovirus. Sequencing of the one PCR-positive dragon showed 95% nucleotide sequence alignment with agamid atadenovirus 1. Further investigation involved testing the breeding population, including the parents of the affected juveniles. Blood and cloacal samples were collected from the adult population, DNA was extracted and tested by PCR for adenovirus. There was a high percentage of positive results from the samples collected from the breeding population. This is the first reported group outbreak of adenoviral disease in bearded dragons in Australia. © 2014 Australian Veterinary Association.

  6. Peptide-Based Technologies to Alter Adenoviral Vector Tropism: Ways and Means for Systemic Treatment of Cancer

    PubMed Central

    Reetz, Julia; Herchenröder, Ottmar; Pützer, Brigitte M.

    2014-01-01

    Due to the fundamental progress in elucidating the molecular mechanisms of human diseases and the arrival of the post-genomic era, increasing numbers of therapeutic genes and cellular targets are available for gene therapy. Meanwhile, the most important challenge is to develop gene delivery vectors with high efficiency through target cell selectivity, in particular under in situ conditions. The most widely used vector system to transduce cells is based on adenovirus (Ad). Recent endeavors in the development of selective Ad vectors that target cells or tissues of interest and spare the alteration of all others have focused on the modification of the virus broad natural tropism. A popular way of Ad targeting is achieved by directing the vector towards distinct cellular receptors. Redirecting can be accomplished by linking custom-made peptides with specific affinity to cellular surface proteins via genetic integration, chemical coupling or bridging with dual-specific adapter molecules. Ideally, targeted vectors are incapable of entering cells via their native receptors. Such altered vectors offer new opportunities to delineate functional genomics in a natural environment and may enable efficient systemic therapeutic approaches. This review provides a summary of current state-of-the-art techniques to specifically target adenovirus-based gene delivery vectors. PMID:24699364

  7. In vivo, cardiac-specific knockdown of target protein, Malic Enzyme-1, in rat via adenoviral delivery of DNA for non-native miRNA

    PubMed Central

    O'Donnell, J. Michael; Kalichira, Asha; Bi, Jian; Lewandowski, E. Douglas

    2013-01-01

    This study examines the feasibility of using the adenoviral delivery of DNA for a non-native microRNA to suppress expression of a target protein (cytosolic NADP+-dependent malic-enzyme 1, ME1) in whole heart in vivo, via an isolated-heart coronary perfusion approach. Complementary DNA constructs for ME1 microRNA were inserted into adenoviral vectors. Viral gene transfer to neonatal rat cardiomyocytes yielded 65% suppression of ME1 protein. This viral package was delivered to rat hearts in vivo (Adv.miR_ME1, 1013 vp/ml PBS) via coronary perfusion, using a cardiac-specific isolation technique. ME1 mRNA was reduced by 73% at 2-6 days post-surgery in heart receiving the Adv.miR_ME1. Importantly, ME1 protein was reduced by 66% (p<0.0002) at 5-6 days relative to sham-operated control hearts. Non-target protein expression for GAPDH, calsequestrin, and mitochondrial malic enzyme, ME3, were all unchanged. The non-target isoform, ME2, was unchanged at 2-5 days and reduced at day 6. This new approach demonstrates for the first time significant and acute silencing of target RNA translation and protein content in whole heart, in vivo, via non-native microRNA expression. PMID:22974418

  8. Virucidal effects of rodent cage-cleaning practices on the viability of adenovirus vectors.

    PubMed

    Porter, Jacqueline D; Lyons, Russette M

    2002-09-01

    Human adenoviruses and adenoviral vectors are classified as Risk Group 2 agents and require BSL2 containment and practices. An additional consideration in using adenoviruses and viral vectors in laboratory animal studies is the possible transmission of these agents to other animals and/or personnel as a result of viral shedding in animal urine and feces. When handling BSL2 agents, cage-wash staff are required to wear appropriate personnel protective equipment, including scrubs, Tyvek suit, hair covering, dust mask, shoes covers, and gloves. Current decontamination procedures are to bag and autoclave soiled rodent cages containing bedding prior to washing in the cage washer to prevent possible adenoviral transmission. However, the practice of autoclaving softens the polycarbonate-based rodent cages, allowing damaging agents or conditions to affect the integrity of the plastic and degrade the cages. The objective of this study was to determine whether current rodent cage-cleaning practices produced virucidal effects for use in lieu of or prior to autoclaving the cages. We found that heating an Av3GFP vector in a test tube to a temperature of 74 degrees C (165 degrees F) for 6 min conditions equivalent to those of the cage washer resulted in greater than an 11-log reduction in infectivity of the vector as evaluated by its cytopathic effect on cells. The combination of heating and a liquid, phosphate-free alkaline detergent produced the same reduction in vector infectivity. However, common cage-cleaning solutions alone possessed no virucidal activity. The high temperatures used in cage-washing procedures alone or in combination with a cleaning solution reduced or eliminated the risk of transmission from viral shedding through urine and feces even at vector concentrations far greater than would ever be expected to be present. Autoclaving cages diminishes the stability and integrity of the polycarbonate cages without providing a further reduction in the risk of virus or

  9. [Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

    PubMed

    Pan, X-X; Zhao, B-X; Teng, Y-M; Xia, W-Y; Wang, J; Li, X-F; Liao, G-Y; Yang, С; Chen, Y-D

    2016-01-01

    Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

  10. Vector platforms for gene therapy of inherited retinopathies

    PubMed Central

    Trapani, Ivana; Puppo, Agostina; Auricchio, Alberto

    2014-01-01

    Inherited retinopathies (IR) are common untreatable blinding conditions. Most of them are inherited as monogenic disorders, due to mutations in genes expressed in retinal photoreceptors (PR) and in retinal pigment epithelium (RPE). The retina’s compatibility with gene transfer has made transduction of different retinal cell layers in small and large animal models via viral and non-viral vectors possible. The ongoing identification of novel viruses as well as modifications of existing ones based either on rational design or directed evolution have generated vector variants with improved transduction properties. Dozens of promising proofs of concept have been obtained in IR animal models with both viral and non-viral vectors, and some of them have been relayed to clinical trials. To date, recombinant vectors based on the adeno-associated virus (AAV) represent the most promising tool for retinal gene therapy, given their ability to efficiently deliver therapeutic genes to both PR and RPE and their excellent safety and efficacy profiles in humans. However, AAVs’ limited cargo capacity has prevented application of the viral vector to treatments requiring transfer of genes with a coding sequence larger than 5 kb. Vectors with larger capacity, i.e. nanoparticles, adenoviral and lentiviral vectors are being exploited for gene transfer to the retina in animal models and, more recently, in humans. This review focuses on the available platforms for retinal gene therapy to fight inherited blindness, highlights their main strengths and examines the efforts to overcome some of their limitations. PMID:25124745

  11. Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

    PubMed

    Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle; Dmitriev, Igor P; Kashentseva, Elena A; Yeh, Anthony J; Cheung, Gordon Y C; Curiel, David T; Leppla, Stephen; Shoemaker, Charles B

    2016-01-06

    Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. Alanine–glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer

    PubMed Central

    Salido, Eduardo C.; Li, Xiao M.; Lu, Yang; Wang, Xia; Santana, Alfredo; Roy-Chowdhury, Namita; Torres, Armando; Shapiro, Larry J.; Roy-Chowdhury, Jayanta

    2006-01-01

    Mutations in the alanine–glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt−/− mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria. PMID:17110443

  13. Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

    PubMed Central

    Gorziglia, M I; Kadan, M J; Yei, S; Lim, J; Lee, G M; Luthra, R; Trapnell, B C

    1996-01-01

    A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy. PMID:8648763

  14. Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

    PubMed Central

    2017-01-01

    ABSTRACT Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required. IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious

  15. A clinical algorithm identifies high risk pediatric oncology and bone marrow transplant patients likely to benefit from treatment of adenoviral infection.

    PubMed

    Williams, Kirsten Marie; Agwu, Allison L; Dabb, Alix A; Higman, Meghan A; Loeb, David M; Valsamakis, Alexandra; Chen, Allen R

    2009-11-01

    Adenoviral infections cause morbidity and mortality in blood and marrow transplantation and pediatric oncology patients. Cidofovir is active against adenovirus, but must be used judiciously because of its nephrotoxicity and unclear indications. Therefore, before introducing cidofovir use during an adenoviral outbreak, we developed a clinical algorithm to distinguish low risk patients from those who merited cidofovir therapy because of significant adenoviral disease and high risk for death. This study was conducted to determine whether the algorithm accurately predicted severe adenovirus disease and whether selective cidofovir treatment was beneficial. A retrospective analysis of a pediatric oncology/blood and marrow transplantation cohort prealgorithm and postalgorithm implementation was performed. Twenty patients with adenovirus infection were identified (14 high risk and 6 low risk). All low-risk patients cleared their infections without treatment. Before algorithm implementation, all untreated high-risk patients died, 4 out of 5 (80%), from adenoviral infection. In contrast, cidofovir reduced adenovirus-related mortality in the high-risk group postalgorithm implementation (9 patients treated, 1 patient died; RR 0.14, P<0.05) and all treated high-risk patients cleared their virus. The clinical algorithm accurately identified patients at high risk for severe fatal adenoviral disease who would benefit from selective use of cidofovir.

  16. A Novel Vaccine Approach for Chagas Disease Using Rare Adenovirus Serotype 48 Vectors

    PubMed Central

    Farrow, Anitra L.; Peng, Binghao J.; Gu, Linlin; Krendelchtchikov, Alexandre; Matthews, Qiana L.

    2016-01-01

    Due to the increasing amount of people afflicted worldwide with Chagas disease and an increasing prevalence in the United States, there is a greater need to develop a safe and effective vaccine for this neglected disease. Adenovirus serotype 5 (Ad5) is the most common adenovirus vector used for gene therapy and vaccine approaches, but its efficacy is limited by preexisting vector immunity in humans resulting from natural infections. Therefore, we have employed rare serotype adenovirus 48 (Ad48) as an alternative choice for adenovirus/Chagas vaccine therapy. In this study, we modified Ad5 and Ad48 vectors to contain T. cruzi’s amastigote surface protein 2 (ASP-2) in the adenoviral early gene. We also modified Ad5 and Ad48 vectors to utilize the “Antigen Capsid-Incorporation” strategy by adding T. cruzi epitopes to protein IX (pIX). Mice that were immunized with the modified vectors were able to elicit T. cruzi-specific humoral and cellular responses. This study indicates that Ad48-modified vectors function comparable to or even premium to Ad5-modified vectors. This study provides novel data demonstrating that Ad48 can be used as a potential adenovirus vaccine vector against Chagas disease. PMID:26978385

  17. In Vivo Stable Transduction of Humanized Liver Tissue in Chimeric Mice via High-Capacity Adenovirus–Lentivirus Hybrid Vector

    PubMed Central

    Kataoka, Miho; Tateno, Chise; Yoshizato, Katsutoshi; Kawasaki, Yoshiko; Kimura, Takahiro; Faure-Kumar, Emmanuelle; Palmer, Donna J.; Ng, Philip; Okamura, Haruki; Kasahara, Noriyuki

    2010-01-01

    Abstract We developed hybrid vectors employing high-capacity adenovirus as a first-stage carrier encoding all the components required for in situ production of a second-stage lentivirus, thereby achieving stable transgene expression in secondary target cells. Such vectors have never previously been tested in normal tissues, because of the scarcity of suitable in vivo systems permissive for second-stage lentivirus assembly. Here we employed a novel murine model in which endogenous liver tissue is extensively reconstituted with engrafted human hepatocytes, and successfully achieved stable transduction by the second-stage lentivirus produced in situ from first-stage adenovirus. This represents the first demonstration of the functionality of adenoviral-lentiviral hybrid vectors in a normal parenchymal organ in vivo. PMID:19725756

  18. Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy

    PubMed Central

    Weymann, Alexander; Arif, Rawa; Weber, Antje; Zaradzki, Marcin; Richter, Karsten; Ensminger, Stephan; Robinson, Peter Nicholas; Wagner, Andreas H.; Karck, Matthias; Kallenbach, Klaus

    2016-01-01

    Objectives Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis. Methods We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM). Results IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1

  19. Age-related pathology after adenoviral overexpression of the leucine-rich repeat kinase 2 in the mouse striatum.

    PubMed

    Kritzinger, Astrid; Ferger, Boris; Gillardon, Frank; Stierstorfer, Birgit; Birk, Gerald; Kochanek, Stefan; Ciossek, Thomas

    2018-06-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) age-dependently cause Parkinson's disease and are associated with several inflammatory diseases. So far, the potential role of LRRK2 expression in glial cells as mediators of neuroinflammation and the influence of aging have not been investigated in viral vector-based LRRK2 animal models. In this study, we compared the effect of striatal injection of high-capacity adenoviral vectors expressing either a kinase-overactive LRRK2 with the familial G2019S mutation or a kinase-inactive LRRK2 variant in young and old C57BL/6J mice. The intrinsic adenovirus tropism guided preferentially glial transduction, and the vector design led to stable expression for at least 6 months. In histopathological analysis, young mice expressing either LRRK2 variant presented with transient vacuolization of striatal white fiber tracts accompanied by accumulation of microglial cells and astrogliosis, but inflammation resolved without permanent damage. Old mice had a stronger and prolonged inflammatory reaction and experienced permanent damage in form of partial neuron loss after 3 months exclusively in case of LRRK2_G2019S expression. The autophagic receptor p62 accumulated in cells with high levels of either LRRK2 variant, even more so in old mice. We conclude that the aging mouse brain is more susceptible to LRRK2-associated pathology, and in this model, glial LRRK2 expression significantly contributed to neuroinflammation, ultimately causing neurodegeneration. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Local Delivery of Gene Vectors From Bare-Metal Stents by Use of a Biodegradable Synthetic Complex Inhibits In-Stent Restenosis in Rat Carotid Arteries

    PubMed Central

    Fishbein, Ilia; Alferiev, Ivan; Bakay, Marina; Stachelek, Stanley J.; Sobolewski, Peter; Lai, Meizan; Choi, Hoon; Chen, I.-W.; Levy, Robert J.

    2012-01-01

    Background Local drug delivery from polymer-coated stents has demonstrated efficacy for preventing in-stent restenosis; however, both the inflammatory effects of polymer coatings and concerns about late outcomes of drug-eluting stent use indicate the need to investigate innovative approaches, such as combining localized gene therapy with stent angioplasty. Thus, we investigated the hypothesis that adenoviral vectors (Ad) could be delivered from the bare-metal surfaces of stents with a synthetic complex for reversible vector binding. Methods and Results We synthesized the 3 components of a gene vector binding complex: (1) A polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethyleneimine (PEI) with pyridyldithio groups for amplification of attachment sites [PEI(PDT)], and (3) a bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond (HL). HL-modified Ad attached to PABT/PEI(PDT)-treated steel surfaces demonstrated both sustained release in vitro over 30 days and localized green fluorescent protein expression in rat arterial smooth muscle cell cultures, which were not sensitive to either inhibition by neutralizing anti-Ad antibodies or inactivation after storage at 37°C. In rat carotid studies, deployment of steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated both site-specific arterial AdGFP expression and adenovirus-luciferase transgene activity per optical imaging. Rat carotid stent delivery of adenovirus encoding inducible nitric oxide synthase resulted in significant inhibition of restenosis. Conclusions Reversible immobilization of adenovirus vectors on the bare-metal surfaces of endovascular stents via a synthetic complex represents an efficient, tunable method for sustained release of gene vectors to the vasculature. PMID:18413497

  1. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

    PubMed Central

    2013-01-01

    Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

  2. Bioengineering a non-genotoxic vector for genetic modification of mesenchymal stem cells.

    PubMed

    Chen, Xuguang; Nomani, Alireza; Patel, Niket; Nouri, Faranak S; Hatefi, Arash

    2018-01-01

    Vectors used for stem cell transfection must be non-genotoxic, in addition to possessing high efficiency, because they could potentially transform normal stem cells into cancer-initiating cells. The objective of this research was to bioengineer an efficient vector that can be used for genetic modification of stem cells without any negative somatic or genetic impact. Two types of multifunctional vectors, namely targeted and non-targeted were genetically engineered and purified from E. coli. The targeted vectors were designed to enter stem cells via overexpressed receptors. The non-targeted vectors were equipped with MPG and Pep1 cell penetrating peptides. A series of commercial synthetic non-viral vectors and an adenoviral vector were used as controls. All vectors were evaluated for their efficiency and impact on metabolic activity, cell membrane integrity, chromosomal aberrations (micronuclei formation), gene dysregulation, and differentiation ability of stem cells. The results of this study showed that the bioengineered vector utilizing VEGFR-1 receptors for cellular entry could transfect mesenchymal stem cells with high efficiency without inducing genotoxicity, negative impact on gene function, or ability to differentiate. Overall, the vectors that utilized receptors as ports for cellular entry (viral and non-viral) showed considerably better somato- and genosafety profiles in comparison to those that entered through electrostatic interaction with cellular membrane. The genetically engineered vector in this study demonstrated that it can be safely and efficiently used to genetically modify stem cells with potential applications in tissue engineering and cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Adenoviral vector gene delivery via the round window membrane in guinea pigs.

    PubMed

    Suzuki, Mitsuya; Yamasoba, Tatsuya; Suzukawa, Keigo; Kaga, Kimitaka

    2003-10-27

    We have found that damage from a local anesthetic solution containing phenol permitted beta-galactosidase (beta-gal) gene delivery to the guinea pig inner ear via the round window membrane (RWM). RWM damage was evident as degeneration of the outer epithelium. After adenovirus lacZ vector was applied to the damaged RWM, immunohistochemistry showed strong beta-gal expression in the RWM, mesothelial cells, organ of Corti, spiral limbus, spiral ligament and spiral ganglion. In the vestibular labyrinth, expression was seen in the sensory and supporting cells, transitional cells, and the dark-cell area. Thus, adenovirus can transfect a variety of inner ear cells in the guinea pig through a damaged RWM.

  4. ROLE OF GENETIC SUSCEPTIBILITY TO LATENT ADENOVIRAL INFECTION AND DECREASED LUNG FUNCTION

    PubMed Central

    Kasuga, Ikuma; Hogg, James C.; Paré, Peter D.; Hayashi, Shizu; Sedgwick, Edward G.; Ruan, Jian; Wallace, Alison M.; He, Jian-Qing; Zhang, Xiaozhu; Sandford, Andrew J.

    2009-01-01

    Background Latent adenoviral infection may amplify cigarette smoke-induced lung inflammation and therefore play an important role in the development of chronic obstructive pulmonary disease (COPD). Adenoviruses can evade the human immune response via their 19-kDa protein (19K) which delays the expression of class I human leukocyte antigen (HLA) proteins. The 19K protein shows higher affinity to HLA-B7 and A2 compared with HLA-A1 and A3. The receptor for adenovirus (CXADR) and integrin β5 (ITGB5) are host factors which might affect adenovirus infection. Therefore, we investigated the contribution of HLA, CXADR, and ITGB5 genetic variants to the presence of the E1A gene and to level of lung function. Methods Study subjects were assayed for HLA-B7, A1, A2 and A3 by PCR-based assays using allele-specific primers. Polymorphisms of the CXADR and ITGB5 genes were genotyped by PCR-based restriction fragment length polymorphism assays. Detection of adenoviral E1A gene was performed by a real-time PCR TaqMan assay. Results E1A positive individuals have a lower FEV1 compared with E1A negative individuals. However, there was no significant difference in E1A positivity rate between the high (HLA-B7 and A2) and low (HLA-A1 and A3) 19K affinity groups. There was also no significant difference in FEV1 level between each affinity group. There was no significant difference in E1A positivity rate or lung function among the CXADR and ITGB5 genotypes. Conclusions Genetic variants in HLA, CXADR and ITGB5 do not influence latent adenoviral infections and are not associated with COPD. PMID:19502044

  5. A Partial E3 Deletion in Replication-Defective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products.

    PubMed

    Haut, Larissa H; Gill, Amanda L; Kurupati, Raj K; Bian, Ang; Li, Yan; Giles-Davis, Wynetta; Xiang, Zhiquan; Zhou, Xiang Yang; Ertl, Hildegund C J

    2016-10-01

    Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.

  6. Anti-tumor function of double-promoter regulated adenovirus carrying SEA gene, in the treatment of bladder cancer.

    PubMed

    Hu, Jianpeng; Xuan, Xujun; Han, Conghui; Hao, Lin; Zhang, Peiying; Chen, Meng; He, Houguang; Fan, Tao; Dong, Binzheng

    2012-03-01

    To construct an adenovirus carrying SEA gene and regulated by telomerase reverse transcriptase (hTERT) and hypoxia-inducible factor (HIF) promoters and investigate its anti-tumor function in vitro, as well as its role in lymphocyte production. hTERT and HIF genes were cloned into adenovirus E1A and E1B shuttle plasmids. The control vector for SEA gene expression is under the regulation of CMV and SV40 promoters. Double regulation was obtained through homologous recombination. The positive clones of replicable adenovirus H2-SEA-Ad were selected by plaque assay. The adenovirus was purified, titrated, and DNA was verified by PCR. The obtained virus was used to infect EJ bladder tumor cells and the SEA Mrna, and protein expression was measured by RT-PCR, western blot, and immunofluorescence microscopy, respectively. Co-culture of lymphocytes and tumor cells was observed dynamically under microscope. The construction of shuttle plasmid p315-CSS-SEA was confirmed by PCR and DNA sequencing. Insertion of superantigen SEA gene in adenovirus (H2-SEA-Ad.SEA) was obtained by homologous recombination. In lymphocytes and tumor cell co-culture, the number of viable tumor cells in test groups was significantly lower than that in control group after 12, 24, and 48 h of treatment. Production of interleukin-2, interleukin-4, and tumor necrosis factor were higher in test groups than in control group. Expression of SEA gene in bladder tumor cells by adenoviral vector caused reduced tumor cell proliferation, as well as stimulation of inflammatory cytokine productions in co-cultures with lymphocytes.

  7. Adenoviral vaccine induction of CD8+ T cell memory inflation: Impact of co-infection and infection order.

    PubMed

    Lee, Lian N; Bolinger, Beatrice; Banki, Zoltan; de Lara, Catherine; Highton, Andrew J; Colston, Julia M; Hutchings, Claire; Klenerman, Paul

    2017-12-01

    The efficacies of many new T cell vaccines rely on generating large populations of long-lived pathogen-specific effector memory CD8 T cells. However, it is now increasingly recognized that prior infection history impacts on the host immune response. Additionally, the order in which these infections are acquired could have a major effect. Exploiting the ability to generate large sustained effector memory (i.e. inflationary) T cell populations from murine cytomegalovirus (MCMV) and human Adenovirus-subtype (AdHu5) 5-beta-galactosidase (Ad-lacZ) vector, the impact of new infections on pre-existing memory and the capacity of the host's memory compartment to accommodate multiple inflationary populations from unrelated pathogens was investigated in a murine model. Simultaneous and sequential infections, first with MCMV followed by Ad-lacZ, generated inflationary populations towards both viruses with similar kinetics and magnitude to mono-infected groups. However, in Ad-lacZ immune mice, subsequent acute MCMV infection led to a rapid decline of the pre-existing Ad-LacZ-specific inflating population, associated with bystander activation of Fas-dependent apoptotic pathways. However, responses were maintained long-term and boosting with Ad-lacZ led to rapid re-expansion of the inflating population. These data indicate firstly that multiple specificities of inflating memory cells can be acquired at different times and stably co-exist. Some acute infections may also deplete pre-existing memory populations, thus revealing the importance of the order of infection acquisition. Importantly, immunization with an AdHu5 vector did not alter the size of the pre-existing memory. These phenomena are relevant to the development of adenoviral vectors as novel vaccination strategies for diverse infections and cancers. (241 words).

  8. Adenoviral vaccine induction of CD8+ T cell memory inflation: Impact of co-infection and infection order

    PubMed Central

    Bolinger, Beatrice; de Lara, Catherine; Hutchings, Claire

    2017-01-01

    The efficacies of many new T cell vaccines rely on generating large populations of long-lived pathogen-specific effector memory CD8 T cells. However, it is now increasingly recognized that prior infection history impacts on the host immune response. Additionally, the order in which these infections are acquired could have a major effect. Exploiting the ability to generate large sustained effector memory (i.e. inflationary) T cell populations from murine cytomegalovirus (MCMV) and human Adenovirus-subtype (AdHu5) 5-beta-galactosidase (Ad-lacZ) vector, the impact of new infections on pre-existing memory and the capacity of the host’s memory compartment to accommodate multiple inflationary populations from unrelated pathogens was investigated in a murine model. Simultaneous and sequential infections, first with MCMV followed by Ad-lacZ, generated inflationary populations towards both viruses with similar kinetics and magnitude to mono-infected groups. However, in Ad-lacZ immune mice, subsequent acute MCMV infection led to a rapid decline of the pre-existing Ad-LacZ-specific inflating population, associated with bystander activation of Fas-dependent apoptotic pathways. However, responses were maintained long-term and boosting with Ad-lacZ led to rapid re-expansion of the inflating population. These data indicate firstly that multiple specificities of inflating memory cells can be acquired at different times and stably co-exist. Some acute infections may also deplete pre-existing memory populations, thus revealing the importance of the order of infection acquisition. Importantly, immunization with an AdHu5 vector did not alter the size of the pre-existing memory. These phenomena are relevant to the development of adenoviral vectors as novel vaccination strategies for diverse infections and cancers. (241 words) PMID:29281733

  9. Adenoviral bcl-2 transfer improves survival and early graft function after ischemia and reperfusion in rat liver transplantation.

    PubMed

    Rentsch, Markus; Kienle, Klaus; Mueller, Thomas; Vogel, Mandy; Jauch, Karl Walter; Püllmann, Kerstin; Obed, Aiman; Schlitt, Hans J; Beham, Alexander

    2005-11-27

    Primary graft dysfunction due to ischemia and reperfusion injury represents a major problem in liver transplantation. The related cell stress may induce apoptosis, which can be suppressed by bcl-2. The purpose of the study was to investigate the effect of adenoviral bcl-2 gene transfer on early graft function and survival in rat liver transplantation. An adenoviral construct that transfers bcl-2 under the control of a tetracycline inducible promoter was generated (advTetOn bcl-2) and used with a second adenovirus that transfers the repressor protein (advCMV Rep). Forty-eight hours before explantation, donor rats were treated with advTetOn bcl-2/ advCMV Rep (n=7) and doxycyclin, with the control adenoviral construct advCMV GFP (n=8) or with doxycyclin alone (n=8). Liver transplantation was performed following 16 hours of cold storage (UW). Bcl-2 expression and intrahepatic apoptosis was assessed. Bile flow was monitored 90 min posttransplantation. The endpoint for survival was 7 days. Bcl-2 was expressed in hepatocytes and sinusoidal lining cells. This was associated with a significant reduction of apoptotic sinusoidal lining cells and hepatocytes after 24 hours and 7 days. Bile production was significantly higher following bcl-2 pretreatment. Furthermore, bcl-2 transfer resulted in significantly improved survival (100% vs. 50% both control groups). Adenoviral bcl-2 transfer results in protein expression in hepatocytes and sinusoidal lining cells resulting in early graft function and survival enhancement after prolonged ischemia and reperfusion injury. The inhibition of apoptosis in the context of liver transplantation might be a reasonable approach in the treatment of graft dysfunction.

  10. Oncolytic Adenovirus: Strategies and Insights for Vector Design and Immuno-Oncolytic Applications

    PubMed Central

    Uusi-Kerttula, Hanni; Hulin-Curtis, Sarah; Davies, James; Parker, Alan L.

    2015-01-01

    Adenoviruses (Ad) are commonly used both experimentally and clinically, including oncolytic virotherapy applications. In the clinical area, efficacy is frequently hampered by the high rates of neutralizing immunity, estimated as high as 90% in some populations that promote vector clearance and limit bioavailability for tumor targeting following systemic delivery. Active tumor targeting is also hampered by the ubiquitous nature of the Ad5 receptor, hCAR, as well as the lack of highly tumor-selective targeting ligands and suitable targeting strategies. Furthermore, significant off-target interactions between the viral vector and cellular and proteinaceous components of the bloodstream have been documented that promote uptake into non-target cells and determine dose-limiting toxicities. Novel strategies are therefore needed to overcome the obstacles that prevent efficacious Ad deployment for wider clinical applications. The use of less seroprevalent Ad serotypes, non-human serotypes, capsid pseudotyping, chemical shielding and genetic masking by heterologous peptide incorporation are all potential strategies to achieve efficient vector escape from humoral immune recognition. Conversely, selective vector arming with immunostimulatory agents can be utilized to enhance their oncolytic potential by activation of cancer-specific immune responses against the malignant tissues. This review presents recent advantages and pitfalls occurring in the field of adenoviral oncolytic therapies. PMID:26610547

  11. Optimization and physicochemical characterization of a cationic lipid-phosphatidylcholine mixed emulsion formulated as a highly efficient vehicle that facilitates adenoviral gene transfer.

    PubMed

    Kim, Soo-Yeon; Lee, Sang-Jin; Kim, Jin-Ki; Choi, Han-Gon; Lim, Soo-Jeong

    2017-01-01

    Cationic lipid-based nanoparticles enhance viral gene transfer by forming electrostatic complexes with adenoviral vectors. We recently demonstrated the superior complexation capabilities of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) emulsion compared with a liposomal counterpart but the cytotoxicity of DOTAP emulsions remained a challenge. The present study is aimed at formulating an emulsion capable of acting as a highly effective viral gene transfer vehicle with reduced cytotoxicity and to physicochemically characterize the structures of virus-emulsion complexes in comparison with virus-liposome complexes when the only difference between emulsions and liposomes was the presence or absence of inner oil core. The emulsion formulation was performed by 1) reducing the content of DOTAP while increasing the content of zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 2) optimizing the oil content. The complexation capability of formulated DOTAP:DMPC mixed emulsions was similar to those of emulsions containing DOTAP alone while displaying significantly lower cytotoxicity. The complexation capabilities of the DOTAP:DMPC mixed emulsion were serum-compatible and were monitored in a variety of cell types, whereas its liposomal counterpart was totally ineffective. Characterization by scanning electron microscopy, transmission electron microscopy, atomic force microscopy, and dynamic light scattering studies indicated that the optimized emulsions spontaneously surrounded the virus particles to generate emulsions that encapsulated the viral particles, whereas viral particles merely attached to the surfaces of the counterpart liposomes to form multiviral aggregates. Overall, these studies demonstrated that optimized DOTAP:DMPC mixed emulsions are potentially useful for adenoviral gene delivery due to less cytotoxicity and the unique ability to encapsulate the viral particle, highlighting the importance of nanoparticle formulation.

  12. Optimization and physicochemical characterization of a cationic lipid-phosphatidylcholine mixed emulsion formulated as a highly efficient vehicle that facilitates adenoviral gene transfer

    PubMed Central

    Kim, Soo-Yeon; Lee, Sang-Jin; Kim, Jin-Ki; Choi, Han-Gon; Lim, Soo-Jeong

    2017-01-01

    Cationic lipid-based nanoparticles enhance viral gene transfer by forming electrostatic complexes with adenoviral vectors. We recently demonstrated the superior complexation capabilities of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) emulsion compared with a liposomal counterpart but the cytotoxicity of DOTAP emulsions remained a challenge. The present study is aimed at formulating an emulsion capable of acting as a highly effective viral gene transfer vehicle with reduced cytotoxicity and to physicochemically characterize the structures of virus-emulsion complexes in comparison with virus–liposome complexes when the only difference between emulsions and liposomes was the presence or absence of inner oil core. The emulsion formulation was performed by 1) reducing the content of DOTAP while increasing the content of zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 2) optimizing the oil content. The complexation capability of formulated DOTAP:DMPC mixed emulsions was similar to those of emulsions containing DOTAP alone while displaying significantly lower cytotoxicity. The complexation capabilities of the DOTAP:DMPC mixed emulsion were serum-compatible and were monitored in a variety of cell types, whereas its liposomal counterpart was totally ineffective. Characterization by scanning electron microscopy, transmission electron microscopy, atomic force microscopy, and dynamic light scattering studies indicated that the optimized emulsions spontaneously surrounded the virus particles to generate emulsions that encapsulated the viral particles, whereas viral particles merely attached to the surfaces of the counterpart liposomes to form multiviral aggregates. Overall, these studies demonstrated that optimized DOTAP:DMPC mixed emulsions are potentially useful for adenoviral gene delivery due to less cytotoxicity and the unique ability to encapsulate the viral particle, highlighting the importance of nanoparticle formulation. PMID:29070949

  13. Adenovirus-Vectored Broadly Neutralizing Antibodies Directed Against gp120 Prevent Human Immunodeficiency Virus Type 1 Acquisition in Humanized Mice.

    PubMed

    Liu, Shan; Jackson, Andrew; Beloor, Jagadish; Kumar, Priti; Sutton, Richard E

    2015-09-01

    Despite nearly three decades of research, a safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) has yet to be achieved. More recently, the discovery of highly potent anti-gp160 broadly neutralizing antibodies (bNAbs) has garnered renewed interest in using antibody-based prophylactic and therapeutic approaches. Here, we encoded bNAbs in first-generation adenoviral (ADV) vectors, which have the distinctive features of a large coding capacity and ease of propagation. A single intramuscular injection of ADV-vectorized bNAbs in humanized mice generated high serum levels of bNAbs that provided protection against multiple repeated challenges with a high dose of HIV-1, prevented depletion of peripheral CD4(+) T cells, and reduced plasma viral loads to below detection limits. Our results suggest that ADV vectors may be a viable option for the prophylactic and perhaps therapeutic use of bNAbs in humans.

  14. Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library

    PubMed Central

    Yoshida, Kimiko; Goto, Naoko; Ohnami, Shumpei; Aoki, Kazunori

    2012-01-01

    The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:23029088

  15. Treatment of collagenase-induced osteoarthritis with a viral vector encoding TSG-6 results in ectopic bone formation.

    PubMed

    Broeren, Mathijs G A; Di Ceglie, Irene; Bennink, Miranda B; van Lent, Peter L E M; van den Berg, Wim B; Koenders, Marije I; Blaney Davidson, Esmeralda N; van der Kraan, Peter M; van de Loo, Fons A J

    2018-01-01

    Tumor necrosis factor-inducible gene 6 (TSG-6) has anti-inflammatory and chondroprotective effects in mouse models of inflammatory arthritis. Because cartilage damage and inflammation are also observed in osteoarthritis (OA), we determined the effect of viral overexpression of TSG-6 in experimental osteoarthritis. Bone marrow-derived cells were differentiated to multinucleated osteoclasts in the presence of recombinant TSG-6 or after transduction with a lentiviral TSG-6 expression vector. Multi-nucleated osteoclasts were analyzed after tartrate resistant acid phosphatase staining and resorption activity was determined on dentin slices. Collagenase-induced osteoarthritis (CIOA) was induced in C57BL/6 mice after intra-articular injection of an adenoviral TSG-6 or control luciferase expression vector. Inflammation-related protease activity was measured using bioluminescent Prosense probes. After a second adenovirus injection, cartilage damage was assessed in histological sections stained with Safranin-O. Ectopic bone formation was scored in X-ray images of the affected knees. TSG-6 did not inhibit the formation of multi-nucleated osteoclasts, but caused a significant reduction in the resorption activity on dentin slices. Adenoviral TSG-6 gene therapy in CIOA could not reduce the cartilage damage compared to the luciferase control virus and no significant difference in inflammation-related protease activity was noted between the TSG-6 and control treated group. Instead, X-ray analysis and histological analysis revealed the presence of ectopic bone formation in the TSG-6 treated group. Gene therapy based on the expression of TSG-6 could not provide cartilage protection in experimental osteoarthritis, but instead resulted in increased ectopic bone formation.

  16. Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

    PubMed

    Carinhas, Nuno; Pais, Daniel A M; Koshkin, Alexey; Fernandes, Paulo; Coroadinha, Ana S; Carrondo, Manuel J T; Alves, Paula M; Teixeira, Ana P

    2016-03-23

    Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

  17. Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration

    PubMed Central

    Zhang, Wenli; Muck-Hausl, Martin; Wang, Jichang; Sun, Chuanbo; Gebbing, Maren; Miskey, Csaba; Ivics, Zoltan; Izsvak, Zsuzsanna; Ehrhardt, Anja

    2013-01-01

    We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR). Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models. PMID:24124483

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

    PubMed

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

    2012-02-10

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

  19. Development of renal-targeted vectors through combined in vivo phage display and capsid engineering of adenoviral fibers from serotype 19p.

    PubMed

    Denby, Laura; Work, Lorraine M; Seggern, Dan J Von; Wu, Eugene; McVey, John H; Nicklin, Stuart A; Baker, Andrew H

    2007-09-01

    The potential efficacy of gene delivery is dictated by the infectivity profile of existing vectors, which is often restrictive. In order to target cells and organs for which no efficient vector is currently available, a promising approach would be to engineer vectors with novel transduction profiles. Applications that involve injecting adenovirus (Ad) vectors into the bloodstream require that native tropism for the liver be removed, and that targeting moieties be engineered into the capsid. We previously reported that pseudotyping the Ad serotype 5 fiber for that of Ad19p results in reduced hepatic transduction. In this study we show that this may be caused, at least in part, by a reduction in the capacity of the Ad19p-based virus to bind blood coagulation factors. It is therefore a potential candidate for vector retargeting, focusing on the kidney as a therapeutic target. We used in vivo phage display in rats, and identified peptides HTTHREP and HITSLLS that homed to the kidneys following intravenous injection. We engineered the HI loop of Ad19p to accommodate peptide insertions and clones. Intravenous delivery of each peptide-modified virus resulted in selective renal targeting, with HTTHREP and HITSLLS-targeted viruses selectively transducing tubular epithelium and glomeruli, respectively. Our study has important implications for the use of genetic engineering of Ad fibers to produce targeted gene delivery vectors.

  20. Surmounting limited gene delivery into primary immune cell populations: Efficient cell type-specific adenoviral transduction by CAR.

    PubMed

    Clausen, Björn E; Brand, Anna; Karram, Khalad

    2015-06-01

    Ectopic gene expression studies in primary immune cells have been notoriously difficult to perform due to the limitations in conventional transfection and viral transduction methods. Although replication-defective adenoviruses provide an attractive alternative for gene delivery, their use has been hampered by the limited susceptibility of murine leukocytes to adenoviral infection, due to insufficient expression of the human coxsackie/adenovirus receptor (CAR). In this issue of the European Journal of Immunology, Heger et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] report the generation of transgenic mice that enable conditional Cre/loxP-mediated expression of human CAR. The authors demonstrate that this R26/CAG-CAR∆1(StopF) mouse strain facilitates the faithful monitoring of Cre activity in situ as well as the specific and efficient adenoviral transduction of primary immune cell populations in vitro. Further tweaking of the system towards more efficient gene transfer in vivo remains a future challenge. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    PubMed

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.

  2. High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.

    PubMed

    Gwiazda, Kamila S; Grier, Alexandra E; Sahni, Jaya; Burleigh, Stephen M; Martin, Unja; Yang, Julia G; Popp, Nicholas A; Krutein, Michelle C; Khan, Iram F; Jacoby, Kyle; Jensen, Michael C; Rawlings, David J; Scharenberg, Andrew M

    2016-09-29

    Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

  3. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    NASA Astrophysics Data System (ADS)

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-06-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  4. Adenovirus Type 5 Viral Particles Pseudotyped with Mutagenized Fiber Proteins Show Diminished Infectivity of Coxsackie B-Adenovirus Receptor-Bearing Cells

    PubMed Central

    Jakubczak, John L.; Rollence, Michele L.; Stewart, David A.; Jafari, Jonathon D.; Von Seggern, Dan J.; Nemerow, Glen R.; Stevenson, Susan C.; Hallenbeck, Paul L.

    2001-01-01

    A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.βgal.ΔF, an E1-, E3-, and fiber-deleted adenoviral vector encoding β-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector. PMID:11222722

  5. Ultrasound-assisted non-viral gene transfer of AQP1 to the irradiated minipig parotid gland restores fluid secretion

    PubMed Central

    Wang, Z; Zourelias, L; Wu, C; Edwards, PC; Trombetta, M; Passineau, MJ

    2015-01-01

    Rationale Xerostomia is a common side effect of ionizing radiation used to treat head and neck cancer. A groundbreaking Phase I human clinical trial utilizing Adenoviral gene transfer of Aquaporin-1 (AQP1) to a single salivary gland of individuals suffering from radiation-induced xerostomia has recently been reported. Unfortunately, the limitations of the Adenoviral vector system utilized in this pioneering trial preclude its advancement to a Phase II trial and we have thus undertaken to evaluate the therapeutic potential of ultrasound-assisted non-viral gene transfer (UAGT) as an alternative means of delivering AQP1 gene therapy to the salivary gland by comparing head-to-head with the canonical Adenoviral vector in a swine model. Findings Swine irradiated unilaterally with a 10Gy electron beam targeted at the parotid gland suffered from significant, sustained hyposalivation that was bilateral, despite irradiation being confined to the targeted gland. Unilateral AQP1 gene therapy with UAGT resulted in bilateral restoration of stimulated salivary flow at 48 hours and one week post-treatment (1.62+/−0.48ml, 1.87+/−0.45ml) to pre-injury levels (1.34+/−0.14ml) in a manner comparable to Adenoviral delivery (2.32+/−0.6ml, 1.33+/−0.97ml). Conclusions UAGT can replace the Adenoviral vector as a means of delivering AQP1 gene therapy in the irradiated swine model and is a candidate for advancement to a Phase I human clinical trial. PMID:25871828

  6. An adenoviral vector expressing lipoprotein A, a major antigen of Mycoplasma mycoides subspecies mycoides, elicits robust immune responses in mice.

    PubMed

    Carozza, Marlène; Rodrigues, Valérie; Unterfinger, Yves; Galea, Sandra; Coulpier, Muriel; Klonjkowski, Bernard; Thiaucourt, François; Totté, Philippe; Richardson, Jennifer

    2015-01-01

    Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC), is a devastating respiratory disease of cattle. In sub-Saharan Africa, where CBPP is enzootic, live attenuated vaccines are deployed but afford only short-lived protection. In cattle, recovery from experimental MmmSC infection has been associated with the presence of CD4(+) T lymphocytes that secrete interferon gamma in response to MmmSC, and in particular to the lipoprotein A (LppA) antigen. In an effort to develop a better vaccine against CBPP, a viral vector (Ad5-LppA) that expressed LppA was generated from human adenovirus type 5. The LppA-specific immune responses elicited by the Ad5-LppA vector were evaluated in mice, and compared to those elicited by recombinant LppA formulated with a potent adjuvant. Notably, a single administration of Ad5-LppA, but not recombinant protein, sufficed to elicit a robust LppA-specific humoral response. After a booster administration, both vector and recombinant protein elicited strong LppA-specific humoral and cell-mediated responses. Ex vivo stimulation of splenocytes induced extensive proliferation of CD4(+) T cells for mice immunized with vector or protein, and secretion of T helper 1-associated and proinflammatory cytokines for mice immunized with Ad5-LppA. Our study - by demonstrating the potential of a viral-vectored prototypic vaccine to elicit prompt and robust immune responses against a major antigen of MmmSC - represents a first step in developing a recombinant vaccine against CBPP. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae.

    PubMed

    Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Pham, Thu Ha; Phan, Tuan-Nghia; Tran, Van-Tuan

    2016-12-01

    Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.

  8. Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma.

    PubMed

    Siurala, Mikko; Havunen, Riikka; Saha, Dipongkor; Lumen, Dave; Airaksinen, Anu J; Tähtinen, Siri; Cervera-Carrascon, Víctor; Bramante, Simona; Parviainen, Suvi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

    2016-08-01

    Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of (111)In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies.

  9. Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma

    PubMed Central

    Siurala, Mikko; Havunen, Riikka; Saha, Dipongkor; Lumen, Dave; Airaksinen, Anu J.; Tähtinen, Siri; Cervera-Carrascon, Víctor; Bramante, Simona; Parviainen, Suvi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

    2016-01-01

    Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of 111In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies. PMID:27357626

  10. Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells

    PubMed Central

    Tielen, Frans; Elstak, Edo; Benschop, Julian; Grimbergen, Max; Stallen, Jan; Janssen, Richard; van Marle, Andre; Essrich, Christian

    2017-01-01

    Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single “all-in-one” Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities. PMID:28800587

  11. STANDARDIZATION AND VALIDATION OF ADENOVIRAL TRANSDUCTION OF AN ANDROGEN RECEPTOR POSITIVE CELL LINE WITH AN MMTV-LUC REPORTER FOR ENDOCRINE SCREENING

    EPA Science Inventory

    Standardization and Validation of Adenoviral Transduction of an Androgen Receptor Positive Cell Line with an MMTV-Luc Reporter for Endocrine Screening P. Hartig, K . Bobseine,
    M. Cardon, C. Lambright and L. E. Gray, Jr. USEPA, Reproductive Toxicology Division, NHEERL, RTP, NC...

  12. Gene Transduction and Cell Entry Pathway of Fiber-Modified Adenovirus Type 5 Vectors Carrying Novel Endocytic Peptide Ligands Selected on Human Tracheal Glandular Cells

    PubMed Central

    Gaden, Florence; Franqueville, Laure; Magnusson, Maria K.; Hong, Saw See; Merten, Marc D.; Lindholm, Leif; Boulanger, Pierre

    2004-01-01

    Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from

  13. A new model for CD8+ T cell memory inflation based upon a recombinant adenoviral vector1

    PubMed Central

    Bolinger, Beatrice; Sims, Stuart; O’Hara, Geraldine; de Lara, Catherine; Tchilian, Elma; Firner, Sonja; Engeler, Daniel; Ludewig, Burkhard; Klenerman, Paul

    2013-01-01

    CD8+ T cell memory inflation, first described in murine cytomegalovirus (MCMV) infection, is characterized by the accumulation of high-frequency, functional antigen-specific CD8+ T cell pools with an effector-memory phenotype and enrichment in peripheral organs. Although persistence of antigen is considered essential, the rules underpinning memory inflation are still unclear. The MCMV model is, however, complicated by the virus’s low-level persistence, and stochastic reactivation. We developed a new model of memory inflation based upon a βgal-recombinant adenovirus vector (Ad-LacZ). After i.v. administration in C57BL/6 mice we observe marked memory inflation in the βgal96 epitope, while a second epitope, βgal497, undergoes classical memory formation. The inflationary T cell responses show kinetics, distribution, phenotype and functions similar to those seen in MCMV and are reproduced using alternative routes of administration. Memory inflation in this model is dependent on MHC Class II. As in MCMV, only the inflating epitope showed immunoproteasome-independence. These data define a new model for memory inflation, which is fully replication-independent, internally controlled and reproduces the key immunologic features of the CD8+ T cell response. This model provides insight into the mechanisms responsible for memory inflation, and since it is based on a vaccine vector, also is relevant to novel T cell-inducing vaccines in humans. PMID:23509359

  14. Sidestream Smoke Exposure Increases the Susceptibility of Airway Epithelia to Adenoviral Infection

    PubMed Central

    Sharma, Priyanka; Kolawole, Abimbola O.; Core, Susan B.; Kajon, Adriana E.; Excoffon, Katherine J. D. A.

    2012-01-01

    Background Although significant epidemiological evidence indicates that cigarette smoke exposure increases the incidence and severity of viral infection, the molecular mechanisms behind the increased susceptibility of the respiratory tract to viral pathogens are unclear. Adenoviruses are non-enveloped DNA viruses and important causative agents of acute respiratory disease. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for many adenoviruses. We hypothesized that cigarette smoke exposure increases epithelial susceptibility to adenovirus infection by increasing the abundance of apical CAR. Methodology and Findings Cultured human airway epithelial cells (CaLu-3) were used as a model to investigate the effect of sidestream cigarette smoke (SSS), mainstream cigarette smoke (MSS), or control air exposure on the susceptibility of polarized respiratory epithelia to adenoviral infection. Using a Cultex air-liquid interface exposure system, we have discovered novel differences in epithelial susceptibility between SSS and MSS exposures. SSS exposure upregulates an eight-exon isoform of CAR and increases adenoviral entry from the apical surface whilst MSS exposure is similar to control air exposure. Additionally, the level of cellular glycogen synthase kinase 3β (GSK3β) is downregulated by SSS exposure and treatment with a specific GSK3β inhibitor recapitulates the effects of SSS exposure on CAR expression and viral infection. Conclusions This is the first time that SSS exposure has been shown to directly enhance the susceptibility of a polarized epithelium to infection by a common respiratory viral pathogen. This work provides a novel understanding of the impact of SSS on the burden of respiratory viral infections and may lead to new strategies to alter viral infections. Moreover, since GSK3β inhibitors are under intense clinical investigation as therapeutics for a diverse range of diseases, studies such as these might provide insight to extend

  15. Intranuclear DNA release is a determinant of transfection activity for a non-viral vector: biocleavable polyrotaxane as a supramolecularly dissociative condenser for efficient intranuclear DNA release.

    PubMed

    Yamada, Yuma; Nomura, Taku; Harashima, Hideyoshi; Yamashita, Atsushi; Katoono, Ryo; Yui, Nobuhiko

    2010-01-01

    It has been believed that nuclear gene delivery is the most important process for gene expression, and various non-viral vectors are currently being developed with this assumption. However, some of our earlier studies revealed a surprising difference in transfection activity between viral and non-viral vectors: this difference is largely due to the result of the intranuclear disposition of DNA rather than its delivery to the nucleus (Hama S. et al. (2006), Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. Mol. Ther., 13, 786-794). Here, we report on some direct evidence that demonstrates the importance of the release of intranuclear DNA on transfection activity. The data show that transfection activity can be substantially enhanced by integrating a multifunctional envelope-type nano device (MEND) and a biocleavable polyrotaxane (DMAE-SS-PRX) as an artificial condenser. Our integration system showed significantly higher transfection activity compared to conventional gene delivery system. Moreover, this system provides a strong support for our hypothesis that intranuclear DNA disposition plays a critical role in gene expression for non-viral vectors.

  16. Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

    PubMed

    Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

    2016-10-01

    The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

  17. Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

    PubMed

    Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

    2018-05-02

    The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

  18. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluatedmore » an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.« less

  19. INGN 201: Ad-p53, Ad5CMV-p53, adenoviral p53, p53 gene therapy--introgen, RPR/INGN 201.

    PubMed

    2007-01-01

    programme to eligible LFS patients who have relapsed after standard treatment as part of physician-sponsored protocols at qualifying institutions in the US.A worldwide, exclusive license to a family of US patents covering a combination therapy comprised of INGN 201 in combination with several inhibitors of epidermal growth factor receptors (EGFr) such as Erbituxtrade mark Vectibixtrade mark and Tarcevatrade mark was granted to Introgen by The University of Texas MD Anderson Cancer Center in November 2006. In February 2006, Introgen exclusively licenced a broad patent (US Patent No. 6 989 375), originally issued to to the Board of Regents of The University of Texas System; the patent covers any therapeutic gene-based therapy when applied in combination with conventional cancer therapy such as radiation or chemotherapy. Introgen Therapeutics was awarded a patent from the US Patent and Trademark Office in June 2005 that directly covers many of the special features of its INGN 201 molecular therapy. US Patent No. 6,905,873 is one of a family of patents that cover INGN 201 that have been issued to the Board of Regents of The University of Texas System and exclusively licensed to Introgen. To date, Introgen controls 30 issued patents relevant to the product covering compositions, therapeutic methods of administering the product in virtually any form, alone and in conjunction with the most widely used chemotherapeutic and radiation treatments, as well as its production, and has a large number of pending patent applications in the US and in foreign countries relating to its ADVEXIN((R)) therapy. In December 2004, the US Patent and Trademark Office issued Patent No. 6,830,749 entitled Recombinant p53 Adenovirus Methods and Compositions. Importantly, the patent is the broadest adenoviral p53 patent to date, covering any adenovirus carrying the p53 gene under the control of any promoter. Previously, Patent No. 6,805,858 covering methods for the administration of INGN 201 to cancer

  20. E1(-)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells.

    PubMed

    Ramalingam, R; Rafii, S; Worgall, S; Brough, D E; Crystal, R G

    1999-05-01

    Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1(-)E4(+) replication-deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1(-)E4(+) Ad vectors provide an "antiapoptotic" signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of "suspended animation," remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a "pro-life" program that modifies cultured endothelial cells to survive for prolonged periods.

  1. Vestibular regeneration--experimental models and clinical implications.

    PubMed

    Albu, Silviu; Muresanu, Dafin F

    2012-09-01

    Therapies aimed at the protection and/or regeneration of inner ear hair cells are of great interest, given the significant monetary and quality of life impact of balance disorders. Different viral vectors have been shown to transfect various cell types in the inner ear. The past decade has provided tremendous advances in the use of adenoviral vectors to achieve targeted treatment delivery. Several routes of delivery have been identified to introduce vectors into the inner ear while minimizing injury to surrounding structures. Recently, the transcription factor Atoh1 was determined to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo has demonstrated the ability to regenerate vestibular hair cells by causing transdifferentiation of neighbouring epithelial-supporting cells. Functional recovery of the vestibular system has also been documented following adenoviral-induced Atoh1 overexpression. Experiments demonstrating gene transfer in human vestibular epithelial cells reveal that the human inner ear is a suitable target for gene therapy. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  2. Showing the Way: Oncolytic Adenoviruses as Chaperones of Immunostimulatory Adjuncts.

    PubMed

    Huang, Jing Li; LaRocca, Christopher J; Yamamoto, Masato

    2016-09-19

    Oncolytic adenoviruses (OAds) are increasingly recognized as vectors for immunotherapy in the treatment of various solid tumors. The myriads of advantages of using adenovirus include targeted specificity upon infection and selective replication, which lead to localized viral burst, exponential spread of OAds, and antitumor effect. OAds can also induce a strong immune reaction due to the massive release of tumor antigens upon cytolysis and the presence of viral antigens. This review will highlight recent advances in adenoviral vectors expressing immunostimulatory effectors, such as GM-CSF (granulocyte macrophage colony-stimulating factor), interferon-α, interleukin-12, and CD40L. We will also discuss the combination of OAds with other immunotherapeutic strategies and describe the current understanding of how adenoviral vectors interact with the immune system to eliminate cancer cells.

  3. Peroxisome Proliferator-Activated Receptor Subtype- and Cell-Type-Specific Activation of Genomic Target Genes upon Adenoviral Transgene Delivery

    PubMed Central

    Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G.; Mandrup, Susanne

    2006-01-01

    Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes. We demonstrate that PPARγ2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARγ2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (α, γ, and β/δ), we show that the subtypes have very different abilities to gain access to target sites and that in general the genomic occupancy correlates with the ability to activate the corresponding target gene. In addition, the specificity and potency of activation by PPAR subtypes are highly dependent on the cell type. Thus, PPAR subtype-specific activation of genomic target genes involves an intricate interplay between the properties of the subtype- and cell-type-specific settings at the individual target loci. PMID:16847324

  4. AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED CU,ZN-SOD AND MN-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED Cu,Zn-SOD AND Mn-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO. JB Smith1, PC Hartig3, MR Blanton3, KK Sulik1,2, and ES Hunter3. 1Department of Cell and Developmental Biology and 2Bowles Cente...

  5. Targeted retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene prevents loss of mouse (twy/twy) anterior horn neurons in vivo sustaining mechanical compression.

    PubMed

    Xu, Kan; Uchida, Kenzo; Nakajima, Hideaki; Kobayashi, Shigeru; Baba, Hisatoshi

    2006-08-01

    Immunohistochemical analysis after adenovirus (AdV)-mediated BDNF gene transfer in and around the area of mechanical compression in the cervical spinal cord of the hyperostotic mouse (twy/twy). To investigate the neuroprotective effect of targeted AdV-BDNF gene transfection in the twy mouse with spontaneous chronic compression of the spinal cord motoneurons. Several studies reported the neuroprotective effects of neurotrophins on injured spinal cord. However, no report has described the effect of targeted retrograde neurotrophic gene delivery on motoneuron survival in chronic compression lesions of the cervical spinal cord resembling lesions of myelopathy. LacZ marker gene using adenoviral vector (AdV-LacZ) was used to evaluate retrograde delivery from the sternomastoid muscle in adult twy mice (16-week-old) and (control). Four weeks after the AdV-LacZ or AdV-BDNF injection, the compressed cervical spinal cord was removed en bloc for immunohistologic investigation of b-galactosidase activity and immunoreactivity and immunoblot analyses of BDNF. The number of anterior horn neurons was counted using Nissl, ChAT and AChE staining. Spinal accessory motoneurons between C1 and C3 segments were successfully transfected by AdV-LacZ in both twy and ICR mice after targeted intramuscular injection. Immunoreactivity to BDNF was significantly stronger in AdV-BDNF-gene transfected twy mice than in AdV-LacZ-gene transfected mice. At the cord level showing the maximum compression in AdV-BDNF-transfected twy mice, the number of anterior horn neurons was sinificantly higher in the topographic neuronal cell counting of Nissl-, ChAT-, and AChE-stained samples than in AdV-LacZ-injected twy mice. Targeted AdV-BDNF-gene delivery significantly increased Nissl-stained anterior horn neurons and enhanced cholinergic enzyme activities in the twy. Our results suggest that targeted retrograde AdV-BDNF-gene in vivo delivery may enhance neuronal survival even under chronic mechanical compression.

  6. Recombinant adeno-associated virus vector carrying the thrombomodulin lectin-like domain for the treatment of abdominal aortic aneurysm.

    PubMed

    Lai, Chao-Han; Wang, Kuan-Chieh; Kuo, Cheng-Hsiang; Lee, Fang-Tzu; Cheng, Tsung-Lin; Chang, Bi-Ing; Yang, Yu-Jen; Shi, Guey-Yueh; Wu, Hua-Lin

    2017-07-01

    Thrombomodulin (TM), through its lectin-like domain (TMD1), sequesters proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that sustains inflammation and tissue damage. Our previous study demonstrated that short-term treatment with recombinant TM containing all the extracellular domains (i.e., rTMD123) inhibits HMGB1-RAGE signaling and confers protection against CaCl 2 -induced AAA formation. In this study, we attempted to further optimize TM domains, as a potential therapeutic agent for AAA, using the recombinant adeno-associated virus (AAV) vector. The therapeutic effects of recombinant TMD1 (rTMD1) and recombinant AAV vectors carrying the lectin-like domain of TM (rAAV-TMD1) were evaluated in the CaCl 2 -induced AAA model and angiotensin II-infused AAA model, respectively. In the CaCl 2 -induced model, treatment with rTMD1 suppressed the tissue levels of HMGB1 and RAGE, macrophage accumulation, elastin destruction and AAA formation, and the effects were comparable to a mole-equivalent dosage of rTMD123. In the angiotensin II-infused model, a single intravenous injection of rAAV-TMD1 (10 11 genome copies), which resulted in a persistently high serum level of TMD1 for at least 12 weeks, effectively attenuated AAA formation with suppression of HMGB1 and RAGE levels and inhibition of proinflammatory cytokine production, macrophage accumulation, matrix metalloproteinase activities and oxidative stress in the aortic wall. These findings corroborate the therapeutic potential of the TM lectin-like domain in AAA. The attenuation of angiotensin II-infused AAA by one-time delivery of rAAV-TMD1 provides a proof-of-concept validation of its application as potential gene therapy for aneurysm development. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Broad patterns in domestic vector-borne Trypanosoma cruzi transmission dynamics: synanthropic animals and vector control.

    PubMed

    Peterson, Jennifer K; Bartsch, Sarah M; Lee, Bruce Y; Dobson, Andrew P

    2015-10-22

    Chagas disease (caused by Trypanosoma cruzi) is the most important neglected tropical disease (NTD) in Latin America, infecting an estimated 5.7 million people in the 21 countries where it is endemic. It is one of the NTDs targeted for control and elimination by the 2020 London Declaration goals, with the first goal being to interrupt intra-domiciliary vector-borne T. cruzi transmission. A key question in domestic T. cruzi transmission is the role that synanthropic animals play in T. cruzi transmission to humans. Here, we ask, (1) do synanthropic animals need to be targeted in Chagas disease prevention policies?, and (2) how does the presence of animals affect the efficacy of vector control? We developed a simple mathematical model to simulate domestic vector-borne T. cruzi transmission and to specifically examine the interaction between the presence of synanthropic animals and effects of vector control. We used the model to explore how the interactions between triatomine bugs, humans and animals impact the number and proportion of T. cruzi-infected bugs and humans. We then examined how T. cruzi dynamics change when control measures targeting vector abundance are introduced into the system. We found that the presence of synanthropic animals slows the speed of T. cruzi transmission to humans, and increases the sensitivity of T. cruzi transmission dynamics to vector control measures at comparable triatomine carrying capacities. However, T. cruzi transmission is amplified when triatomine carrying capacity increases with the abundance of syntathoropic hosts. Our results suggest that in domestic T. cruzi transmission scenarios where no vector control measures are in place, a reduction in synanthropic animals may slow T. cruzi transmission to humans, but it would not completely eliminate transmission. To reach the 2020 goal of interrupting intra-domiciliary T. cruzi transmission, it is critical to target vector populations. Additionally, where vector control measures

  8. Hexon modification to improve the activity of oncolytic adenovirus vectors against neoplastic and stromal cells in pancreatic cancer.

    PubMed

    Lucas, Tanja; Benihoud, Karim; Vigant, Frédéric; Schmidt, Christoph Q; Schmidt, Christoph Q Andreas; Wortmann, Andreas; Bachem, Max G; Simmet, Thomas; Kochanek, Stefan

    2015-01-01

    Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFβ receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety.

  9. Suppression of adenoviral gene expression in the liver: role of innate vs adaptive immunity and their cell lysis mechanisms.

    PubMed

    Minagawa, Masahiro; Kawamura, Hiroki; Liu, Zhangxu; Govindarajan, Sugantha; Dennert, Gunther

    2005-06-01

    Injection of adenoviral constructs causes liver infection prompting immunity, which suppress viral gene expression. Innate and adaptive immunity mediate these processes raising the question which pathways are the most prominent. Adenovirus expressing the beta-galactosidase (beta-gal) gene was injected into normal and immunodeficient mice. Elimination of beta-gal-expressing hepatocytes and increases in liver enzymes were assayed. Major histocompatibility complex (MHC) class I densities, perforin channel insertion and apoptosis by Fas and tumor necrosis factor (TNF)-alpha were assayed. At high virus doses, suppression of viral gene expression was as efficient in immunodeficient as in normal mice, while at low doses effects of cytotoxic T lymphocytes (CTL) were demonstrable. Despite CTL priming and elimination of infected hepatocytes no liver injury is detected. Hepatocyte MHC I densities were able to trigger CTL granule exocytosis and perforin lysis in vitro but not in vivo. This is we show is because of decreased sensitivity of hepatocytes from infected mice to perforin and increased sensitivity to Fas and TNF-alpha lysis. Effector cells of the innate immune system are exceedingly effective in suppressing adenoviral gene expression. Perforin-independent pathways, those mediated by TNF-alpha and Fas are very efficient in hepatocytes from virus-infected livers.

  10. Adenoviral hemorrhagic disease in California mule deer, 1990-2014.

    PubMed

    Woods, Leslie W; Schumaker, Brant A; Pesavento, Patricia A; Crossley, Beate M; Swift, Pamela K

    2018-03-01

    We reviewed case records from the California Animal Health and Food Safety (CAHFS) laboratory and the California Department of Fish and Wildlife (CDFW) spanning 25 years (1990-2014) for all deer accessions submitted to CAHFS for pathology and/or histopathology, with and without a diagnosis of adenoviral hemorrhagic disease (AHD), in order to determine the prevalence of AHD in California. We also examined spatial and temporal distribution, age, and mule deer subspecies in deer that died from AHD. Of 483 deer submitted to CAHFS for diagnostic testing in 1990-2014, 17.2% were diagnosed with confirmed AHD, and 26.5% were confirmed plus suspected cases of AHD. Columbian black-tailed deer ( Odocoileus hemionus columbianus), particularly fawns and juveniles, were most frequently affected. Deer adenovirus ( Odocoileus adenovirus 1; OdAdV-1) was detected by immunohistochemistry in archived CDFW formalin-fixed, paraffin-embedded tissues from deer that died in mortality events in 1981, 1983, and 1986-1987. OdAdV-1 is a common cause of hemorrhagic disease mortality events in California deer, and mortality as a result of AHD is documented as early as 1981.

  11. JPRS Report Science & Technology USSR: Life Sciences.

    DTIC Science & Technology

    1988-12-15

    genetic engi- neering studies employed in studies on the effects of antisense RNA ( asRNA ) on the reproduction of type 5 adenovirus in cultured...monkey Cos I cells. The asRNA was targeted against the EIA region of the adenoviral genome, which has a key role in the regulation of other adenoviral...genes. A recombinant vector designated ppS- NEO containing Moloney leukemia genome was employed in the synthesis of asRNA , as well as recom- binant

  12. JPRS Report, Science & Technology, USSR: Life Sciences

    DTIC Science & Technology

    1988-12-15

    neering studies employed in studies on the effects of antisense RNA ( asRNA ) on the reproduction of type 5 adenovirus in cultured monkey Cos I cells...The asRNA was targeted against the EIA region of the adenoviral genome, which has a key role in the regulation of other adenoviral genes. A...recombinant vector designated ppS- NEO containing Moloney leukemia genome was employed in the synthesis of asRNA , as well as recom- binant plasmid ppA in

  13. Immunological thresholds in neurological gene therapy: highly efficient elimination of transduced cells might be related to the specific formation of immunological synapses between T cells and virus-infected brain cells

    PubMed Central

    Barcia, Carlos; Gerdes, Christian; Xiong, Wei-Dong; Thomas, Clare E.; Liu, Chunyan; Kroeger, Kurt M.; Castro, Maria G.; Lowenstein, Pedro R.

    2007-01-01

    First-generation adenovirus can be engineered with powerful promoters to drive expression of therapeutic transgenes. Numerous clinical trials for glioblastoma multiforme using first generation adenoviral vectors have either been performed or are ongoing, including an ongoing, Phase III, multicenter trial in Europe and Israel (Ark Therapeutics, Inc.). Although in the absence of anti-adenovirus immune responses expression in the brain lasts 6–18 months, systemic infection with adenovirus induces immune responses that inhibit dramatically therapeutic transgene expression from first generation adenoviral vectors, thus, potentially compromising therapeutic efficacy. Here, we show evidence of an immunization threshold for the dose that generates an immune response strong enough to eliminate transgene expression from the CNS. For the systemic immunization to eliminate transgene expression from the brain, ≥1 × 107 infectious units (iu) of adenovirus need to be used as immunogen. Furthermore, this immune response eliminates >90% of transgene expression from 1 × 107–1 × 10³ iu of vector injected into the striatum 60 days earlier. Importantly, elimination of transgene expression is independent of the nature of the promoter that drives transgene expression and is accompanied by brain infiltration of CD8+ T cells and macrophages. In conclusion, once the threshold for systemic immunization (i.e. 1 × 107 iu) is crossed, the immune response eliminates transgene expression by >90% even from brains that receive as little as 1000 iu of adenoviral vectors, independently of the type of promoter that drives expression. PMID:18084640

  14. Detectable reporter gene expression following transduction of adenovirus and adeno-associated virus serotype 2 vectors within full-thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo.

    PubMed

    Santangelo, Kelly S; Baker, Sarah A; Nuovo, Gerard; Dyce, Jonathan; Bartlett, Jeffrey S; Bertone, Alicia L

    2010-02-01

    This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p < or = 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo. (c) 2009 Orthopaedic Research Society.

  15. In vivo anti-tumour activity of recombinant MVM parvoviral vectors carrying the human interleukin-2 cDNA.

    PubMed

    El Bakkouri, Karim; Servais, Charlotte; Clément, Nathalie; Cheong, Siew Chiat; Franssen, Jean-Denis; Velu, Thierry; Brandenburger, Annick

    2005-02-01

    The natural oncotropism and oncotoxicity of vectors derived from the autonomous parvovirus, minute virus of mice (prototype strain) [MVM(p)], combined with the immunotherapeutic properties of cytokine transgenes, make them interesting candidates for cancer gene therapy. The in vivo anti-tumour activity of a recombinant parvoviral vector, MVM-IL2, was evaluated in a syngeneic mouse melanoma model that is relatively resistant in vitro to the intrinsic cytotoxicity of wild-type MVM(p). In vitro infection of the K1735 melanoma cells prior to their injection resulted in loss of tumorigenicity in 70% of mice (7/10). Tumour-free mice were protected against a challenge with non-infected parental cells. In addition, MVM-IL2-infected tumour cells induced an anti-tumour activity on parental cells injected at a distant location. These non-infected tumour cells were injected either at the same time or 7 days before the injection of MVM-IL2-infected cells. In the latter setting, which mimics a therapeutic model for small tumours, 4/10 mice were still tumour-free after 4 months. Our results show that (i) the MVM-IL2 parvoviral vector efficiently transduces tumour cells; and (ii) the low multiplicity of infection (MOI = 1) used in our experiments was sufficient to elicit an anti-tumour effect on distant cells, which supports further studies on this vector as a new tool for cancer gene therapy. Copyright (c) 2004 John Wiley & Sons, Ltd.

  16. Construction of human artificial chromosome vectors by recombineering.

    PubMed

    Kotzamanis, George; Cheung, Wing; Abdulrazzak, Hassan; Perez-Luz, Sara; Howe, Steven; Cooke, Howard; Huxley, Clare

    2005-05-23

    Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking approximately 70 kb of alphoid DNA onto a 156-kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56-65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the approximately 70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination.

  17. [Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene].

    PubMed

    Dang, Yin-li; Yan, Yan; Zhang, Xiao-xiao; Li, Pu-yuan; Yu, Lan; Zhang, Lei; Zhang, Fang-lin; Xu, Zhi-kai; Wu, Xing-an

    2011-05-01

    To stably express herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in Chinese hamster ovary cells (CHO-K1). The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by Lipofectamine 2000. The transfected cells were selected by G418 and methotrexate (MTX). The expression of HSV-1 gC was analyzed by Slot blot. HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot. The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully. CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot. The HSV-1 gC proteins have been expressed successfully and have good bioactivity. The results make it possible for further study and clinical use of HSV-1 gC.

  18. Adenoviral short hairpin RNA therapy targeting phosphodiesterase 5a relieves cardiac remodeling and dysfunction following myocardial infarction.

    PubMed

    Li, Longhu; Haider, Husnain Kh; Wang, Linlin; Lu, Gang; Ashraf, Muhammad

    2012-05-15

    We previously showed that treatment with tadalafil, a long-acting phosphodiesterase-5a (PDE5a) inhibitor, effectively prevented adverse left ventricular (LV) remodeling of the infarcted heart. We hypothesized that short-hairpin RNA (shRNA) therapy targeting PDE5a would simulate the effects of pharmacological intervention for treatment of postinfarction LV remodeling and dysfunction. Experimental model of myocardial infarction was developed in female mice by permanent ligation of left coronary artery. Immediately after that, an adenoviral vector encoding for shRNA sequence targeting PDE5a (Ad-shPDE5a) was injected intramyocardially, which specifically inhibited PDE5a in the heart. Four weeks later, Ad-shPDE5a treated mice showed significant mitigation of the left ventricle (LV) dilatation and dysfunction as indicated by smaller LV cavity and more preserved ejection fraction and fractional shortening. Infarction size and fibrosis were significantly reduced in Ad-shPDE5a-treated mice. Additionally, more salvaged cardiomyocytes, significantly reduced collagen contents, and higher blood vessel density were observed in Ad-shPDE5a-treated mice. The cytoprotective effects of Ad-shPDE5a were demonstrated in vitro in Ad-shPDE5a transfected cardiomyocytes cultured under oxygen glucose deprivation. Among downstream mediators of PDE5a signaling, cyclic GMP (cGMP) and cGMP-dependent protein kinase G (PKG) were activated with concomitant reduction in caspase-3 activity. However, no significant change in PKA and cAMP activities were observed in Ad-shPDE5a-treated hearts. Inhibition with shRNA improved cardiac remodeling and dysfunction by reducing infarction size and cardiac fibrosis and increased cGMP and PKG activity. These findings suggest that PDE5 inhibition with Ad-shPDE5a is a novel approach for treatment of myocardial infarction.

  19. Adenoviral short hairpin RNA therapy targeting phosphodiesterase 5a relieves cardiac remodeling and dysfunction following myocardial infarction

    PubMed Central

    Li, Longhu; Haider, Husnain Kh.; Wang, Linlin; Lu, Gang

    2012-01-01

    We previously showed that treatment with tadalafil, a long-acting phosphodiesterase-5a (PDE5a) inhibitor, effectively prevented adverse left ventricular (LV) remodeling of the infarcted heart. We hypothesized that short-hairpin RNA (shRNA) therapy targeting PDE5a would simulate the effects of pharmacological intervention for treatment of postinfarction LV remodeling and dysfunction. Experimental model of myocardial infarction was developed in female mice by permanent ligation of left coronary artery. Immediately after that, an adenoviral vector encoding for shRNA sequence targeting PDE5a (Ad-shPDE5a) was injected intramyocardially, which specifically inhibited PDE5a in the heart. Four weeks later, Ad-shPDE5a treated mice showed significant mitigation of the left ventricle (LV) dilatation and dysfunction as indicated by smaller LV cavity and more preserved ejection fraction and fractional shortening. Infarction size and fibrosis were significantly reduced in Ad-shPDE5a-treated mice. Additionally, more salvaged cardiomyocytes, significantly reduced collagen contents, and higher blood vessel density were observed in Ad-shPDE5a-treated mice. The cytoprotective effects of Ad-shPDE5a were demonstrated in vitro in Ad-shPDE5a transfected cardiomyocytes cultured under oxygen glucose deprivation. Among downstream mediators of PDE5a signaling, cyclic GMP (cGMP) and cGMP-dependent protein kinase G (PKG) were activated with concomitant reduction in caspase-3 activity. However, no significant change in PKA and cAMP activities were observed in Ad-shPDE5a-treated hearts. Inhibition with shRNA improved cardiac remodeling and dysfunction by reducing infarction size and cardiac fibrosis and increased cGMP and PKG activity. These findings suggest that PDE5 inhibition with Ad-shPDE5a is a novel approach for treatment of myocardial infarction. PMID:22447941

  20. Vector adaptive predictive coder for speech and audio

    NASA Technical Reports Server (NTRS)

    Chen, Juin-Hwey (Inventor); Gersho, Allen (Inventor)

    1990-01-01

    A real-time vector adaptive predictive coder which approximates each vector of K speech samples by using each of M fixed vectors in a first codebook to excite a time-varying synthesis filter and picking the vector that minimizes distortion. Predictive analysis for each frame determines parameters used for computing from vectors in the first codebook zero-state response vectors that are stored at the same address (index) in a second codebook. Encoding of input speech vectors s.sub.n is then carried out using the second codebook. When the vector that minimizes distortion is found, its index is transmitted to a decoder which has a codebook identical to the first codebook of the decoder. There the index is used to read out a vector that is used to synthesize an output speech vector s.sub.n. The parameters used in the encoder are quantized, for example by using a table, and the indices are transmitted to the decoder where they are decoded to specify transfer characteristics of filters used in producing the vector s.sub.n from the receiver codebook vector selected by the vector index transmitted.

  1. Adenoviral Gene Therapy Vectors Targeted to Prostate Cancer

    DTIC Science & Technology

    2004-06-01

    results from pre- clinical models into clinical trials . This problem has also been highlighted in Ad5 capsid mutation studies. Mutation of CAR and integrin...infectious eye disease in hospitals and eye 21. Harnett, G. B., and W. A. Newnham. 1981. Isolation of adenovirus type 19 clinics , from the male and female...promi- units or of large cDNAs such as the 7.1-kb ABCR gene nent in iris and ciliary body, with scattered positive cells involved in Stargardt disease

  2. Multineuronal vectorization is more efficient than time-segmental vectorization for information extraction from neuronal activities in the inferior temporal cortex.

    PubMed

    Kaneko, Hidekazu; Tamura, Hiroshi; Tate, Shunta; Kawashima, Takahiro; Suzuki, Shinya S; Fujita, Ichiro

    2010-08-01

    In order for patients with disabilities to control assistive devices with their own neural activity, multineuronal spike trains must be efficiently decoded because only limited computational resources can be used to generate prosthetic control signals in portable real-time applications. In this study, we compare the abilities of two vectorizing procedures (multineuronal and time-segmental) to extract information from spike trains during the same total neuron-seconds. In the multineuronal vectorizing procedure, we defined a response vector whose components represented the spike counts of one to five neurons. In the time-segmental vectorizing procedure, a response vector consisted of components representing a neuron's spike counts for one to five time-segment(s) of a response period of 1 s. Spike trains were recorded from neurons in the inferior temporal cortex of monkeys presented with visual stimuli. We examined whether the amount of information of the visual stimuli carried by these neurons differed between the two vectorizing procedures. The amount of information calculated with the multineuronal vectorizing procedure, but not the time-segmental vectorizing procedure, significantly increased with the dimensions of the response vector. We conclude that the multineuronal vectorizing procedure is superior to the time-segmental vectorizing procedure in efficiently extracting information from neuronal signals. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  3. Determinants of Outcomes of Adenoviral Keratoconjunctivitis.

    PubMed

    Lee, Cecilia S; Lee, Aaron Y; Akileswaran, Lakshmi; Stroman, David; Najafi-Tagol, Kathryn; Kleiboeker, Steve; Chodosh, James; Magaret, Amalia; Wald, Anna; Van Gelder, Russell N

    2018-03-27

    To determine host and pathogen factors predictive of outcomes in a large clinical cohort with keratoconjunctivitis. Retrospective analyses of the clinical and molecular data from a randomized, controlled, masked trial for auricloscene for keratoconjunctivitis (NVC-422 phase IIB, NovaBay; clinicaltrials.gov identifier, NCT01877694). Five hundred participants from United States, India, Brazil, and Sri Lanka with clinical diagnosis of keratoconjunctivitis and positive rapid test results for adenovirus. Clinical signs and symptoms and bilateral conjunctival swabs were obtained on days 1, 3, 6, 11, and 18. Polymerase chain reaction (PCR) analysis was performed to detect and quantify adenovirus in all samples. Regression models were used to evaluate the association of various variables with keratoconjunctivitis outcomes. Time to resolution of each symptom or sign was assessed by adenoviral species with Cox regression. The difference in composite scores of clinical signs between days 1 and 18, mean visual acuity change between days 1 and 18, and time to resolution of each symptom or sign. Of 500 participants, 390 (78%) showed evidence of adenovirus by PCR. Among adenovirus-positive participants, adenovirus D species was most common (63% of total cases), but a total of 4 species and 21 different types of adenovirus were detected. Adenovirus D was associated with more severe signs and symptoms, a higher rate of subepithelial infiltrate development, and a slower decline in viral load compared with all other adenovirus species. The clinical courses of all patients with non-adenovirus D species infection and adenovirus-negative keratoconjunctivitis were similar. Mean change in visual acuity between days 1 and 18 was a gain of 1.9 letters; worse visual outcome was associated with older age. A substantial proportion of keratoconjunctivitis is not associated with a detectable adenovirus. The clinical course of those with adenovirus D keratoconjunctivitis is significantly more

  4. Production of SV40-derived vectors.

    PubMed

    Strayer, David S; Mitchell, Christine; Maier, Dawn A; Nichols, Carmen N

    2010-06-01

    Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Here we describe a protocol for production and purification of these vectors. These procedures require only packaging cells (e.g., COS-7) and circular vector genome DNA. Amplification involves repeated infection of packaging cells with vector produced by transfection. Cotransfection is not required in any step. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients and resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. These approaches are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. These vectors are best applied to long-term expression of proteins normally encoded by mammalian cells or by viruses that infect mammalian cells, or of untranslated RNAs (e.g., RNA interference). The preparative approaches described facilitate application of these vectors and allow almost any laboratory to exploit their strengths for diverse gene delivery applications.

  5. The Development of the Differential MEMS Vector Hydrophone

    PubMed Central

    Zhang, Guojun; Liu, Mengran; Shen, Nixin; Wang, Xubo; Zhang, Wendong

    2017-01-01

    To solve the problem that MEMS vector hydrophones are greatly interfered with by the vibration of the platform and flow noise in applications, this paper describes a differential MEMS vector hydrophone that could simultaneously receive acoustic signals and reject acceleration signals. Theoretical and simulation analyses have been carried out. Lastly, a prototype of the differential MEMS vector hydrophone has been created and tested using a standing wave tube and a vibration platform. The results of the test show that this hydrophone has a high sensitivity, Mv = −185 dB (@ 500 Hz, 0 dB reference 1 V/μPa), which is almost the same as the previous MEMS vector hydrophones, and has a low acceleration sensitivity, Mv = −58 dB (0 dB reference 1 V/g), which has decreased by 17 dB compared with the previous MEMS vector hydrophone. The differential MEMS vector hydrophone basically meets the requirements of acoustic vector detection when it is rigidly fixed to a working platform, which lays the foundation for engineering applications of MEMS vector hydrophones. PMID:28594384

  6. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

    PubMed

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. PCR diagnostics and monitoring of adenoviral infections in hematopoietic stem cell transplantation recipients

    PubMed Central

    Ussowicz, Marek; Rybka, Blanka; Wendycz-Domalewska, Danuta; Ryczan, Renata; Gorczyńska, Ewa; Kałwak, Krzysztof; Woźniak, Mieczysław

    2010-01-01

    After stem cell transplantation, human patients are prone to life-threatening opportunistic infections with a plethora of microorganisms. We report a retrospective study on 116 patients (98 children, 18 adults) who were transplanted in a pediatric bone marrow transplantation unit. Blood, urine and stool samples were collected and monitored for adenovirus (AdV) DNA using polymerase chain reaction (PCR) and real-time PCR (RT-PCR) on a regular basis. AdV DNA was detected in 52 (44.8%) patients, with mortality reaching 19% in this subgroup. Variables associated with adenovirus infection were transplantations from matched unrelated donors and older age of the recipient. An increased seasonal occurrence of adenoviral infections was observed in autumn and winter. Analysis of immune reconstitution showed a higher incidence of AdV infections during periods of low T-lymphocyte count. This study also showed a strong interaction between co-infections of AdV and BK polyomavirus in patients undergoing hematopoietic stem cell transplantations. PMID:20848295

  8. Declining Prevalence of Disease Vectors Under Climate Change

    NASA Astrophysics Data System (ADS)

    Escobar, Luis E.; Romero-Alvarez, Daniel; Leon, Renato; Lepe-Lopez, Manuel A.; Craft, Meggan E.; Borbor-Cordova, Mercy J.; Svenning, Jens-Christian

    2016-12-01

    More than half of the world population is at risk of vector-borne diseases including dengue fever, chikungunya, zika, yellow fever, leishmaniasis, chagas disease, and malaria, with highest incidences in tropical regions. In Ecuador, vector-borne diseases are present from coastal and Amazonian regions to the Andes Mountains; however, a detailed characterization of the distribution of their vectors has never been carried out. We estimate the distribution of 14 vectors of the above vector-borne diseases under present-day and future climates. Our results consistently suggest that climate warming is likely threatening some vector species with extinction, locally or completely. These results suggest that climate change could reduce the burden of specific vector species. Other vector species are likely to shift and constrain their geographic range to the highlands in Ecuador potentially affecting novel areas and populations. These forecasts show the need for development of early prevention strategies for vector species currently absent in areas projected as suitable under future climate conditions. Informed interventions could reduce the risk of human exposure to vector species with distributional shifts, in response to current and future climate changes. Based on the mixed effects of future climate on human exposure to disease vectors, we argue that research on vector-borne diseases should be cross-scale and include climatic, demographic, and landscape factors, as well as forces facilitating disease transmission at fine scales.

  9. Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries.

    PubMed

    Hernandez-Hurtado, Adelina A; Borrego-Soto, Gissela; Marino-Martinez, Ivan A; Lara-Arias, Jorge; Romero-Diaz, Viktor J; Abrego-Guerra, Adalberto; Vilchez-Cavazos, Jose F; Elizondo-Riojas, Guillermo; Martinez-Rodriguez, Herminia G; Espinoza-Juarez, Marcela A; Lopez-Romero, Gloria C; Robles-Zamora, Alejandro; Mendoza Lemus, Oscar F; Ortiz-Lopez, Rocio; Rojas-Martinez, Augusto

    2016-01-01

    Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

  10. Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries

    PubMed Central

    Hernandez-Hurtado, Adelina A.; Lara-Arias, Jorge; Romero-Diaz, Viktor J.; Abrego-Guerra, Adalberto; Vilchez-Cavazos, Jose F.; Elizondo-Riojas, Guillermo; Martinez-Rodriguez, Herminia G.; Espinoza-Juarez, Marcela A.; Mendoza Lemus, Oscar F.

    2016-01-01

    Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology. PMID:27818692

  11. Water-soluble polymers bearing phosphorylcholine group and other zwitterionic groups for carrying DNA derivatives.

    PubMed

    Lin, Xiaojie; Ishihara, Kazuhiko

    2014-01-01

    Water-soluble polymers with equal positive and negative charges in the same monomer unit, such as the phosphorylcholine group and other zwitterionic groups, exhibit promising potential in gene delivery with appreciable transfection efficiency, compared with the traditional poly(ethylene glycol)-based polycation-gene complexes. These zwitterionic polymers with various architectural structures and properties have been synthesized by various polymerization methods, such as conventional radical polymerization, atom-transfer radical-polymerization, reversible addition-fragmentation chain-transfer polymerization, and nitroxide-mediated radical polymerization. These techniques have been used to efficiently facilitate gene therapy by fabrication of non-viral vectors with high cytocompatibility, large gene-carrying capacity, effective cell-membrane permeability, and in vivo gene-loading/releasing functionality. Zwitterionic polymer-based gene delivery vectors systems can be categorized into soluble-polymer/gene mixing, molecular self-assembly, and polymer-gene conjugation systems. This review describes the preparation and characterization of various zwitterionic polymer-based gene delivery vectors, specifically water-soluble phospholipid polymers for carrying gene derivatives.

  12. RGD capsid modification enhances mucosal protective immunity of a non-human primate adenovirus vector expressing Pseudomonas aeruginosa OprF

    PubMed Central

    Krause, A; Whu, W Z; Qiu, J; Wafadari, D; Hackett, N R; Sharma, A; Crystal, R G; Worgall, S

    2013-01-01

    Replication-deficient adenoviral (Ad) vectors of non-human serotypes can serve as Ad vaccine platforms to circumvent pre-existing anti-human Ad immunity. We found previously that, in addition to that feature, a non-human primate-based AdC7 vector expressing outer membrane protein F of P. aeruginosa (AdC7OprF) was more potent in inducing lung mucosal and protective immunity compared to a human Ad5-based vector. In this study we analysed if genetic modification of the AdC7 fibre to display an integrin-binding arginine–glycine–aspartic acid (RGD) sequence can further enhance lung mucosal immunogenicity of AdC7OprF. Intratracheal immunization of mice with either AdC7OprF.RGD or AdC7OprF induced robust serum levels of anti-OprF immunoglobulin (Ig)G up to 12 weeks that were higher compared to immunization with the human vectors Ad5OprF or Ad5OprF.RGD. OprF-specific cellular responses in lung T cells isolated from mice immunized with AdC7OprF.RGD and AdC7OprF were similar for T helper type 1 (Th1) [interferon (IFN)-γ in CD8+ and interleukin (IL)-12 in CD4+], Th2 (IL-4, IL-5 and IL-13 in CD4+) and Th17 (IL-17 in CD4+). Interestingly, AdC7OprF.RGD induced more robust protective immunity against pulmonary infection with P. aeruginosa compared to AdC7OprF or the control Ad5 vectors. The enhanced protective immunity induced by AdC7OprF.RGD was maintained in the absence of alveolar macrophages (AM) or CD1d natural killer T cells. Together, the data suggest that addition of RGD to the fibre of an AdC7-based vaccine is useful to enhance its mucosal protective immunogenicity. PMID:23607394

  13. Air travel and vector-borne disease movement.

    PubMed

    Tatem, A J; Huang, Z; Das, A; Qi, Q; Roth, J; Qiu, Y

    2012-12-01

    Recent decades have seen substantial expansions in the global air travel network and rapid increases in traffic volumes. The effects of this are well studied in terms of the spread of directly transmitted infections, but the role of air travel in the movement of vector-borne diseases is less well understood. Increasingly however, wider reaching surveillance for vector-borne diseases and our improving abilities to map the distributions of vectors and the diseases they carry, are providing opportunities to better our understanding of the impact of increasing air travel. Here we examine global trends in the continued expansion of air transport and its impact upon epidemiology. Novel malaria and chikungunya examples are presented, detailing how geospatial data in combination with information on air traffic can be used to predict the risks of vector-borne disease importation and establishment. Finally, we describe the development of an online tool, the Vector-Borne Disease Airline Importation Risk (VBD-Air) tool, which brings together spatial data on air traffic and vector-borne disease distributions to quantify the seasonally changing risks for importation to non-endemic regions. Such a framework provides the first steps towards an ultimate goal of adaptive management based on near real time flight data and vector-borne disease surveillance.

  14. Optical Associative Memory Model With Threshold Modification Using Complementary Vector

    NASA Astrophysics Data System (ADS)

    Bian, Shaoping; Xu, Kebin; Hong, Jing

    1989-02-01

    A new criterion to evaluate the similarity between two vectors in associative memory is presented. According to it, an experimental research about optical associative memory model with threshold modification using complementary vector is carried out. This model is capable of eliminating the posibility to recall erroneously. Therefore the accuracy of reading out is improved.

  15. DNA transformations of Candida tropicalis with replicating and integrative vectors.

    PubMed

    Sanglard, D; Fiechter, A

    1992-12-01

    The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

  16. Applications of lentiviral vectors in molecular imaging.

    PubMed

    Chatterjee, Sushmita; De, Abhijit

    2014-06-01

    Molecular imaging provides the ability of simultaneous visual and quantitative estimation of long term gene expression directly from living organisms. To reveal the kinetics of gene expression by imaging method, often sustained expression of the transgene is required. Lentiviral vectors have been extensively used over last fifteen years for delivery of a transgene in a wide variety of cell types. Lentiviral vectors have the well known advantages such as sustained transgene delivery through stable integration into the host genome, the capability of infecting non-dividing and dividing cells, broad tissue tropism, a reasonably large carrying capacity for delivering therapeutic and reporter gene combinations. Additionally, they do not express viral proteins during transduction, have a potentially safe integration site profile, and a relatively easy system for vector manipulation and infective viral particle production. As a result, lentiviral vector mediated therapeutic and imaging reporter gene delivery to various target organs holds promise for the future treatment. In this review, we have conducted a brief survey of important lentiviral vector developments in diverse biomedical fields including reproductive biology.

  17. Targeting a Newly Established Spontaneous Feline Fibrosarcoma Cell Line by Gene Transfer

    PubMed Central

    Nande, Rounak; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D.; Denvir, Jim; Valluri, Jagan; Duncan, Gary C.; Claudio, Pier Paolo

    2012-01-01

    Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas. PMID:22666387

  18. Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer.

    PubMed

    Nande, Rounak; Di Benedetto, Altomare; Aimola, Pierpaolo; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D; Denvir, Jim; Valluri, Jagan; Duncan, Gary C; Claudio, Pier Paolo

    2012-01-01

    Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.

  19. T Cell Responses Induced by Adenoviral Vectored Vaccines Can Be Adjuvanted by Fusion of Antigen to the Oligomerization Domain of C4b-Binding Protein

    PubMed Central

    Forbes, Emily K.; de Cassan, Simone C.; Llewellyn, David; Biswas, Sumi; Goodman, Anna L.; Cottingham, Matthew G.; Long, Carole A.; Pleass, Richard J.; Hill, Adrian V. S.; Hill, Fergal; Draper, Simon J.

    2012-01-01

    Viral vectored vaccines have been shown to induce both T cell and antibody responses in animals and humans. However, the induction of even higher level T cell responses may be crucial in achieving vaccine efficacy against difficult disease targets, especially in humans. Here we investigate the oligomerization domain of the α-chain of C4b-binding protein (C4 bp) as a candidate T cell “molecular adjuvant” when fused to malaria antigens expressed by human adenovirus serotype 5 (AdHu5) vectored vaccines in BALB/c mice. We demonstrate that i) C-terminal fusion of an oligomerization domain can enhance the quantity of antigen-specific CD4+ and CD8+ T cell responses induced in mice after only a single immunization of recombinant AdHu5, and that the T cells maintain similar functional cytokine profiles; ii) an adjuvant effect is observed for AdHu5 vectors expressing either the 42 kDa C-terminal domain of Plasmodium yoelii merozoite surface protein 1 (PyMSP142) or the 83 kDa ectodomain of P. falciparum strain 3D7 apical membrane antigen 1 (PfAMA1), but not a candidate 128kDa P. falciparum MSP1 biallelic fusion antigen; iii) following two homologous immunizations of AdHu5 vaccines, antigen-specific T cell responses are further enhanced, however, in both BALB/c mice and New Zealand White rabbits no enhancement of functional antibody responses is observed; and iv) that the T cell adjuvant activity of C4 bp is not dependent on a functional Fc-receptor γ-chain in the host, but is associated with the oligomerization of small (<80 kDa) antigens expressed by recombinant AdHu5. The oligomerization domain of C4 bp can thus adjuvant T cell responses induced by AdHu5 vectors against selected antigens and its clinical utility as well as mechanism of action warrant further investigation. PMID:22984589

  20. Phosphodiesterase 5a Inhibition with Adenoviral Short Hairpin RNA Benefits Infarcted Heart Partially through Activation of Akt Signaling Pathway and Reduction of Inflammatory Cytokines.

    PubMed

    Li, Longhu; Zhao, Dong; Jin, Zhe; Zhang, Jian; Paul, Christian; Wang, Yigang

    2015-01-01

    Treatment with short hairpin RNA (shRNA) interference therapy targeting phosphodiesterase 5a after myocardial infarction (MI) has been shown to mitigate post-MI heart failure. We investigated the mechanisms that underpin the beneficial effects of PDE5a inhibition through shRNA on post-MI heart failure. An adenoviral vector with an shRNA sequence inserted was adopted for the inhibition of phosphodiesterase 5a (Ad-shPDE5a) in vivo and in vitro. Myocardial infarction (MI) was induced in male C57BL/6J mice by left coronary artery ligation, and immediately after that, the Ad-shPDE5a was injected intramyocardially around the MI region and border areas. Four weeks post-MI, the Ad-shPDE5a-treated mice showed significant mitigation of the left ventricular (LV) dilatation and dysfunction compared to control mice. Infarction size and fibrosis were also significantly reduced in Ad-shPDE5a-treated mice. Additionally, Ad-shPDE5a treatment decreased the MI-induced inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and transforming growth factor-β1, which was confirmed in vitro in Ad-shPDE5a transfected myofibroblasts cultured under oxygen glucose deprivation. Finally, Ad-shPDE5a treatment was found to activate the myocardial Akt signaling pathway in both in vivo and in vitro experiments. These findings indicate that PDE5a inhibition by Ad-shPDE5a via the Akt signal pathway could be of significant value in the design of future therapeutics for post-MI heart failure.

  1. Measurement and assessment of carrying capacity of the environment in Ningbo, China.

    PubMed

    Liu, R Z; Borthwick, Alistair G L

    2011-08-01

    Carrying Capacity of the Environment (CCE) provides a useful measure of the sustainable development of a region. Approaches that use integrated assessment instead of measurement can lead to misinterpretation of sustainable development because of confusion between Environmental Stress (ES) indexes and CCE indexes, and the selection of over-simple linear plus models. The present paper proposes a comprehensive measurement system for CCE which comprises models of natural resources capacity, environmental assimilative capacity, ecosystem services capacity, and society supporting capacity. The corresponding measurable indexes are designed to assess CCE using a carrying capacity surplus ratio model and a vector of surplus ratio of carrying capacity model. The former aims at direct comparison of ES and CCE based on the values of basic indexes, and the latter uses a Euclidean vector to assess CCE states. The measurement and assessment approaches are applicable to Strategic Environmental Assessment (SEA) and environmental planning and management. A case study is presented for Ningbo, China, whereby all the basic indexes of ECC are measured and the CCE states assessed for 2005 and 2010. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. What is the 'true' effect of Trypanosoma rangeli on its triatomine bug vector?

    PubMed

    Peterson, Jennifer K; Graham, Andrea L

    2016-06-01

    The phrase, "T. rangeli is pathogenic to its insect vector," is commonly found in peer-reviewed publications on the matter, such that it has become the orthodox view of this interaction. In a literature survey, we identified over 20 papers with almost the exact phrase and several others alluding to it. The idea is of particular importance in triatomine population dynamics and the study of vector-borne T. cruzi transmission, as it could mean that triatomines infected with T. rangeli have lower fitness than uninfected insects. Trypanosoma rangeli pathogenicity was first observed in a series of studies carried out over fifty years ago using the triatomine species Rhodnius prolixus. However, there are few studies of the effect of T. rangeli on its other vector species, and several of the studies were carried out with R. prolixus under non-physiological conditions. Here, we re-evaluate the published studies that led to the conclusion that T. rangeli is pathogenic to its vector, to determine whether or not this indeed is the "true" effect of T. rangeli on its triatomine vector. © 2016 The Society for Vector Ecology.

  3. Enhanced Peptide of Prostate Cancer Using Targeted Adenoviral Vectors

    DTIC Science & Technology

    2005-06-01

    receptor subtype 2 has been constructed and evaluated in-human prostate cancer cells with regard to binding: of 64Cu - octreotide. In vivo experiments...of 64CU -octreotide.. The mice wer.e. sacrificed 1. h after peptide injection for biodistribution analysis. In vivo biodistribution studies showed...similar uptake of 64Cu - octreotide in both DU-145 and PC-3 tumors after infection with-AdSSTR2. (2.5. and 2.7% ID/g, respectively). This uptake was

  4. Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

    PubMed Central

    2013-01-01

    Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the

  5. Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression.

    PubMed

    Unzu, C; Melero, I; Hervás-Stubbs, S; Sampedro, A; Mancheño, U; Morales-Kastresana, A; Serrano-Mendioroz, I; de Salamanca, R E; Benito, A; Fontanellas, A

    2015-11-01

    Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles kg(-1) (10(10) infective units kg(-1)) of HDA only resulted in transient (≈14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.

  6. A method to rapidly and accurately compare relative efficacies of non-invasive imaging reporter genes in a mouse model, and its application to luciferase reporters

    PubMed Central

    Gil, Jose S.; Machado, Hidevaldo B.; Herschman, Harvey R.

    2013-01-01

    Purpose Our goal is to develop a simple, quantitative, robust method to compare the efficacy of imaging reporter genes in culture and in vivo. We describe an adenoviral vector-liver transduction procedure, and compare the luciferase reporter efficacies. Procedures Alternative reporter genes are expressed in a common adenoviral vector. Vector amounts used in vivo are based on cell culture titrations, ensuring the same transduction efficacy is used for each vector. After imaging, in vivo and in vitro values are normalized to hepatic vector transduction using quantitative real-time PCR. Results We assayed standard firefly luciferase (FLuc), enhanced firefly luciferase (EFLuc), luciferase 2 (Luc2), humanized Renilla luciferase (hRLuc), Renilla luciferase 8.6-535 (RLuc8.6), and a membrane-bound Gaussia luciferase variant (extGLuc) in cell culture and in vivo. We observed a greater that 100-fold increase in bioluminescent signal for both EFLuc and Luc2 when compared to FLuc, and a greater than 106-fold increase for RLuc8.6 when compared to hRLuc. ExtGLuc was not detectable in liver. Conclusions Our findings contrast, in some cases, with conclusions drawn in prior comparisons of these reporter genes, and demonstrate the need for a standardized method to evaluate alternative reporter genes in vivo. Our procedure can be adapted for reporter genes that utilize alternative imaging modalities (fluorescence, bioluminescence, MRI, SPECT, PET). PMID:21850545

  7. Clinical and Antiviral Efficacy of an Ophthalmic Formulation of Dexamethasone Povidone-Iodine in a Rabbit Model of Adenoviral Keratoconjunctivitis

    PubMed Central

    Clement, Christian; Capriotti, Joseph A.; Kumar, Manish; Hobden, Jeffery A.; Foster, Timothy P.; Bhattacharjee, Partha S.; Thompson, Hilary W.; Mahmud, Rashed; Liang, Bo

    2011-01-01

    Purpose. To determine the efficacy of a new formulation of topical dexamethasone 0.1%/povidone-iodine 0.4% (FST-100) in reducing clinical symptoms and infectious viral titers in a rabbit model of adenoviral keratoconjunctivitis. Methods. Rabbit corneas were inoculated bilaterally with 2 × 106 plaque-forming-units (PFU) of adenovirus type 5 (Ad5) after corneal scarification. Animals were randomized 1:1:1:1 (five rabbits per group) to FST-100, 0.5% cidofovir, tobramycin/dexamethasone (Tobradex; Alcon Laboratories, Fort Worth, TX) ophthalmic suspension, and balanced salt solution (BSS; Alcon Laboratories). Treatment began 12 hours after viral inoculation and continued for 7 consecutive days. The eyes were clinically scored daily for scleral inflammation (injection), ocular neovascularization, eyelid inflammation (redness), friability of vasculature, inflammatory discharge (pus), and epiphora (excessive tearing). Eye swabs were collected daily before treatment for the duration of the study. Virus was eluted from the swabs and PFU determined by titration on human A549 cells, according to standard procedures. Results. The FST-100 treatment resulted in significantly lower clinical scores (P < 0.05) than did the other treatments. The 0.5% cidofovir exhibited the most ocular toxicity compared with FST-100, tobramycin/dexamethasone, and balanced salt solution treatments. FST-100 and 0.5% cidofovir significantly (P < 0.05) reduced viral titers compared with tobramycin/dexamethasone or balanced salt solution. Conclusions. FST-100 was the most efficacious in minimizing the clinical symptoms of adenovirus infection in rabbit eyes. FST-100 and 0.5% cidofovir were both equally effective in reducing viral titers and decreasing the duration of viral shedding. By providing symptomatic relief in addition to reducing infectious virus titers, FST-100 should be a valuable addition to treatment of epidemic adenoviral keratoconjunctivitis. PMID:20702820

  8. Doxycycline-Regulated 3T3-L1 Preadipocyte Cell Line with Inducible, Stable Expression of Adenoviral E4orf1 Gene: A Cell Model to Study Insulin-Independent Glucose Disposal

    PubMed Central

    Krishnapuram, Rashmi; Dhurandhar, Emily J.; Dubuisson, Olga; Hegde, Vijay; Dhurandhar, Nikhil V.

    2013-01-01

    Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1). Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin. PMID:23544159

  9. Doxycycline-regulated 3T3-L1 preadipocyte cell line with inducible, stable expression of adenoviral E4orf1 gene: a cell model to study insulin-independent glucose disposal.

    PubMed

    Krishnapuram, Rashmi; Dhurandhar, Emily J; Dubuisson, Olga; Hegde, Vijay; Dhurandhar, Nikhil V

    2013-01-01

    Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1). Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin.

  10. Assessment of Climate Change and Vector-borne Diseases in the United States

    NASA Astrophysics Data System (ADS)

    Monaghan, A. J.; Beard, C. B.; Eisen, R. J.; Barker, C. M.; Garofalo, J.; Hahn, M.; Hayden, M.; Ogden, N.; Schramm, P.

    2016-12-01

    Vector-borne diseases are illnesses that are transmitted by vectors, which include mosquitoes, ticks, and fleas. The seasonality, distribution, and prevalence of vector-borne diseases are influenced significantly by climate factors, primarily high and low temperature extremes and precipitation patterns. In this presentation we summarize key findings from Chapter 5 ("Vector-borne Diseases") of the recently published USGCRP Scientific Assessment of the Impacts of Climate Change on Human Health in the United States. Climate change is expected to alter geographic and seasonal distributions of vectors and vector-borne diseases, leading to earlier activity and northward range expansion of ticks capable of carrying the bacteria that cause Lyme disease and other pathogens, and influencing the distribution, abundance and prevalence of infection in mosquitoes that transmit West Nile virus and other pathogens. The emergence or reemergence of vector-borne pathogens is also likely.

  11. Novel mechanism of JNK pathway activation by adenoviral E1A

    PubMed Central

    Morrison, Helen; Pospelova, Tatiana V.; Pospelov, Valery A.; Herrlich, Peter

    2014-01-01

    The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action. PMID:24742962

  12. [Construction and expression of a recombinant adenovirus with LZP3].

    PubMed

    Chen, Bang-dang; Zhang, Fu-chun; Sun, Mei-yu; Li, Yi-jie; Ma, Zheng-hai

    2007-08-01

    To explore a new immunocontraceptive vaccine and construct an attenuated recombinant adenoviral vaccine against Lagurus lagurus zona pellucida 3(LZP3). LZP3 gene was subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with LZP3, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. Finally, HeLa cells were infected by the recombinant adenovirus. LZP3 gene was detected from the recombinant virus by PCR, and its transcription and expression were analyzed by RT-PCR and Western blot. Recombinant adenovirus vector pAd-LZP3 with LZP3 gene was constructed by homologous recombination in E.coli, and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAd-LZP3. PCR test indicated that LZP3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2x10(10) pfu/L. The transcription and expression of LZP3 gene in the infected HeLa cells were confirmed by RT-PCR and Western blot. The recombinant adenovirus RAd-LZP3 can be successfully expressed in the infected HeLa cells, which lays the foundation for further researches into immunizing animals with RAd-LZP3.

  13. Tick-borne pathogens and the vector potential of ticks in China.

    PubMed

    Yu, Zhijun; Wang, Hui; Wang, Tianhong; Sun, Wenying; Yang, Xiaolong; Liu, Jingze

    2015-01-14

    Ticks, as obligate blood-sucking ectoparasites, attack a broad range of vertebrates and transmit a great diversity of pathogenic microorganisms. They are considered second only to mosquitoes as vectors of human disease, and the most important vector of pathogens of domestic and wild animals. Of the 117 described species in the Chinese tick fauna, 60 are known to transmit one or more diseases: 36 species isolated within China and 24 species isolated outside China. Moreover, 38 of these species carry multiple pathogens, indicating the potentially vast role of these vectors in transmitting pathogens. Spotted fever is the most common tick-borne disease, and is carried by at least 27 tick species, with Lyme disease and human granulocytic anaplasmosis ranked as the second and third most widespread tick-borne diseases, carried by 13 and 10 species, respectively. Such knowledge provides us with clues for the identification of tick-associated pathogens and suggests ideas for the control of tick-borne diseases in China. However, the numbers of tick-associated pathogens and tick-borne diseases in China are probably underestimated because of the complex distribution and great diversity of tick species in this country.

  14. Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs

    PubMed Central

    Geisbert, Joan B; Shedlock, Devon J; Xu, Ling; Lamoreaux, Laurie; Custers, Jerome H. H. V; Popernack, Paul M; Yang, Zhi-Yong; Pau, Maria G; Roederer, Mario; Koup, Richard A; Goudsmit, Jaap; Jahrling, Peter B; Nabel, Gary J

    2006-01-01

    Background Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine. Methods and Findings To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 1010 particles, two logs lower than that used previously. Conclusions Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 1010 rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate. PMID:16683867

  15. Enhancement or inhibition of PLCγ2 expression in rat hepatocytes by recombinant adenoviral vectors that contain full-length gene or siRNA.

    PubMed

    Chen, X G; Liu, Y M; Lv, Q X; Ma, J

    2017-01-01

    We investigated the effects of recombinant adenovirus vectors that overexpress or silence PLCγ2 on the expression of this gene during hepatocyte proliferation. Hepatocytes were isolated, identified by immunofluorescent cytochemical staining and infected by previously constructed Ad-PLCγ2 and Ad-PLCγ2 siRNA1, siRNA2 and siRNA3. Green fluorescent protein (GFP) expression was observed by fluorescence microscopy. Infection percentage was calculated by flow cytometry. mRNA and protein levels of PLCγ2 were detected by quantitative reverse transcription-PCR (qRT-PCR) and western blotting, respectively. The viability of the infected hepatocytes was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We found that nearly 97% of cells were positive for the hepatocyte marker, CK18. After infection of Ad-PLCγ2 and Ad-PLCγ2 siRNA, more than 99% of hepatocytes expressed GFP significantly, and mRNA and protein expression of PLCγ2 was up-regulated significantly in Ad-PLCγ2 infected hepatocytes, but down-regulated in Ad-PLCγ2 siRNA2 infected cells. The cell proliferation rate decreased in PLCγ2-overexpressing cells, while the rate increased in PLCγ2-silencing cells. We verified that recombinant Ad-PLCγ2 and Ad-PLCγ2 siRNA2 were constructed successfully. These two recombinant vectors promoted or decreased the expression of PLCγ2 in rat hepatocytes and affected the cell proliferation rate, which provides a useful tool for further investigation of the role of PLCγ2 in hepatocyte apoptosis.

  16. Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells.

    PubMed

    Li, L; Yang, L; Scudiero, D A; Miller, S A; Yu, Z-X; Stukenberg, P T; Shoemaker, R H; Kotin, R M

    2007-05-01

    Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.

  17. Nephron segment-specific gene expression using AAV vectors.

    PubMed

    Asico, Laureano D; Cuevas, Santiago; Ma, Xiaobo; Jose, Pedro A; Armando, Ines; Konkalmatt, Prasad R

    2018-02-26

    AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; Na + /glucose co-transporter (SGLT2) gene promoter for expression in the S1 and S2 segments of the proximal tubule; sodium, potassium, 2 chloride co-transporter (NKCC2) gene promoter for expression in the thick ascending limb of Henle's loop (TALH); E-cadherin (ECAD) gene promoter for expression in the collecting duct (CD); and cytomegalovirus (CMV) early promoter that provides expression in most of the mammalian cells, as control. We tested the specificity of the promoter constructs in vitro for cell type-specific expression in mouse kidney cells in primary culture, followed by retrograde infusion of the AAV vectors via the ureter in the mouse. Our data show that AAV9 vector, in combination with the segment-specific promoters administered by retrograde infusion via the ureter, provides renal nephron segment-specific gene expression. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Adenovirus E1B 19-Kilodalton Protein Modulates Innate Immunity through Apoptotic Mimicry

    PubMed Central

    Grigera, Fernando; Ucker, David S.; Cook, James L.

    2014-01-01

    ABSTRACT Cells that undergo apoptosis in response to chemical or physical stimuli repress inflammatory reactions, but cells that undergo nonapoptotic death in response to such stimuli lack this activity. Whether cells dying from viral infection exhibit a cell death-type modulatory effect on inflammatory reactions is unknown. We compared the effects on macrophage inflammatory responses of cells dying an apoptotic or a nonapoptotic death as a result of adenoviral infection. The results were exactly opposite to the predictions from the conventional paradigm. Cells dying by apoptosis induced by infection with an adenovirus type 5 (Ad5) E1B 19-kilodalton (E1B 19K) gene deletion mutant did not repress macrophage NF-κB activation or cytokine responses to proinflammatory stimuli, whereas cells dying a nonapoptotic death from infection with E1B 19K-competent, wild-type Ad5 repressed these macrophage inflammatory responses as well as cells undergoing classical apoptosis in response to chemical injury. The immunorepressive, E1B 19K-related cell death activity depended upon direct contact of the virally infected corpses with responder macrophages. Replacement of the viral E1B 19K gene with the mammalian Bcl-2 gene in cis restored the nonapoptotic, immunorepressive cell death activity of virally infected cells. These results define a novel function of the antiapoptotic, adenoviral E1B 19K protein that may limit local host innate immune inflammation during accumulation of virally infected cells at sites of infection and suggest that E1B 19K-deleted, replicating adenoviral vectors might induce greater inflammatory responses to virally infected cells than E1B 19K-positive vectors, because of the net effect of their loss-of-function mutation. IMPORTANCE We observed that cells dying a nonapoptotic cell death induced by adenovirus infection repressed macrophage proinflammatory responses while cells dying by apoptosis induced by infection with an E1B 19K deletion mutant virus did not

  19. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    PubMed

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

  20. Acceleration of convergence of vector sequences

    NASA Technical Reports Server (NTRS)

    Sidi, A.; Ford, W. F.; Smith, D. A.

    1983-01-01

    A general approach to the construction of convergence acceleration methods for vector sequence is proposed. Using this approach, one can generate some known methods, such as the minimal polynomial extrapolation, the reduced rank extrapolation, and the topological epsilon algorithm, and also some new ones. Some of the new methods are easier to implement than the known methods and are observed to have similar numerical properties. The convergence analysis of these new methods is carried out, and it is shown that they are especially suitable for accelerating the convergence of vector sequences that are obtained when one solves linear systems of equations iteratively. A stability analysis is also given, and numerical examples are provided. The convergence and stability properties of the topological epsilon algorithm are likewise given.

  1. Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease.

    PubMed

    Shen, Jin-Song; Meng, Xing-Li; Yokoo, Takashi; Sakurai, Ken; Watabe, Kazuhiko; Ohashi, Toya; Eto, Yoshikatsu

    2005-05-01

    Brain-directed prenatal gene therapy may benefit some lysosomal storage diseases that affect the central nervous system (CNS) before birth. Our previous study showed that intrauterine introduction of recombinant adenoviruses into cerebral ventricles results in efficient gene transfer to the CNS in the mouse. However, transgene expression decreased with time due to the non-integrative property of adenoviral vectors. In this study, in order to obtain permanent gene transduction, we investigated the feasibility of retrovirus-mediated in utero gene transduction. Concentrated retrovirus encoding the LacZ gene was injected into the cerebral ventricles of the embryos of normal and twitcher mice (a murine model of Krabbe disease) at embryonic day 12. The distribution and maintenance of the transgene expression in the recipient brain were analyzed histochemically, biochemically and by the quantitative polymerase chain reaction method pre- and postnatally. Efficient and highly persistent gene transduction to the brain was achieved both in normal and the twitcher mouse. Transduced neurons, astrocytes and oligodendrocytes were distributed throughout the brain. The transduced LacZ gene, its transcript and protein expression in the brain were maintained for 14 months without decrement. In addition, gene transduction to multiple tissues other than the brain was also detected at low levels. This study suggests that brain-directed in utero gene transfer using retrovirus vector may be beneficial to the treatment of lysosomal storage diseases with severe brain damage early in life, such as Krabbe disease. Copyright (c) 2005 John Wiley & Sons, Ltd.

  2. [The virological and epidemiological aspects of human adenoviral conjunctivitis in Tunisia].

    PubMed

    Fedaoui, N; Ben Ayed, N; Ben Yahia, A; Matri, L; Nacef, L; Triki, H

    2017-01-01

    Human adenoviruses (HAdV) are the main cause of viral conjunctivitis. In Tunisia and North Africa more generally, there is no regular nationwide surveillance program that monitors viruses causing conjunctivitis and keratoconjunctivitis. In this study, we report the results of HAdV screening in conjunctival samples collected for over 14 years in Tunisia. A total of 282 conjunctival samples received between 2000 and 2013 were investigated. Detection and identification of genotype were performed by PCR-sequencing at the hexon gene; 64.5% of samples (n=182) revealed positive by PCR detection without correlation noted between infection, age, sex, social class or clinical manifestations of viral conjunctivitis. HAdV-D8 was the largely predominant genotype in Tunisia, representing 81.3% of all isolates, and was detected continuously from 2000 to 2013. Minor co-circulating genotypes were also identified - HAdV-E4, HAdV-B3, B55 and HAdV-B7 - accounting for 10.7%, 4.9%, 1.9% and 0.9% of isolates, respectively. In conclusion, this work reports epidemiological data on adenoviral conjunctivitis from a region where such information is very scarce and contributes to a better knowledge of the worldwide distribution of causative genotypes. It also presents an approach for the identification of circulating HAdV in the country and demonstrates the importance of molecular tools for both detection and identification of genotypes, which allow rapid virological investigation, especially during epidemics. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-10-01

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

  5. Syngeneic AAV pseudo-vectors potentiates full vector transduction

    USDA-ARS?s Scientific Manuscript database

    An excessive amount of empty capsids are generated during regular AAV vector production process. These pseudo-vectors often remain in final vectors used for animal studies or clinical trials. The potential effects of these pseudo-vectors on AAV transduction have been a major concern. In the current ...

  6. Refined human artificial chromosome vectors for gene therapy and animal transgenesis

    PubMed Central

    Kazuki, Y; Hoshiya, H; Takiguchi, M; Abe, S; Iida, Y; Osaki, M; Katoh, M; Hiratsuka, M; Shirayoshi, Y; Hiramatsu, K; Ueno, E; Kajitani, N; Yoshino, T; Kazuki, K; Ishihara, C; Takehara, S; Tsuji, S; Ejima, F; Toyoda, A; Sakaki, Y; Larionov, V; Kouprina, N; Oshimura, M

    2011-01-01

    Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis. PMID:21085194

  7. Ad35 and Ad26 Vaccine Vectors Induce Potent and Cross-Reactive Antibody and T-Cell Responses to Multiple Filovirus Species

    PubMed Central

    Zahn, Roland; Gillisen, Gert; Roos, Anna; Koning, Marina; van der Helm, Esmeralda; Spek, Dirk; Weijtens, Mo; Grazia Pau, Maria; Radošević, Katarina; Weverling, Gerrit Jan; Custers, Jerome; Vellinga, Jort; Schuitemaker, Hanneke; Goudsmit, Jaap; Rodríguez, Ariane

    2012-01-01

    Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species (Ebola Zaire and Ebola Sudan/Gulu,), two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26–Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years. PMID:23236343

  8. Emerging Vector-Borne Diseases - Incidence through Vectors.

    PubMed

    Savić, Sara; Vidić, Branka; Grgić, Zivoslav; Potkonjak, Aleksandar; Spasojevic, Ljubica

    2014-01-01

    Vector-borne diseases use to be a major public health concern only in tropical and subtropical areas, but today they are an emerging threat for the continental and developed countries also. Nowadays, in intercontinental countries, there is a struggle with emerging diseases, which have found their way to appear through vectors. Vector-borne zoonotic diseases occur when vectors, animal hosts, climate conditions, pathogens, and susceptible human population exist at the same time, at the same place. Global climate change is predicted to lead to an increase in vector-borne infectious diseases and disease outbreaks. It could affect the range and population of pathogens, host and vectors, transmission season, etc. Reliable surveillance for diseases that are most likely to emerge is required. Canine vector-borne diseases represent a complex group of diseases including anaplasmosis, babesiosis, bartonellosis, borreliosis, dirofilariosis, ehrlichiosis, and leishmaniosis. Some of these diseases cause serious clinical symptoms in dogs and some of them have a zoonotic potential with an effect to public health. It is expected from veterinarians in coordination with medical doctors to play a fundamental role at primarily prevention and then treatment of vector-borne diseases in dogs. The One Health concept has to be integrated into the struggle against emerging diseases. During a 4-year period, from 2009 to 2013, a total number of 551 dog samples were analyzed for vector-borne diseases (borreliosis, babesiosis, ehrlichiosis, anaplasmosis, dirofilariosis, and leishmaniasis) in routine laboratory work. The analysis was done by serological tests - ELISA for borreliosis, dirofilariosis, and leishmaniasis, modified Knott test for dirofilariosis, and blood smear for babesiosis, ehrlichiosis, and anaplasmosis. This number of samples represented 75% of total number of samples that were sent for analysis for different diseases in dogs. Annually, on average more then half of the samples

  9. Expression of BCL-2 in liver grafts after adenoviral transfer improves survival following prolonged ischemia and reperfusion in rat liver transplantation.

    PubMed

    Kienle, K; Rentsch, M; Müller, T; Engelhard, N; Vogel, M; Jauch, K W; Beham, A

    2005-01-01

    Apoptosis represents a crucial mechanism of ischemia-reperfusion injury after liver transplantation. Bcl-2 may inhibit apoptosis. This study investigates the effect on ischemia/reperfusion injury and survival after rat liver transplantation of adenoviral bcl-2 transfer into donor livers. A nonreplicative adenovirus, expressing bcl-2 under control of a tetracyclin-inducible promoter (adv TetOn bcl-2) was used to treat male Lewis rats in combination with a second adenovirus transferring the TetOn repressor protein under control of a cytomegalovirus promoter (advCMVRep). Virus induction was achieved by addition of doxycyclin to the drinking water. Controls were pretreated with a control adenovirus (advCMV GFP) or with doxycycline. Liver transplantations were performed after 16-hour graft storage. Bcl-2 expression was evaluated by Western blot and immunohistology. Survival was monitored for 7 days, and tissue specimens were collected at 24 hours and 7 days post reperfusion. After pretreatment with advTetOn bcl-2/adv CMVRep, intrahepatic bcl-2 expression was evident at 24 hours and 7 days but was absent among controls. Bcl-2 expression was detected in hepatocytes and, to a high degree, in sinusoidal lining cells. TUNEL-positive sinusoidal lining cells were strikingly reduced after bcl-2 transfer (0.1 +/- 0.3 cells/hpf, mean +/- SD) compared to control virus (4.8 +/- 2.3) or doxycyclin-treated grafts (1.3 +/- 0.2); P < .05. After bcl-2 treatment, survival after transplantation was 100%, whereas it was 50% in both control groups (P = .035). The study shows the feasibility of transient, doxycyclin-controlled adenoviral gene transfer in a transplantation model. Bcl-2 expression increased survival after ischemia/reperfusion in rat liver transplantation, potentially through protection of sinusoidal lining cells.

  10. Adenovirus-mediated suppression of HMGI(Y) protein synthesis as potential therapy of human malignant neoplasias

    PubMed Central

    Scala, Stefania; Portella, Giuseppe; Fedele, Monica; Chiappetta, Gennaro; Fusco, Alfredo

    2000-01-01

    High mobility group I (HMGI) proteins are overexpressed in several human malignant tumors. We previously demonstrated that inhibition of HMGI synthesis prevents thyroid cell transformation. Here, we report that an adenovirus carrying the HMGI(Y) gene in an antisense orientation (Ad-Yas) induced programmed cell death of two human thyroid anaplastic carcinoma cell lines (ARO and FB-1), but not normal thyroid cells. The Ad-Yas virus led to death of lung, colon, and breast carcinoma cells. A control adenovirus carrying the lacZ gene did not inhibit the growth of either normal or neoplastic cells. Ad-Yas treatment of tumors induced in athymic mice by ARO cells caused a drastic reduction in tumor size. Therefore, suppression of HMGI(Y) protein synthesis by an HMGI(Y) antisense adenoviral vector may be a useful treatment strategy in a variety of human malignant neoplasias, in which HMGI(Y) gene overexpression is a general event. PMID:10759549

  11. Detection and phylogenetic analysis of a new adenoviral polymerase gene in reptiles in Korea.

    PubMed

    Bak, Eun-Jung; Jho, Yeonsook; Woo, Gye-Hyeong

    2018-06-01

    Over a period of 7 years (2004-2011), samples from 34 diseased reptiles provided by local governments, zoos, and pet shops were tested for viral infection. Animals were diagnosed based on clinical signs, including loss of appetite, diarrhea, rhinorrhea, and unexpected sudden death. Most of the exotic animals had gastrointestinal problems, such as mucosal redness and ulcers, while the native animals had no clinical symptoms. Viral sequences were found in seven animals. Retroviral genes were amplified from samples from five Burmese pythons (Python molurus bivittatus), an adenovirus was detected in a panther chameleon (Furcifer pardalis), and an adenovirus and a paramyxovirus were detected in a tropical girdled lizard (Cordylus tropidosternum). Phylogenetic analysis of retroviruses and paramyxoviruses showed the highest sequence identity to both a Python molurus endogenous retrovirus and a Python curtus endogenous retrovirus and to a lizard isolate, respectively. Partial sequencing of an adenoviral DNA polymerase gene from the lizard isolate suggested that the corresponding virus was a novel isolate different from the reference strain (accession no. AY576677.1). The virus was not isolated but was detected, using molecular genetic techniques, in a lizard raised in a pet shop. This animal was also coinfected with a paramyxovirus.

  12. VectorBase: a home for invertebrate vectors of human pathogens

    PubMed Central

    Lawson, Daniel; Arensburger, Peter; Atkinson, Peter; Besansky, Nora J.; Bruggner, Robert V.; Butler, Ryan; Campbell, Kathryn S.; Christophides, George K.; Christley, Scott; Dialynas, Emmanuel; Emmert, David; Hammond, Martin; Hill, Catherine A.; Kennedy, Ryan C.; Lobo, Neil F.; MacCallum, M. Robert; Madey, Greg; Megy, Karine; Redmond, Seth; Russo, Susan; Severson, David W.; Stinson, Eric O.; Topalis, Pantelis; Zdobnov, Evgeny M.; Birney, Ewan; Gelbart, William M.; Kafatos, Fotis C.; Louis, Christos; Collins, Frank H.

    2007-01-01

    VectorBase () is a web-accessible data repository for information about invertebrate vectors of human pathogens. VectorBase annotates and maintains vector genomes providing an integrated resource for the research community. Currently, VectorBase contains genome information for two organisms: Anopheles gambiae, a vector for the Plasmodium protozoan agent causing malaria, and Aedes aegypti, a vector for the flaviviral agents causing Yellow fever and Dengue fever. PMID:17145709

  13. Aptamer modification improves the adenoviral transduction of malignant glioma cells.

    PubMed

    Chen, Hao; Zheng, Xiaojing; Di, BingYan; Wang, Dongyang; Zhang, Yaling; Xia, Haibin; Mao, Qinwen

    2013-12-01

    Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  14. The Attenuation of Central Angiotensin II-dependent Pressor Response and Intra-neuronal Signaling by Intracarotid Injection of Nanoformulated Copper/Zinc Superoxide Dismutase

    PubMed Central

    Rosenbaugh, Erin G.; Roat, James; Gao, Lie; Yang, Rui-Fang; Manickam, Devika S.; Yin, Jing-Xiang; Schultz, Harold D.; Bronich, Tatiana K.; Batrakova, Elena V.; Kabanov, Alexander V.; Zucker, Irving H.; Zimmerman, Matthew C.

    2010-01-01

    Adenoviral-mediated overexpression of the intracellular superoxide (O2•−) scavenging enzyme copper/zinc superoxide dismutase (CuZnSOD) in the brain attenuates central angiotensin II (AngII)-induced cardiovascular responses. However, the therapeutic potential for adenoviral vectors is weakened by toxicity and the inability of adenoviral vectors to target the brain following peripheral administration. Therefore, we developed a non-viral delivery system in which CuZnSOD protein is electrostatically bound to a synthetic poly(ethyleneimine)-poly(ethyleneglycol) (PEI-PEG) polymer to form a polyion complex (CuZnSOD nanozyme). We hypothesized that PEI-PEG polymer increases transport of functional CuZnSOD to neurons, which inhibits AngII intra-neuronal signaling. The AngII-induced increase in O2•−, as measured by dihydroethidium fluorescence and electron paramagnetic resonance spectroscopy, was significantly inhibited in CuZnSOD nanozyme-treated neurons compared to free CuZnSOD- and non-treated neurons. CuZnSOD nanozyme also attenuated the AngII-induced inhibition of K+ current in neurons. Intracarotid injection of CuZnSOD nanozyme into rabbits significantly inhibited the pressor response of intracerebroventricular-delivered AngII; however, intracarotid injection of free CuZnSOD or PEI-PEG polymer alone failed to inhibit this response. Importantly, neither the PEI-PEG polymer alone nor the CuZnSOD nanozyme induced neuronal toxicity. These findings indicate that CuZnSOD nanozyme inhibits AngII intra-neuronal signaling in vitro and in vivo. PMID:20378166

  15. Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis.

    PubMed

    Santosuosso, Michael; McCormick, Sarah; Zhang, Xizhong; Zganiacz, Anna; Xing, Zhou

    2006-08-01

    Parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. We examined a heterologous prime-boost regimen utilizing BCG as a prime vaccine and our recently described adenoviral vector expressing Ag85A (AdAg85A) as a boost vaccine. Since we recently demonstrated that a single intranasal but not intramuscular immunization with AdAg85A was able to induce potent protection from pulmonary Mycobacterium tuberculosis challenge in a mouse model, we compared the protective effects of parenteral and mucosal booster immunizations following subcutaneous BCG priming. Protection by BCG prime immunization was not effectively boosted by subcutaneous BCG or intramuscular AdAg85A. In contrast, protection by BCG priming was remarkably boosted by intranasal AdAg85A. Such enhanced protection by intranasal AdAg85A was correlated to the numbers of gamma interferon-positive CD4 and CD8 T cells residing in the airway lumen of the lung. Our study demonstrates that intranasal administration of AdAg85A represents an effective way to boost immune protection by parenteral BCG vaccination.

  16. Insulin-like growth factor-I gene delivery to astrocytes reduces their inflammatory response to lipopolysaccharide

    PubMed Central

    2011-01-01

    Background Insulin-like growth factor-I (IGF-I) exerts neuroprotective actions in the central nervous system that are mediated at least in part by control of activation of astrocytes. In this study we have assessed the efficacy of exogenous IGF-I and IGF-I gene therapy in reducing the inflammatory response of astrocytes from cerebral cortex. Methods An adenoviral vector harboring the rat IGF-I gene and a control adenoviral vector harboring a hybrid gene encoding the herpes simplex virus type 1 thymidine kinase fused to Aequorea victoria enhanced green fluorescent protein were used in this study. Primary astrocytes from mice cerebral cortex were incubated for 24 h or 72 h with vehicle, IGF-I, the IGF-I adenoviral vector, or control vector; and exposed to bacterial lipopolysaccharide to induce an inflammatory response. IGF-I levels were measured by radioimmunoassay. Levels of interleukin 6, tumor necrosis factor-α, interleukin-1β and toll-like receptor 4 mRNA were assessed by quantitative real-time polymerase chain reaction. Levels of IGF-I receptor and IGF binding proteins 2 and 3 were assessed by western blotting. The subcellular distribution of nuclear factor κB (p65) was assessed by immunocytochemistry. Statistical significance was assessed by one way analysis of variance followed by the Bonferroni pot hoc test. Results IGF-I gene therapy increased IGF-I levels without affecting IGF-I receptors or IGF binding proteins. Exogenous IGF-I, and IGF-I gene therapy, decreased expression of toll-like receptor 4 and counteracted the lipopolysaccharide-induced inflammatory response of astrocytes. In addition, IGF-I gene therapy decreased lipopolysaccharide-induced translocation of nuclear factor κB (p65) to the cell nucleus. Conclusion These findings demonstrate efficacy of exogenous IGF-I and of IGF-I gene therapy in reducing the inflammatory response of astrocytes. IGF-I gene therapy may represent a new approach to reduce inflammatory reactions in glial cells. PMID

  17. Charged Particle Dynamics in the Magnetic Field of a Long Straight Current-Carrying Wire

    ERIC Educational Resources Information Center

    Prentice, A.; Fatuzzo, M.; Toepker, T.

    2015-01-01

    By describing the motion of a charged particle in the well-known nonuniform field of a current-carrying long straight wire, a variety of teaching/learning opportunities are described: 1) Brief review of a standard problem; 2) Vector analysis; 3) Dimensionless variables; 4) Coupled differential equations; 5) Numerical solutions.

  18. Current status of genome editing in vector mosquitoes: A review.

    PubMed

    Reegan, Appadurai Daniel; Ceasar, Stanislaus Antony; Paulraj, Michael Gabriel; Ignacimuthu, Savarimuthu; Al-Dhabi, Naif Abdullah

    2017-01-16

    Mosquitoes pose a major threat to human health as they spread many deadly diseases like malaria, dengue, chikungunya, filariasis, Japanese encephalitis and Zika. Identification and use of novel molecular tools are essential to combat the spread of vector borne diseases. Genome editing tools have been used for the precise alterations of the gene of interest for producing the desirable trait in mosquitoes. Deletion of functional genes or insertion of toxic genes in vector mosquitoes will produce either knock-out or knock-in mutants that will check the spread of vector-borne diseases. Presently, three types of genome editing tools viz., zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) are widely used for the editing of the genomes of diverse organisms. These tools are also applied in vector mosquitoes to control the spread of vector-borne diseases. A few studies have been carried out on genome editing to control the diseases spread by vector mosquitoes and more studies need to be performed with the utilization of more recently invented tools like CRISPR/Cas9 to combat the spread of deadly diseases by vector mosquitoes. The high specificity and flexibility of CRISPR/Cas9 system may offer possibilities for novel genome editing for the control of important diseases spread by vector mosquitoes. In this review, we present the current status of genome editing research on vector mosquitoes and also discuss the future applications of vector mosquito genome editing to control the spread of vectorborne diseases.

  19. Detection of neuroendocrine tumors using promoter-specific secreted Gaussia luciferase.

    PubMed

    Tseng, Alan Wei-Shun; Akerstrom, Victoria; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

    2016-01-01

    Accurate detection of neuroendocrine (NE) tumors is critically important for better prognosis and treatment outcomes in patients. To demonstrate the efficacy of using an adenoviral vector for the detection of NE tumors, we have constructed a pair of adenoviral vectors which, in combination, can conditionally replicate and release Gaussia luciferase into the circulation after infecting the NE tumors. The expression of these two vectors is regulated upstream by an INSM1-promoter (insulinoma-associated-1) that is specifically active in NE tumors and developing NE tissues, but silenced in normal adult tissues. In order to retain the tumor-specificity of the INSM1 promoter, we have modified the promoter using the core insulator sequence from the chicken β-globin HS4 insulator and the neuronal restrictive silencing element (NRSE). This modified INSM1-promoter can retain NE tumor specificity in an adenoviral construct while driving a mutated adenovirus E1A gene (∆24E1A), the Metridia, or Gaussia luciferase gene. The in vitro cell line and mouse xenograft human tumor studies revealed the NE specificity of the INSM1-promoter in NE lung cancer, neuroblastoma, medulloblastoma, retinoblastoma, and insulinoma. When we combined the INSM1-promoter driven Gaussia luciferase with ∆24E1A, the co-infected NE tumor secreted higher levels of Gaussia luciferase as compared to the INSM1p-Gaussia virus alone. In a mouse subcutaneous xenograft tumor model, the combination viruses secreted detectable level of Gaussia luciferase after infecting an INSM1-positive NE lung tumor for ≥12 days. Therefore, the INSM1-promoter specific conditional replicating adenovirus represents a sensitive diagnostic tool to aid clinicians in the detection of NE tumors.

  20. Enhanced Peptide Radiotherapy of Prostate Cancer Using Targeted Adenoviral Vectors

    DTIC Science & Technology

    2004-06-01

    regard to binding of 64Cu - octreotide. In vitro experiments were performed with DU-145 and PC-3 human prostate cancer cells. Expression levels of SSTR2...were determined using a 64Cu -octreotide saturation binding assay on cell membrane preparations. In vivo experiments were conducted in scid mice bearing...subcutaneous DU-l45 or PC-3 cells. AdSSTR2 was injected intratumorally followed 48 h later by an i.v. injection of 64Cu -octreotide. The mice were

  1. Treatment of adenoviral keratoconjunctivitis with a combination of povidone-iodine 1.0% and dexamethasone 0.1% drops: a clinical prospective controlled randomized study.

    PubMed

    Kovalyuk, Natalya; Kaiserman, Igor; Mimouni, Michael; Cohen, Ornit; Levartovsky, Shmuel; Sherbany, Hilda; Mandelboim, Michal

    2017-12-01

    To determine the efficacy of combination povidone-iodine (PVP-I) 1.0% eyedrops and dexamethasone 0.1% eyedrops in the treatment of adenoviral keratoconjunctivitis. In a prospective, randomized, controlled, double-blinded clinical trial patients with recent adenoviral keratoconjunctivitis (diagnosed clinically and confirmed by PCR), we randomly divided into three treatment groups: study group - received PVP-I 1.0% and dexamethasone 0.1%, control 1 group - received dexamethasone 0.1% and control 2 group - received lubricating eyedrops (hypromellose 0.3%). The treatment was administered four times a day in each group. All patients were examined and filled a questionnaire before treatment and on the 3rd, 5th and 7th days of treatment. We included in the study 78 eyes (26 in each group). Adenovirus type 8 was the most common pathogen (83% of cases). The fastest improvement in patients red eyes, discharge, superficial punctate keratitis and pseudomembranes was observed in the study group (p < 0.001). Those patients reached a near complete recovery in 5-7 days, which was also confirmed by reduction in Adenovirus titres by PCR. The slowest improvement was in the control 2 group. Subepithelial infiltrates (SEI) were observed in 44% of the control 1 group, 20% of the control 2 group and in 0% of the study group. The rate of reduction in Adenovirus titres was the slowest in the control 1 group. The combination of PVP-I 1.0% and dexamethasone 0.1% four times a day can reduce symptoms and expedite recovery in epidemic keratoconjunctivitis patients. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  2. CD11c identifies a subset of murine liver natural killer cells that responds to adenoviral hepatitis

    PubMed Central

    Burt, Bryan M.; Plitas, George; Stableford, Jennifer A.; Nguyen, Hoang M.; Bamboat, Zubin M.; Pillarisetty, Venu G.; DeMatteo, Ronald P.

    2008-01-01

    The liver contains a unique repertoire of immune cells and a particular abundance of NK cells. We have found that CD11c defines a distinct subset of NK cells (NK1.1+CD3−) in the murine liver whose function was currently unknown. In naïve animals, CD11c+ liver NK cells displayed an activated phenotype and possessed enhanced effector functions when compared with CD11c− liver NK cells. During the innate response to adenovirus infection, CD11c+ NK cells were the more common IFN-γ-producing NK cells in the liver, demonstrated enhanced lytic capability, and gained a modest degree of APC function. The mechanism of IFN-γ production in vivo depended on TLR9 ligation as well as IL-12 and -18. Taken together, our findings demonstrate that CD11c+ NK cells are a unique subset of NK cells in the murine liver that contribute to the defense against adenoviral hepatitis. PMID:18664530

  3. Safety and immunogenicity of adenovirus-vectored near-consensus HIV type 1 clade B gag vaccines in healthy adults.

    PubMed

    Harro, Clayton D; Robertson, Michael N; Lally, Michelle A; O'Neill, Lori D; Edupuganti, Srilatha; Goepfert, Paul A; Mulligan, Mark J; Priddy, Frances H; Dubey, Sheri A; Kierstead, Lisa S; Sun, Xiao; Casimiro, Danilo R; DiNubile, Mark J; Shiver, John W; Leavitt, Randi Y; Mehrotra, Devan V

    2009-01-01

    Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-gamma ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus > or =200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus > or = 200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen

  4. Safety and Immunogenicity of Adenovirus-Vectored Near-Consensus HIV Type 1 Clade B gag Vaccines in Healthy Adults

    PubMed Central

    Robertson, Michael N.; Lally, Michelle A.; O'Neill, Lori D.; Edupuganti, Srilatha; Goepfert, Paul A.; Mulligan, Mark J.; Priddy, Frances H.; Dubey, Sheri A.; Kierstead, Lisa S.; Sun, Xiao; Casimiro, Danilo R.; DiNubile, Mark J.; Shiver, John W.; Leavitt, Randi Y.; Mehrotra, Devan V.

    2009-01-01

    Abstract Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-γ ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus ≥200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus ≥200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen the

  5. VectorBase: a data resource for invertebrate vector genomics

    PubMed Central

    Lawson, Daniel; Arensburger, Peter; Atkinson, Peter; Besansky, Nora J.; Bruggner, Robert V.; Butler, Ryan; Campbell, Kathryn S.; Christophides, George K.; Christley, Scott; Dialynas, Emmanuel; Hammond, Martin; Hill, Catherine A.; Konopinski, Nathan; Lobo, Neil F.; MacCallum, Robert M.; Madey, Greg; Megy, Karine; Meyer, Jason; Redmond, Seth; Severson, David W.; Stinson, Eric O.; Topalis, Pantelis; Birney, Ewan; Gelbart, William M.; Kafatos, Fotis C.; Louis, Christos; Collins, Frank H.

    2009-01-01

    VectorBase (http://www.vectorbase.org) is an NIAID-funded Bioinformatic Resource Center focused on invertebrate vectors of human pathogens. VectorBase annotates and curates vector genomes providing a web accessible integrated resource for the research community. Currently, VectorBase contains genome information for three mosquito species: Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus, a body louse Pediculus humanus and a tick species Ixodes scapularis. Since our last report VectorBase has initiated a community annotation system, a microarray and gene expression repository and controlled vocabularies for anatomy and insecticide resistance. We have continued to develop both the software infrastructure and tools for interrogating the stored data. PMID:19028744

  6. Detection of osteoclastic cell-cell fusion through retroviral vector packaging.

    PubMed

    Kondo, Takako; Ikeda, Kyoji; Matsuo, Koichi

    2004-11-01

    Cell-cell fusion generates multinucleated cells such as osteoclasts in bone, myotubes in muscle, and trophoblasts in placenta. Molecular details governing these fusion processes are still largely unknown. As a step toward identification of fusogenic genes, we tested the concept that retroviral vectors can be packaged as a result of cell-cell fusion. First, we introduced replication-deficient retroviral vectors expressing mCAT-1, which mediates fusogenic interaction with the retroviral envelope protein Env, into Chinese hamster ovary (CHO) cells to generate vector cells. Plasmids expressing virion proteins Gag, Pol, and Env were introduced into a separate culture of CHO cells to generate packaging cells. Co-culturing vector and packaging cells resulted in production of infectious retroviruses carrying the mCAT-1 gene as a consequence of cell-cell fusion. Second, we introduced a retroviral vector into primary osteoclast precursors and co-cultured them with established osteoclast precursor RAW264.7 cells, which turned out to harbor packaging activity. Packaged retroviral vector was detected in culture supernatants only where the osteoclast differentiation factor receptor activator for NF-kappaB ligand (RANKL) induced fusion between these two cell types. These data suggest that retrovirus production can occur as a result of cell-cell fusion. This provides a novel approach for isolating and characterizing fusogenic genes using retroviral expression vectors.

  7. Emerging Vector-Borne Diseases – Incidence through Vectors

    PubMed Central

    Savić, Sara; Vidić, Branka; Grgić, Zivoslav; Potkonjak, Aleksandar; Spasojevic, Ljubica

    2014-01-01

    Vector-borne diseases use to be a major public health concern only in tropical and subtropical areas, but today they are an emerging threat for the continental and developed countries also. Nowadays, in intercontinental countries, there is a struggle with emerging diseases, which have found their way to appear through vectors. Vector-borne zoonotic diseases occur when vectors, animal hosts, climate conditions, pathogens, and susceptible human population exist at the same time, at the same place. Global climate change is predicted to lead to an increase in vector-borne infectious diseases and disease outbreaks. It could affect the range and population of pathogens, host and vectors, transmission season, etc. Reliable surveillance for diseases that are most likely to emerge is required. Canine vector-borne diseases represent a complex group of diseases including anaplasmosis, babesiosis, bartonellosis, borreliosis, dirofilariosis, ehrlichiosis, and leishmaniosis. Some of these diseases cause serious clinical symptoms in dogs and some of them have a zoonotic potential with an effect to public health. It is expected from veterinarians in coordination with medical doctors to play a fundamental role at primarily prevention and then treatment of vector-borne diseases in dogs. The One Health concept has to be integrated into the struggle against emerging diseases. During a 4-year period, from 2009 to 2013, a total number of 551 dog samples were analyzed for vector-borne diseases (borreliosis, babesiosis, ehrlichiosis, anaplasmosis, dirofilariosis, and leishmaniasis) in routine laboratory work. The analysis was done by serological tests – ELISA for borreliosis, dirofilariosis, and leishmaniasis, modified Knott test for dirofilariosis, and blood smear for babesiosis, ehrlichiosis, and anaplasmosis. This number of samples represented 75% of total number of samples that were sent for analysis for different diseases in dogs. Annually, on average more then half of the samples

  8. Fluidic Vectoring of a Planar Incompressible Jet Flow

    NASA Astrophysics Data System (ADS)

    Mendez, Miguel Alfonso; Scelzo, Maria Teresa; Enache, Adriana; Buchlin, Jean-Marie

    2018-06-01

    This paper presents an experimental, a numerical and a theoretical analysis of the performances of a fluidic vectoring device for controlling the direction of a turbulent, bi-dimensional and low Mach number (incompressible) jet flow. The investigated design is the co-flow secondary injection with Coanda surface, which allows for vectoring angles up to 25° with no need of moving mechanical parts. A simple empirical model of the vectoring process is presented and validated via experimental and numerical data. The experiments consist of flow visualization and image processing for the automatic detection of the jet centerline; the numerical simulations are carried out solving the Unsteady Reynolds Average Navier- Stokes (URANS) closed with the k - ω SST turbulence model, using the PisoFoam solver from OpenFOAM. The experimental validation on three different geometrical configurations has shown that the model is capable of providing a fast and reliable evaluation of the device performance as a function of the operating conditions.

  9. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse.

    PubMed

    Guan, Yuting; Ma, Yanlin; Li, Qi; Sun, Zhenliang; Ma, Lie; Wu, Lijuan; Wang, Liren; Zeng, Li; Shao, Yanjiao; Chen, Yuting; Ma, Ning; Lu, Wenqing; Hu, Kewen; Han, Honghui; Yu, Yanhong; Huang, Yuanhua; Liu, Mingyao; Li, Dali

    2016-05-01

    The X-linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas-mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  10. Transcriptional Enhancers Induce Insertional Gene Deregulation Independently From the Vector Type and Design

    PubMed Central

    Maruggi, Giulietta; Porcellini, Simona; Facchini, Giulia; Perna, Serena K; Cattoglio, Claudia; Sartori, Daniela; Ambrosi, Alessandro; Schambach, Axel; Baum, Christopher; Bonini, Chiara; Bovolenta, Chiara; Mavilio, Fulvio; Recchia, Alessandra

    2009-01-01

    The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) γ-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase—PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a γ-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design. PMID:19293778

  11. Development of an Expression Vector to Overexpress or Downregulate Genes in Curvularia protuberata.

    PubMed

    Liu, Chengke; Cleckler, Blake; Morsy, Mustafa

    2018-05-05

    Curvularia protuberata , an endophytic fungus in the Ascomycota, provides plants with thermotolerance only when it carries a mycovirus known as Curvularia thermotolerance virus (CThTV), and forms a three-way symbiotic relationship among these organisms. Under heat stress, several genes are expressed differently between virus-free C. protuberata (VF) and C. protuberata carrying CThTV (AN). We developed an expression vector, pM2Z-fun, carrying a zeocin resistance gene driven by the ToxA promoter, to study gene functions in C. protuberata to better understand this three-way symbiosis. Using this new 3.7-kb vector, five genes that are differentially expressed in C. protuberata —including genes involved in the trehalose, melanin, and catalase biosynthesis pathways—were successfully overexpressed or downregulated in VF or AN C. protuberata strains, respectively. The VF overexpression lines showed higher metabolite and enzyme activity than in the control VF strain. Furthermore, downregulation of expression of the same genes in the AN strain resulted in lower metabolite and enzyme activity than in the control AN strain. The newly generated expression vector, pM2Z-fun, has been successfully used to express target genes in C. protuberata and will be useful in further functional expression studies in other Ascomycota fungi.

  12. Schools as Potential Risk Sites for Vector-Borne Disease Transmission: Mosquito Vectors in Rural Schools in Two Municipalities in Colombia.

    PubMed

    Olano, Víctor Alberto; Matiz, María Inés; Lenhart, Audrey; Cabezas, Laura; Vargas, Sandra Lucía; Jaramillo, Juan Felipe; Sarmiento, Diana; Alexander, Neal; Stenström, Thor Axel; Overgaard, Hans J

    2015-09-01

    Dengue and other vector-borne diseases are of great public health importance in Colombia. Vector surveillance and control activities are often focused at the household level. Little is known about the importance of nonhousehold sites, including schools, in maintaining vector-borne disease transmission. The objectives of this paper were to determine the mosquito species composition in rural schools in 2 municipalities in Colombia and to assess the potential risk of vector-borne disease transmission in school settings. Entomological surveys were carried out in rural schools during the dry and rainy seasons of 2011. A total of 12 mosquito species were found: Aedes aegypti, Anopheles pseudopunctipennis, Culex coronator, Cx. quinquefasciatus, and Limatus durhamii in both immature and adult forms; Ae. fluviatilis, Cx. nigripalpus, Cx. corniger, and Psorophora ferox in immature forms only; and Ae. angustivittatus, Haemagogus equinus, and Trichoprosopon lampropus in adult forms only. The most common mosquito species was Cx. quinquefasciatus. Classrooms contained the greatest abundance of adult female Ae. aegypti and Cx. quinquefasciatus. The most common Ae. aegypti breeding sites were containers classified as "others" (e.g., cans), followed by containers used for water storage. A high level of Ae. aegypti infestation was found during the wet season. Our results suggest that rural schools are potentially important foci for the transmission of dengue and other mosquito-borne diseases. We propose that public health programs should be implemented in rural schools to prevent vector-borne diseases.

  13. Generation of cylindrically polarized vector vortex beams with digital micromirror device

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gong, Lei; Liu, Weiwei; Wang, Meng

    We propose a novel technique to directly transform a linearly polarized Gaussian beam into vector-vortex beams with various spatial patterns. Full high-quality control of amplitude and phase is implemented via a Digital Micro-mirror Device (DMD) binary holography for generating Laguerre-Gaussian, Bessel-Gaussian, and helical Mathieu–Gaussian modes, while a radial polarization converter (S-waveplate) is employed to effectively convert the optical vortices into cylindrically polarized vortex beams. Additionally, the generated vector-vortex beams maintain their polarization symmetry after arbitrary polarization manipulation. Due to the high frame rates of DMD, rapid switching among a series of vector modes carrying different orbital angular momenta paves themore » way for optical microscopy, trapping, and communication.« less

  14. Weather, host and vector — their interplay in the spread of insect-borne animal virus diseases

    PubMed Central

    Sellers, R. F.

    1980-01-01

    The spread of insect-borne animal virus diseases is influenced by a number of factors. Hosts migrate, move or are conveyed over long distances: vectors are carried on the wind for varying distances in search of hosts and breeding sites; weather and climate affect hosts and vectors through temperature, moisture and wind. As parasites of host and vector, viruses are carried by animals, birds and insects, and their spread can be correlated with the migration of hosts and the carriage of vectors on winds associated with the movements of the Intertropical Convergence Zone (ITCZ) and warm winds to the north and south of the limits of the ITCZ. The virus is often transmitted from a local cycle to a migratory cycle and back again. Examples of insect-borne virus diseases and their spread are analysed. Japanese, Murray Valley, Western equine, Eastern equine and St Louis encephalitis represent viruses transmitted by mosquito—bird or pig cycles. The areas experiencing infection with these viruses can be divided into a number of zones: A, B, C, D, E and F. In zone A there is a continuous cycle of virus in host and vector throughout the year; in zone B, there is an upsurge in the cycle during the wet season, but the cycle continues during the dry season; there is movement of infected vectors between and within zones A and B on the ITCZ and the virus is introduced to zone C by infected vectors on warm winds; persistence may occur in zone C if conditions are right. In zone D, virus is introduced each year by infected vectors on warm winds and the arrival of the virus coincides with the presence of susceptible nestling birds and susceptible piglets. The disappearance of virus occurs at the time when migrating mosquitoes and birds are returning to warmer climates. The virus is introduced to zone E only on occasions every 5-10 years when conditions are suitable. Infected hosts introduced to zone F do not lead to circulation of virus, since the climate is unsuitable for vectors

  15. Are Bred Vectors The Same As Lyapunov Vectors?

    NASA Astrophysics Data System (ADS)

    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

  16. Determination of the axial-vector weak coupling constant with ultracold neutrons.

    PubMed

    Liu, J; Mendenhall, M P; Holley, A T; Back, H O; Bowles, T J; Broussard, L J; Carr, R; Clayton, S; Currie, S; Filippone, B W; García, A; Geltenbort, P; Hickerson, K P; Hoagland, J; Hogan, G E; Hona, B; Ito, T M; Liu, C-Y; Makela, M; Mammei, R R; Martin, J W; Melconian, D; Morris, C L; Pattie, R W; Pérez Galván, A; Pitt, M L; Plaster, B; Ramsey, J C; Rios, R; Russell, R; Saunders, A; Seestrom, S J; Sondheim, W E; Tatar, E; Vogelaar, R B; VornDick, B; Wrede, C; Yan, H; Young, A R

    2010-10-29

    A precise measurement of the neutron decay β asymmetry A₀ has been carried out using polarized ultracold neutrons from the pulsed spallation ultracold neutron source at the Los Alamos Neutron Science Center. Combining data obtained in 2008 and 2009, we report A₀ = -0.119 66±0.000 89{-0.001 40}{+0.001 23}, from which we determine the ratio of the axial-vector to vector weak coupling of the nucleon g{A}/g{V}=-1.275 90{-0.004 45}{+0.004 09}.

  17. Design and construction of targeted AAVP vectors for mammalian cell transduction.

    PubMed

    Hajitou, Amin; Rangel, Roberto; Trepel, Martin; Soghomonyan, Suren; Gelovani, Juri G; Alauddin, Mian M; Pasqualini, Renata; Arap, Wadih

    2007-01-01

    Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targeting to a specific receptor. We have recently reported a new generation of hybrid prokaryotic-eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and construction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells in vitro and to target tissues in vivo after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammalian transgene cassette is inserted into an intergenomic region. High-titer suspensions (approximately 10(10)-10(11) transducing units per microl) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.

  18. Slugs: potential novel vectors of Escherichia coli O157.

    PubMed

    Sproston, Emma L; Macrae, M; Ogden, Iain D; Wilson, Michael J; Strachan, Norval J C

    2006-01-01

    Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157.

  19. Virus Database and Online Inquiry System Based on Natural Vectors.

    PubMed

    Dong, Rui; Zheng, Hui; Tian, Kun; Yau, Shek-Chung; Mao, Weiguang; Yu, Wenping; Yin, Changchuan; Yu, Chenglong; He, Rong Lucy; Yang, Jie; Yau, Stephen St

    2017-01-01

    We construct a virus database called VirusDB (http://yaulab.math.tsinghua.edu.cn/VirusDB/) and an online inquiry system to serve people who are interested in viral classification and prediction. The database stores all viral genomes, their corresponding natural vectors, and the classification information of the single/multiple-segmented viral reference sequences downloaded from National Center for Biotechnology Information. The online inquiry system serves the purpose of computing natural vectors and their distances based on submitted genomes, providing an online interface for accessing and using the database for viral classification and prediction, and back-end processes for automatic and manual updating of database content to synchronize with GenBank. Submitted genomes data in FASTA format will be carried out and the prediction results with 5 closest neighbors and their classifications will be returned by email. Considering the one-to-one correspondence between sequence and natural vector, time efficiency, and high accuracy, natural vector is a significant advance compared with alignment methods, which makes VirusDB a useful database in further research.

  20. Polar decomposition for attitude determination from vector observations

    NASA Technical Reports Server (NTRS)

    Bar-Itzhack, Itzhack Y.

    1993-01-01

    This work treats the problem of weighted least squares fitting of a 3D Euclidean-coordinate transformation matrix to a set of unit vectors measured in the reference and transformed coordinates. A closed-form analytic solution to the problem is re-derived. The fact that the solution is the closest orthogonal matrix to some matrix defined on the measured vectors and their weights is clearly demonstrated. Several known algorithms for computing the analytic closed form solution are considered. An algorithm is discussed which is based on the polar decomposition of matrices into the closest unitary matrix to the decomposed matrix and a Hermitian matrix. A somewhat longer improved algorithm is suggested too. A comparison of several algorithms is carried out using simulated data as well as real data from the Upper Atmosphere Research Satellite. The comparison is based on accuracy and time consumption. It is concluded that the algorithms based on polar decomposition yield a simple although somewhat less accurate solution. The precision of the latter algorithms increase with the number of the measured vectors and with the accuracy of their measurement.

  1. A new parallel-vector finite element analysis software on distributed-memory computers

    NASA Technical Reports Server (NTRS)

    Qin, Jiangning; Nguyen, Duc T.

    1993-01-01

    A new parallel-vector finite element analysis software package MPFEA (Massively Parallel-vector Finite Element Analysis) is developed for large-scale structural analysis on massively parallel computers with distributed-memory. MPFEA is designed for parallel generation and assembly of the global finite element stiffness matrices as well as parallel solution of the simultaneous linear equations, since these are often the major time-consuming parts of a finite element analysis. Block-skyline storage scheme along with vector-unrolling techniques are used to enhance the vector performance. Communications among processors are carried out concurrently with arithmetic operations to reduce the total execution time. Numerical results on the Intel iPSC/860 computers (such as the Intel Gamma with 128 processors and the Intel Touchstone Delta with 512 processors) are presented, including an aircraft structure and some very large truss structures, to demonstrate the efficiency and accuracy of MPFEA.

  2. Vector Addition: Effect of the Context and Position of the Vectors

    NASA Astrophysics Data System (ADS)

    Barniol, Pablo; Zavala, Genaro

    2010-10-01

    In this article we investigate the effect of: 1) the context, and 2) the position of the vectors, on 2D vector addition tasks. We administered a test to 512 students completing introductory physics courses at a private Mexican university. In the first part, we analyze students' responses in three isomorphic problems: displacements, forces, and no physical context. Students were asked to draw two vectors and the vector sum. We analyzed students' procedures detecting the difficulties when drawing the vector addition and proved that the context matters, not only compared to the context-free case but also between the contexts. In the second part, we analyze students' responses with three different arrangements of the sum of two vectors: tail-to-tail, head-to-tail and separated vectors. We compared the frequencies of the errors in the three different positions to deduce students' conceptions in the addition of vectors.

  3. Decoding the Ubiquitin-Mediated Pathway of Arthropod Disease Vectors

    PubMed Central

    Choy, Anthony; Severo, Maiara S.; Sun, Ruobai; Girke, Thomas; Gillespie, Joseph J.; Pedra, Joao H. F.

    2013-01-01

    Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector. PMID:24205097

  4. Covariantized vector Galileons

    NASA Astrophysics Data System (ADS)

    Hull, Matthew; Koyama, Kazuya; Tasinato, Gianmassimo

    2016-03-01

    Vector Galileons are ghost-free systems containing higher derivative interactions of vector fields. They break the vector gauge symmetry, and the dynamics of the longitudinal vector polarizations acquire a Galileon symmetry in an appropriate decoupling limit in Minkowski space. Using an Arnowitt-Deser-Misner approach, we carefully reconsider the coupling with gravity of vector Galileons, with the aim of studying the necessary conditions to avoid the propagation of ghosts. We develop arguments that put on a more solid footing the results previously obtained in the literature. Moreover, working in analogy with the scalar counterpart, we find indications for the existence of a "beyond Horndeski" theory involving vector degrees of freedom that avoids the propagation of ghosts thanks to secondary constraints. In addition, we analyze a Higgs mechanism for generating vector Galileons through spontaneous symmetry breaking, and we present its consistent covariantization.

  5. Generation of vector beams using a double-wedge depolarizer: Non-quantum entanglement

    NASA Astrophysics Data System (ADS)

    Samlan, C. T.; Viswanathan, Nirmal K.

    2016-07-01

    Propagation of horizontally polarized Gaussian beam through a double-wedge depolarizer generates vector beams with spatially varying state of polarization. Jones calculus is used to show that such beams are maximally nonseparable on the basis of even (Gaussian)-odd (Hermite-Gaussian) mode parity and horizontal-vertical polarization state. The maximum nonseparability in the two degrees of freedom of the vector beam at the double wedge depolarizer output is verified experimentally using a modified Sagnac interferometer and linear analyser projected interferograms to measure the concurrence 0.94±0.002 and violation of Clauser-Horne-Shimony-Holt form of Bell-like inequality 2.704±0.024. The investigation is carried out in the context of the use of vector beams for metrological applications.

  6. LY294002 enhances expression of proteins encoded by recombinant replication-defective adenoviruses via mTOR- and non-mTOR-dependent mechanisms.

    PubMed

    Shepelev, Mikhail V; Korobko, Elena V; Vinogradova, Tatiana V; Kopantsev, Eugene P; Korobko, Igor V

    2013-03-04

    Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery.

  7. Water-soluble fullerene (C60) derivatives as nonviral gene-delivery vectors.

    PubMed

    Sitharaman, Balaji; Zakharian, Tatiana Y; Saraf, Anita; Misra, Preeti; Ashcroft, Jared; Pan, Su; Pham, Quynh P; Mikos, Antonios G; Wilson, Lon J; Engler, David A

    2008-01-01

    A new class of water-soluble C60 transfecting agents has been prepared using Hirsch-Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both

  8. Adenovirus‑mediated overexpression of cystic fibrosis transmembrane conductance regulator enhances invasiveness and motility of serous ovarian cancer cells.

    PubMed

    Xu, Jiao; Lin, Liangbo; Yong, Min; Dong, Xiaojing; Yu, Tinghe; Hu, Lina

    2016-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the adenosine triphosphate‑binding cassette transporter family, members of which are involved in several types of cancer. Previous studies by our group reported that CFTR was highly expressed in serous ovarian cancer (SOC) tissues, and that knockdown of CFTR suppressed the proliferation of ovarian cancer in vitro and in vivo. Thus, the aim of the present study was to construct a recombinant adenoviral vector for the expression of the human CFTR gene in order to study the role of CFTR overexpression in the malignant invasion and migration of SOC cells in vitro. The present study then focused on the mechanisms of the role of CFTR in the migratory and invasive malignant properties of SOC cells. The CFTR gene was inserted into an adenoviral vector by using the AdEasy system in order to obtain the Ad‑CFTR overexpression vector, which was used to transfect the A2780 SOC cell line. Reverse-transcription polymerase chain reaction, western blot analysis and immunofluorescence were performed to detect the expression and localization of CFTR. Cell invasion and motility of the transfected cells compared with those of control cells were observed using Transwell and wound healing assays. A ~4,700 bp fragment of the CFTR gene was confirmed to be correctly cloned in the adenoviral vector and amplification of Ad‑CFTR was observed in HEK293 cells during package. After 48 h of transfection with Ad‑CFTR, ~90% of A2780 cells were red fluorescence protein‑positive. Immunofluorescence showed that following transfection, CFTR expression was increased and CFTR was located in the cell membrane and cytoplasm. CFTR overexpression was shown to enhance the invasion and motility of A2780 cells in vitro. Furthermore, the effects of CFTR overexpression on the activation c‑Src signaling were observed by western blot analysis. CFTR overexpressing cells showed the lowest activity of phospho‑Src (Tyr530

  9. Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages.

    PubMed

    Maler, Mareike D; Nielsen, Peter J; Stichling, Nicole; Cohen, Idan; Ruzsics, Zsolt; Wood, Connor; Engelhard, Peggy; Suomalainen, Maarit; Gyory, Ildiko; Huber, Michael; Müller-Quernheim, Joachim; Schamel, Wolfgang W A; Gordon, Siamon; Jakob, Thilo; Martin, Stefan F; Jahnen-Dechent, Willi; Greber, Urs F; Freudenberg, Marina A; Fejer, György

    2017-08-01

    The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors. IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages

  10. Mapping of Malaria Vectors at District Level in India: Changing Scenario and Identified Gaps.

    PubMed

    Singh, Poonam; Lingala, Mercy Aparna L; Sarkar, Soma; Dhiman, Ramesh C

    2017-02-01

    Malaria is one of the six major vector-borne diseases in India, the endemicity of which changes with changes in ecological, climatic, and sociodevelopmental conditions. The anopheline vectors are greatly affected by ecological conditions such as deforestation, urbanization, climate and lifestyle. Despite the advent of tools such as Geographic Information System (GIS), the updated information on the distribution of anopheline vectors of malaria is not available. In India, the plan for vector control is organized at subcentral level but information about vectors is unavailable even at the district level. Therefore, a systematic presentation of vector distribution has been made to provide maps in respect of major vector species. A search of the literature for major vector species, that is, Anopheles culicifacies, Anopheles fluviatilis, Anopheles stephensi, Anopheles minimus, and Anopheles dirus sensu lato, since 1927 till 2015 was carried out. Data have been presented as present, absent, and no information about vector species during pre-eradication (1927-1958), posteradication (1959-1999), and current scenario (2000-2015). Vectors' distribution and malaria endemicity were mapped using Arc GIS. Of 630 districts of India, major vectors An. culicifacies, An. fluviatilis, and An. stephensi were present in 420, 241, and 243 districts, respectively. In 183 districts, there is no information on any major malaria vector species although 27 of them from the states of Arunachal Pradesh, Jharkhand, Manipur, and Mizoram are highly endemic for malaria, having incidences of 2-40 cases/1000/year. The identified gaps in vector distribution, particularly in malaria endemic areas, necessitate further surveys so as to generate the missing information.

  11. Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.

    PubMed

    Rivera-Meza, Mario; Quintanilla, María Elena; Tampier, Lutske; Mura, Casilda V; Sapag, Amalia; Israel, Yedy

    2010-01-01

    Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The V(max) of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.

  12. The Absolute Vector Magnetometers on Board Swarm, Lessons Learned From Two Years in Space.

    NASA Astrophysics Data System (ADS)

    Hulot, G.; Leger, J. M.; Vigneron, P.; Brocco, L.; Olsen, N.; Jager, T.; Bertrand, F.; Fratter, I.; Sirol, O.; Lalanne, X.

    2015-12-01

    ESA's Swarm satellites carry 4He absolute magnetometers (ASM), designed by CEA-Léti and developed in partnership with CNES. These instruments are the first-ever space-born magnetometers to use a common sensor to simultaneously deliver 1Hz independent absolute scalar and vector readings of the magnetic field. They have provided the very high accuracy scalar field data nominally required by the mission (for both science and calibration purposes, since each satellite also carries a low noise high frequency fluxgate magnetometer designed by DTU), but also very useful experimental absolute vector data. In this presentation, we will report on the status of the instruments, as well as on the various tests and investigations carried out using these experimental data since launch in November 2013. In particular, we will illustrate the advantages of flying ASM instruments on space-born magnetic missions for nominal data quality checks, geomagnetic field modeling and science objectives.

  13. An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity.

    PubMed

    Zhang, Wenli; Fu, Jun; Liu, Jing; Wang, Hailong; Schiwon, Maren; Janz, Sebastian; Schaffarczyk, Lukas; von der Goltz, Lukas; Ehrke-Schulz, Eric; Dörner, Johannes; Solanki, Manish; Boehme, Philip; Bergmann, Thorsten; Lieber, Andre; Lauber, Chris; Dahl, Andreas; Petzold, Andreas; Zhang, Youming; Stewart, A Francis; Ehrhardt, Anja

    2017-05-23

    Adenoviruses (Ads) are large human-pathogenic double-stranded DNA (dsDNA) viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR) and linear-circular homologous recombination (LCHR). We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS). We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS) of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Versatile generation of optical vector fields and vector beams using a non-interferometric approach.

    PubMed

    Tripathi, Santosh; Toussaint, Kimani C

    2012-05-07

    We present a versatile, non-interferometric method for generating vector fields and vector beams which can produce all the states of polarization represented on a higher-order Poincaré sphere. The versatility and non-interferometric nature of this method is expected to enable exploration of various exotic properties of vector fields and vector beams. To illustrate this, we study the propagation properties of some vector fields and find that, in general, propagation alters both their intensity and polarization distribution, and more interestingly, converts some vector fields into vector beams. In the article, we also suggest a modified Jones vector formalism to represent vector fields and vector beams.

  15. Slugs: Potential Novel Vectors of Escherichia coli O157

    PubMed Central

    Sproston, Emma L.; Macrae, M.; Ogden, Iain D.; Wilson, Michael J.; Strachan, Norval J. C.

    2006-01-01

    Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157. PMID:16391036

  16. Vector-borne diseases models with residence times - A Lagrangian perspective.

    PubMed

    Bichara, Derdei; Castillo-Chavez, Carlos

    2016-11-01

    A multi-patch and multi-group modeling framework describing the dynamics of a class of diseases driven by the interactions between vectors and hosts structured by groups is formulated. Hosts' dispersal is modeled in terms of patch-residence times with the nonlinear dynamics taking into account the effective patch-host size. The residence times basic reproduction number R 0 is computed and shown to depend on the relative environmental risk of infection. The model is robust, that is, the disease free equilibrium is globally asymptotically stable (GAS) if R 0 ≤1 and a unique interior endemic equilibrium is shown to exist that is GAS whenever R 0 >1 whenever the configuration of host-vector interactions is irreducible. The effects of patchiness and groupness, a measure of host-vector heterogeneous structure, on the basic reproduction number R 0 , are explored. Numerical simulations are carried out to highlight the effects of residence times on disease prevalence. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Gene therapy using retrovirus vectors: vector development and biosafety at clinical trials.

    PubMed

    Doi, Knayo; Takeuchi, Yasuhiro

    2015-01-01

    Retrovirus vectors (gammaretroviral and lentiviral vectors) have been considered as promising tools to transfer therapeutic genes into patient cells because they can permanently integrate into host cellular genome. To treat monogenic, inherited diseases, retroviral vectors have been used to add correct genes into patient cells. Conventional gammaretroviral vectors achieved successful results in clinical trials: treated patients had therapeutic gene expression in target cells and had improved symptoms of diseases. However, serious side-effects of leukemia occurred, caused by retroviral insertional mutagenesis (IM). These incidences stressed the importance of monitoring vector integration sites in patient cells as well as of re-consideration on safer vectors. More recently lentiviral vectors which can deliver genes into non-dividing cells started to be used in clinical trials including neurological disorders, showing their efficacy. Vector integration site analysis revealed that lentiviruses integrate less likely to near promoter regions of oncogenes than gammaretroviruses and no adverse events have been reported in lentiviral vector-mediated gene therapy clinical trials. Therefore lentiviral vectors have promises to be applied to a wide range of common diseases in near future. For example, T cells from cancer patients were transduced to express chimeric T cell receptors recognizing their tumour cells enhancing patients' anti-cancer immunity.

  18. On sufficient statistics of least-squares superposition of vector sets.

    PubMed

    Konagurthu, Arun S; Kasarapu, Parthan; Allison, Lloyd; Collier, James H; Lesk, Arthur M

    2015-06-01

    The problem of superposition of two corresponding vector sets by minimizing their sum-of-squares error under orthogonal transformation is a fundamental task in many areas of science, notably structural molecular biology. This problem can be solved exactly using an algorithm whose time complexity grows linearly with the number of correspondences. This efficient solution has facilitated the widespread use of the superposition task, particularly in studies involving macromolecular structures. This article formally derives a set of sufficient statistics for the least-squares superposition problem. These statistics are additive. This permits a highly efficient (constant time) computation of superpositions (and sufficient statistics) of vector sets that are composed from its constituent vector sets under addition or deletion operation, where the sufficient statistics of the constituent sets are already known (that is, the constituent vector sets have been previously superposed). This results in a drastic improvement in the run time of the methods that commonly superpose vector sets under addition or deletion operations, where previously these operations were carried out ab initio (ignoring the sufficient statistics). We experimentally demonstrate the improvement our work offers in the context of protein structural alignment programs that assemble a reliable structural alignment from well-fitting (substructural) fragment pairs. A C++ library for this task is available online under an open-source license.

  19. Enhancement of Gag-specific but reduction of Env- and Pol-specific CD8+ T cell responses by simian immunodeficiency virus nonstructural proteins in mice.

    PubMed

    Zhang, Yinfeng; Sun, Caijun; Feng, Liqiang; Xiao, Lijun; Chen, Ling

    2012-04-01

    Accessory and regulatory proteins (nonstructural proteins) have received increasing attention as components in novel HIV/SIV vaccine design. However, the complicated interactions between nonstructural proteins and structural proteins remain poorly understood, especially their effects on immunogenicity. In this study, the immunogenicity of structural proteins in the presence and absence of nonstructural proteins was compared. First, a series of recombinant plasmids and adenoviral vectors carrying various SIVmac239 nonstructural and structural genes was constructed. Then mice were primed with DNA plasmids and boosted with corresponding Ad5 vectors of different combinations, and the resulting immune responses were measured. Our results demonstrated that when the individual Gag, Pol, or Env gene products were coimmunized with the whole repertoire of nonstructural proteins, the Gag-specific CD8(+) T response was greatly enhanced, while the Env- and Pol-specific CD8(+) T responses were significantly reduced. The same pattern was not observed in CD4(+) T cell responses. Antibody responses against both the Gag and Env proteins were elicited more effectively when these structural antigens were immunized together with nonstructural antigens. These findings may provide helpful insights into the development of novel HIV/SIV vaccines.

  20. Insulated Foamy Viral Vectors

    PubMed Central

    Browning, Diana L.; Collins, Casey P.; Hocum, Jonah D.; Leap, David J.; Rae, Dustin T.; Trobridge, Grant D.

    2016-01-01

    Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34+ cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy. PMID:26715244

  1. Early Error Detection: An Action-Research Experience Teaching Vector Calculus

    ERIC Educational Resources Information Center

    Añino, María Magdalena; Merino, Gabriela; Miyara, Alberto; Perassi, Marisol; Ravera, Emiliano; Pita, Gustavo; Waigandt, Diana

    2014-01-01

    This paper describes an action-research experience carried out with second year students at the School of Engineering of the National University of Entre Ríos, Argentina. Vector calculus students played an active role in their own learning process. They were required to present weekly reports, in both oral and written forms, on the topics studied,…

  2. Vector independent transmission of the vector-borne bluetongue virus.

    PubMed

    van der Sluijs, Mirjam Tineke Willemijn; de Smit, Abraham J; Moormann, Rob J M

    2016-01-01

    Bluetongue is an economically important disease of ruminants. The causative agent, Bluetongue virus (BTV), is mainly transmitted by insect vectors. This review focuses on vector-free BTV transmission, and its epizootic and economic consequences. Vector-free transmission can either be vertical, from dam to fetus, or horizontal via direct contract. For several BTV-serotypes, vertical (transplacental) transmission has been described, resulting in severe congenital malformations. Transplacental transmission had been mainly associated with live vaccine strains. Yet, the European BTV-8 strain demonstrated a high incidence of transplacental transmission in natural circumstances. The relevance of transplacental transmission for the epizootiology is considered limited, especially in enzootic areas. However, transplacental transmission can have a substantial economic impact due to the loss of progeny. Inactivated vaccines have demonstrated to prevent transplacental transmission. Vector-free horizontal transmission has also been demonstrated. Since direct horizontal transmission requires close contact of animals, it is considered only relevant for within-farm spreading of BTV. The genetic determinants which enable vector-free transmission are present in virus strains circulating in the field. More research into the genetic changes which enable vector-free transmission is essential to better evaluate the risks associated with outbreaks of new BTV serotypes and to design more appropriate control measures.

  3. Vectors and Rotations in 3-Dimensions: Vector Algebra for the C++ Programmer

    DTIC Science & Technology

    2016-12-01

    Proving Ground, MD 21005-5068 This report describes 2 C++ classes: a Vector class for performing vector algebra in 3-dimensional space ( 3D ) and a Rotation...class for performing rotations of vectors in 3D . Each class is self-contained in a single header file (Vector.h and Rotation.h) so that a C...vector, rotation, 3D , quaternion, C++ tools, rotation sequence, Euler angles, yaw, pitch, roll, orientation 98 Richard Saucier 410-278-6721Unclassified

  4. The Magsat precision vector magnetometer

    NASA Technical Reports Server (NTRS)

    Acuna, M. H.

    1980-01-01

    This paper examines the Magsat precision vector magnetometer which is designed to measure projections of the ambient field in three orthogonal directions. The system contains a highly stable and linear triaxial fluxgate magnetometer with a dynamic range of + or - 2000 nT (1 nT = 10 to the -9 weber per sq m). The magnetometer electronics, analog-to-digital converter, and digitally controlled current sources are implemented with redundant designs to avoid a loss of data in case of failures. Measurements are carried out with an accuracy of + or - 1 part in 64,000 in magnitude and 5 arcsec in orientation (1 arcsec = 0.00028 deg).

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

    DOEpatents

    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).

  6. Using adenovirus armed short hairpin RNA targeting transforming growth factor β1 inhibits melanoma growth and metastasis in an ex vivo animal model.

    PubMed

    Tai, Kuo-Feng; Wang, Chien-Hsing

    2013-12-01

    The transforming growth factor β (TGF-β) is the key molecule implicated in impaired immune function in human patients with malignant melanoma. TGF-β can promote tumor growth, invasion, and metastasis in advanced stages of melanoma. Blocking these tumor-promoting effects of TGF-β provides a potentially important therapeutic strategy for the treatment of melanoma. In this study, we used an adenovirus-based shRNA expression system and successfully constructed Ad/TGF-β1-RNA interference (RNAi) which mediated the RNAi for TGF-β1 gene silencing. We examined the effects of TGF-β1 protein knockdown by RNAi on the growth and metastasis of melanoma in C57BL/6 mice induced by the B16F0 cell line. The TGF-β1 hairpin oligonucleotide was cloned into adenoviral vector. The resulting recombinant adenoviruses infected murine melanoma cell line, B16F0, and designated as B16F0/TGF-β1-RNAi cells. The blank adenoviral vector also infected B16F0 cells and designed as B16F0/vector-control cells served as a control. TGF-β1 expression was reduced in B16F0/TGF-β1-RNAi cells compared with B16F0 cells and B16F0/vector-control cells. Three million wild-type B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-β1-RNAi cells were injected subcutaneously into the right flanks of adult female syngeneic mice C57BL/6. The tumor sizes were 756.09 (65.35), 798.48 (78.77), and 203.55 (24.56) mm at the 14th day in the mice receiving B16F0 cells, B16F0/vector-control cells, and B16F0/TGFβ1-RNAi cells, respectively. The P value was less than 0.01 by 1-way analysis of variance. TGF-β1 knockdown in B16F0 cells enhanced the infiltration of CD4 and CD8 T cells in the tumor regions. C57BL/6 mice were evaluated for pulmonary metastasis after tail vein injection of 2 million B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-β1-RNAi cells. The pulmonary metastasis also reduced significantly on days 14 day and 21 in mice injected with B16F0/TGF-β1-RNAi tumors. The blood vessel density of the

  7. Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats.

    PubMed

    Huang, Shun-Ping; Lin, Po-Kang; Liu, Jorn-Hon; Khor, Chin-Ni; Lee, Yih-Jing

    2004-01-01

    Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats. Copyright 2004 National Science Council, ROC and S. Karger AG, Basel

  8. Vector-borne Infections

    PubMed Central

    Ben Beard, C.

    2011-01-01

    Infections with vector-borne pathogens are a major source of emerging diseases. The ability of vectors to bridge spatial and ecologic gaps between animals and humans increases opportunities for emergence. Small adaptations of a pathogen to a vector can have profound effects on the rate of transmission to humans. PMID:21529382

  9. Peridomestic Aedes malayensis and Aedes albopictus are capable vectors of arboviruses in cities.

    PubMed

    Mendenhall, Ian H; Manuel, Menchie; Moorthy, Mahesh; Lee, Theodore T M; Low, Dolyce H W; Missé, Dorothée; Gubler, Duane J; Ellis, Brett R; Ooi, Eng Eong; Pompon, Julien

    2017-06-01

    Dengue and chikungunya are global re-emerging mosquito-borne diseases. In Singapore, sustained vector control coupled with household improvements reduced domestic mosquito populations for the past 45 years, particularly the primary vector Aedes aegypti. However, while disease incidence was low for the first 30 years following vector control implementation, outbreaks have re-emerged in the past 15 years. Epidemiological observations point to the importance of peridomestic infection in areas not targeted by control programs. We investigated the role of vectors in peri-domestic areas. We carried out entomological surveys to identify the Aedes species present in vegetated sites in highly populated areas and determine whether mosquitoes were present in open-air areas frequented by people. We compared vector competence of Aedes albopictus and Aedes malayensis with Ae. aegypti after oral infection with sympatric dengue serotype 2 and chikungunya viruses. Mosquito saliva was tested for the presence of infectious virus particles as a surrogate for transmission following oral infection. We identified Aedes albopictus and Aedes malayensis throughout Singapore and quantified their presence in forested and opened grassy areas. Both Ae. albopictus and Ae. malayensis can occupy sylvatic niches and were highly susceptible to both arboviruses. A majority of saliva of infected Ae. malayensis contained infectious particles for both viruses. Our study reveals the prevalence of competent vectors in peri-domestic areas, including Ae. malayensis for which we established the vector status. Epidemics can be driven by infection foci, which are epidemiologically enhanced in the context of low herd immunity, selective pressure on arbovirus transmission and the presence of infectious asymptomatic persons, all these conditions being present in Singapore. Learning from Singapore's vector control success that reduced domestic vector populations, but has not sustainably reduced arboviral incidence

  10. Peridomestic Aedes malayensis and Aedes albopictus are capable vectors of arboviruses in cities

    PubMed Central

    Manuel, Menchie; Low, Dolyce H. W.; Missé, Dorothée; Gubler, Duane J.; Ellis, Brett R.; Ooi, Eng Eong; Pompon, Julien

    2017-01-01

    Background Dengue and chikungunya are global re-emerging mosquito-borne diseases. In Singapore, sustained vector control coupled with household improvements reduced domestic mosquito populations for the past 45 years, particularly the primary vector Aedes aegypti. However, while disease incidence was low for the first 30 years following vector control implementation, outbreaks have re-emerged in the past 15 years. Epidemiological observations point to the importance of peridomestic infection in areas not targeted by control programs. We investigated the role of vectors in peri-domestic areas. Methods We carried out entomological surveys to identify the Aedes species present in vegetated sites in highly populated areas and determine whether mosquitoes were present in open-air areas frequented by people. We compared vector competence of Aedes albopictus and Aedes malayensis with Ae. aegypti after oral infection with sympatric dengue serotype 2 and chikungunya viruses. Mosquito saliva was tested for the presence of infectious virus particles as a surrogate for transmission following oral infection. Results We identified Aedes albopictus and Aedes malayensis throughout Singapore and quantified their presence in forested and opened grassy areas. Both Ae. albopictus and Ae. malayensis can occupy sylvatic niches and were highly susceptible to both arboviruses. A majority of saliva of infected Ae. malayensis contained infectious particles for both viruses. Conclusions Our study reveals the prevalence of competent vectors in peri-domestic areas, including Ae. malayensis for which we established the vector status. Epidemics can be driven by infection foci, which are epidemiologically enhanced in the context of low herd immunity, selective pressure on arbovirus transmission and the presence of infectious asymptomatic persons, all these conditions being present in Singapore. Learning from Singapore’s vector control success that reduced domestic vector populations, but has

  11. Advancing vector biology research: a community survey for future directions, research applications and infrastructure requirements

    PubMed Central

    Kohl, Alain; Pondeville, Emilie; Schnettler, Esther; Crisanti, Andrea; Supparo, Clelia; Christophides, George K.; Kersey, Paul J.; Maslen, Gareth L.; Takken, Willem; Koenraadt, Constantianus J. M.; Oliva, Clelia F.; Busquets, Núria; Abad, F. Xavier; Failloux, Anna-Bella; Levashina, Elena A.; Wilson, Anthony J.; Veronesi, Eva; Pichard, Maëlle; Arnaud Marsh, Sarah; Simard, Frédéric; Vernick, Kenneth D.

    2016-01-01

    Vector-borne pathogens impact public health, animal production, and animal welfare. Research on arthropod vectors such as mosquitoes, ticks, sandflies, and midges which transmit pathogens to humans and economically important animals is crucial for development of new control measures that target transmission by the vector. While insecticides are an important part of this arsenal, appearance of resistance mechanisms is increasingly common. Novel tools for genetic manipulation of vectors, use of Wolbachia endosymbiotic bacteria, and other biological control mechanisms to prevent pathogen transmission have led to promising new intervention strategies, adding to strong interest in vector biology and genetics as well as vector–pathogen interactions. Vector research is therefore at a crucial juncture, and strategic decisions on future research directions and research infrastructure investment should be informed by the research community. A survey initiated by the European Horizon 2020 INFRAVEC-2 consortium set out to canvass priorities in the vector biology research community and to determine key activities that are needed for researchers to efficiently study vectors, vector-pathogen interactions, as well as access the structures and services that allow such activities to be carried out. We summarize the most important findings of the survey which in particular reflect the priorities of researchers in European countries, and which will be of use to stakeholders that include researchers, government, and research organizations. PMID:27677378

  12. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yu Hui; Wu Jihong; Li Huiming

    The interaction of vascular endothelial growth factor (VEGF) and its receptors (Flt-1, Flk-1/KDR) is correlated with neovascularization in the eyes. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for inhibiting corneal neovascularization. In this study, we have expressed the cDNA for sFlk-1 under the control of cytomegalovirus immediate-early promoter (CMV) from an E1/partial E3 deleted replication defective recombinant adenovirus, and Ad.sflk-1 expression was determined by Western blotting. We have shown that conditioned media from Ad.sflk-1-infected ARPE-19 cells significantly reduced VEGF-induced human umbilical vein endothelial cells (HUVEC) and murine endothelial cells (SVEC) proliferation in vitro comparedmore » with the control vector. In vivo, adenoviral vectors expressing green fluorescent protein alone (Ad.GFP) were utilized to monitor gene transfer to the cornea. Moreover, in the models of corneal neovascularization, the injection of Ad.sflk-1 (10{sup 8} PFU) into the anterior chamber could significantly inhibit angiogenic changes compared with Ad.null-injected and vehicle-injected models. Immunohistochemical analysis showed that corneal endothelial cells and corneal stroma of cauterized rat eyes were efficiently transduced and expressed sFlk-1. These results not only support that adenoviral vectors are capable of high-level transgene expression but also demonstrate that Ad.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization.« less

  13. Implementing a vector surveillance-response system for chagas disease control: a 4-year field trial in Nicaragua.

    PubMed

    Yoshioka, Kota; Tercero, Doribel; Pérez, Byron; Nakamura, Jiro; Pérez, Lenin

    2017-03-06

    Chagas disease is one of the neglected tropical diseases (NTDs). International goals for its control involve elimination of vector-borne transmission. Central American countries face challenges in establishing sustainable vector control programmes, since the main vector, Triatoma dimidiata, cannot be eliminated. In 2012, the Ministry of Health in Nicaragua started a field test of a vector surveillance-response system to control domestic vector infestation. This paper reports the main findings from this pilot study. This study was carried out from 2012 to 2015 in the Municipality of Totogalpa. The Japan International Cooperation Agency provided technical cooperation in designing and monitoring the surveillance-response system until 2014. This system involved 1) vector reports by householders to health facilities, 2) data analysis and planning of responses at the municipal health centre and 3) house visits or insecticide spraying by health personnel as a response. We registered all vector reports and responses in a digital database. The collected data were used to describe and analyse the system performance in terms of amount of vector reports as well as rates and timeliness of responses. During the study period, T. dimidiata was reported 396 times. Spatiotemporal analysis identified some high-risk clusters. All houses reported to be infested were visited by health personnel in 2013 and this response rate dropped to 39% in 2015. Rates of insecticide spraying rose above 80% in 2013 but no spraying was carried out in the following 2 years. The timeliness of house visits improved significantly after the responsibility was transferred from a vector control technician to primary health care staff. We argue that the proposed vector surveillance-response system is workable within the resource-constrained health system in Nicaragua. Integration to the primary health care services was a key to improve the system performance. Continual efforts are necessary to keep adapting

  14. A Kalman Filter for SINS Self-Alignment Based on Vector Observation.

    PubMed

    Xu, Xiang; Xu, Xiaosu; Zhang, Tao; Li, Yao; Tong, Jinwu

    2017-01-29

    In this paper, a self-alignment method for strapdown inertial navigation systems based on the q -method is studied. In addition, an improved method based on integrating gravitational apparent motion to form apparent velocity is designed, which can reduce the random noises of the observation vectors. For further analysis, a novel self-alignment method using a Kalman filter based on adaptive filter technology is proposed, which transforms the self-alignment procedure into an attitude estimation using the observation vectors. In the proposed method, a linear psuedo-measurement equation is adopted by employing the transfer method between the quaternion and the observation vectors. Analysis and simulation indicate that the accuracy of the self-alignment is improved. Meanwhile, to improve the convergence rate of the proposed method, a new method based on parameter recognition and a reconstruction algorithm for apparent gravitation is devised, which can reduce the influence of the random noises of the observation vectors. Simulations and turntable tests are carried out, and the results indicate that the proposed method can acquire sound alignment results with lower standard variances, and can obtain higher alignment accuracy and a faster convergence rate.

  15. A Kalman Filter for SINS Self-Alignment Based on Vector Observation

    PubMed Central

    Xu, Xiang; Xu, Xiaosu; Zhang, Tao; Li, Yao; Tong, Jinwu

    2017-01-01

    In this paper, a self-alignment method for strapdown inertial navigation systems based on the q-method is studied. In addition, an improved method based on integrating gravitational apparent motion to form apparent velocity is designed, which can reduce the random noises of the observation vectors. For further analysis, a novel self-alignment method using a Kalman filter based on adaptive filter technology is proposed, which transforms the self-alignment procedure into an attitude estimation using the observation vectors. In the proposed method, a linear psuedo-measurement equation is adopted by employing the transfer method between the quaternion and the observation vectors. Analysis and simulation indicate that the accuracy of the self-alignment is improved. Meanwhile, to improve the convergence rate of the proposed method, a new method based on parameter recognition and a reconstruction algorithm for apparent gravitation is devised, which can reduce the influence of the random noises of the observation vectors. Simulations and turntable tests are carried out, and the results indicate that the proposed method can acquire sound alignment results with lower standard variances, and can obtain higher alignment accuracy and a faster convergence rate. PMID:28146059

  16. Multi-color incomplete Cholesky conjugate gradient methods for vector computers

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Poole, E.L.

    1986-01-01

    This research is concerned with the solution on vector computers of linear systems of equations. Ax = b, where A is a large, sparse symmetric positive definite matrix with non-zero elements lying only along a few diagonals of the matrix. The system is solved using the incomplete Cholesky conjugate gradient method (ICCG). Multi-color orderings are used of the unknowns in the linear system to obtain p-color matrices for which a no-fill block ICCG method is implemented on the CYBER 205 with O(N/p) length vector operations in both the decomposition of A and, more importantly, in the forward and back solvesmore » necessary at each iteration of the method. (N is the number of unknowns and p is a small constant). A p-colored matrix is a matrix that can be partitioned into a p x p block matrix where the diagonal blocks are diagonal matrices. The matrix is stored by diagonals and matrix multiplication by diagonals is used to carry out the decomposition of A and the forward and back solves. Additionally, if the vectors across adjacent blocks line up, then some of the overhead associated with vector startups can be eliminated in the matrix vector multiplication necessary at each conjugate gradient iteration. Necessary and sufficient conditions are given to determine which multi-color orderings of the unknowns correspond to p-color matrices, and a process is indicated for choosing multi-color orderings.« less

  17. A comparison of in situ measurements of vector-E and - vector-V x vector-B from Dynamics Explorer 2

    NASA Technical Reports Server (NTRS)

    Hanson, W. B.; Coley, W. R.; Heelis, R. A.; Maynard, N. C.; Aggson, T. L.

    1993-01-01

    Dynamics Explorer-2 provided the first opportunity to make a direct comparison of in situ measurements of the high-latitude convection electric field by two distinctly different techniques. The vector electric field instrument (VEFI) used antennae to measure the intrinsic electric fields and the ion drift meter (IDM) and retarding potential analyzer (RPA) measured the ion drift velocity vector, from which the convection electric field can be deduced. The data from three orbits having large electric fields at high latitude are presented, one at high, one at medium, and one at low altitudes. The general agreement between the two measurements of electric field is very good, with typical differences at high latitudes of the order of a few millivolts per meter, but there are some regions where the particle fluxes are extremely large (e.g., the cusp) and the disagreement is worse, probably because of IDM difficulties. The auroral zone potential patterns derived from the two devices are in excellent agreement for two of the cases, but not in the third, where bad attitude data may be the problem. At low latitudes there are persistent differences in the measurements of a few millivolts per meter, though these differences are quite constant from orbit to orbit. This problem seems to arise from some shortcoming in the VEFI measurments. Overall, however, these measurements confirm the concept of `frozen-in' plasma that drifts with velocity vector-E x vector-B/B(exp 2) within the measurement errors of the two techniques.

  18. Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

    PubMed Central

    2013-01-01

    Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

  19. [Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].

    PubMed

    Yang, Xian-Xian; Zhang, Mei; Yan, Zhao-Wen; Zhang, Ru-Hong; Mu, Xiong-Zheng

    2008-01-01

    To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.

  20. Rotations with Rodrigues' Vector

    ERIC Educational Resources Information Center

    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…

  1. Orthogonal vector algorithm to obtain the solar vector using the single-scattering Rayleigh model.

    PubMed

    Wang, Yinlong; Chu, Jinkui; Zhang, Ran; Shi, Chao

    2018-02-01

    Information obtained from a polarization pattern in the sky provides many animals like insects and birds with vital long-distance navigation cues. The solar vector can be derived from the polarization pattern using the single-scattering Rayleigh model. In this paper, an orthogonal vector algorithm, which utilizes the redundancy of the single-scattering Rayleigh model, is proposed. We use the intersection angles between the polarization vectors as the main criteria in our algorithm. The assumption that all polarization vectors can be considered coplanar is used to simplify the three-dimensional (3D) problem with respect to the polarization vectors in our simulation. The surface-normal vector of the plane, which is determined by the polarization vectors after translation, represents the solar vector. Unfortunately, the two-directionality of the polarization vectors makes the resulting solar vector ambiguous. One important result of this study is, however, that this apparent disadvantage has no effect on the complexity of the algorithm. Furthermore, two other universal least-squares algorithms were investigated and compared. A device was then constructed, which consists of five polarized-light sensors as well as a 3D attitude sensor. Both the simulation and experimental data indicate that the orthogonal vector algorithms, if used with a suitable threshold, perform equally well or better than the other two algorithms. Our experimental data reveal that if the intersection angles between the polarization vectors are close to 90°, the solar-vector angle deviations are small. The data also support the assumption of coplanarity. During the 51 min experiment, the mean of the measured solar-vector angle deviations was about 0.242°, as predicted by our theoretical model.

  2. Cloning vector

    DOEpatents

    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.

  3. Cloning vector

    DOEpatents

    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.

  4. Rhotrix Vector Spaces

    ERIC Educational Resources Information Center

    Aminu, Abdulhadi

    2010-01-01

    By rhotrix we understand an object that lies in some way between (n x n)-dimensional matrices and (2n - 1) x (2n - 1)-dimensional matrices. Representation of vectors in rhotrices is different from the representation of vectors in matrices. A number of vector spaces in matrices and their properties are known. On the other hand, little seems to be…

  5. Vector 33: A reduce program for vector algebra and calculus in orthogonal curvilinear coordinates

    NASA Astrophysics Data System (ADS)

    Harper, David

    1989-06-01

    This paper describes a package with enables REDUCE 3.3 to perform algebra and calculus operations upon vectors. Basic algebraic operations between vectors and between scalars and vectors are provided, including scalar (dot) product and vector (cross) product. The vector differential operators curl, divergence, gradient and Laplacian are also defined, and are valid in any orthogonal curvilinear coordinate system. The package is written in RLISP to allow algebra and calculus to be performed using notation identical to that for operations. Scalars and vectors can be mixed quite freely in the same expression. The package will be of interest to mathematicians, engineers and scientists who need to perform vector calculations in orthogonal curvilinear coordinates.

  6. Emergence and Prevalence of Human Vector-Borne Diseases in Sink Vector Populations

    PubMed Central

    Rascalou, Guilhem; Pontier, Dominique; Menu, Frédéric; Gourbière, Sébastien

    2012-01-01

    Vector-borne diseases represent a major public health concern in most tropical and subtropical areas, and an emerging threat for more developed countries. Our understanding of the ecology, evolution and control of these diseases relies predominantly on theory and data on pathogen transmission in large self-sustaining ‘source’ populations of vectors representative of highly endemic areas. However, there are numerous places where environmental conditions are less favourable to vector populations, but where immigration allows them to persist. We built an epidemiological model to investigate the dynamics of six major human vector borne-diseases in such non self-sustaining ‘sink’ vector populations. The model was parameterized through a review of the literature, and we performed extensive sensitivity analysis to look at the emergence and prevalence of the pathogen that could be encountered in these populations. Despite the low vector abundance in typical sink populations, all six human diseases were able to spread in 15–55% of cases after accidental introduction. The rate of spread was much more strongly influenced by vector longevity, immigration and feeding rates, than by transmission and virulence of the pathogen. Prevalence in humans remained lower than 5% for dengue, leishmaniasis and Japanese encephalitis, but substantially higher for diseases with longer duration of infection; malaria and the American and African trypanosomiasis. Vector-related parameters were again the key factors, although their influence was lower than on pathogen emergence. Our results emphasize the need for ecology and evolution to be thought in the context of metapopulations made of a mosaic of sink and source habitats, and to design vector control program not only targeting areas of high vector density, but working at a larger spatial scale. PMID:22629337

  7. Novel Minicircle Vector for Gene Therapy in Murine Myocardial Infarction

    PubMed Central

    Huang, Mei; Chen, ZhiYing; Hu, Shijun; Jia, Fangjun; Li, Zongjin; Hoyt, Grant; Robbins, Robert C.; Kay, Mark A.; Wu, Joseph C.

    2011-01-01

    Background Conventional plasmids for gene therapy produce low-level and short-term gene expression. In this study, we develop a novel non-viral vector which robustly and persistently expresses the hypoxia inducible factor-1 alpha (HIF-1α) therapeutic gene in the heart, leading to functional benefits following myocardial infarction (MI). Methods and Results We first created minicircles carrying double fusion (MC-DF) reporter gene consisting of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) for noninvasive measurement of transfection efficiency. Mouse C2C12 myoblasts and normal FVB mice were used for in vitro and in vivo confirmation, respectively. Bioluminescence imaging (BLI) showed stable minicircle gene expression in the heart for >12 weeks and the activity level was 5.6±1.2 fold stronger than regular plasmid at day 4 (P<0.01). Next, we created minicircles carrying hypoxia inducible factor-1 alpha (MC-HIF-1α) therapeutic gene for treatment of MI. Adult FVB mice underwent LAD ligation and were injected intramyocardially with (1) MC-HIF-1α, (2) regular plasmid carrying HIF-1α (PL-HIF-1α) as positive control, and (3) PBS as negative control (n=10/group). Echocardiographic study showed a significantly greater improvement of left ventricular ejection fraction (LVEF) in the minicircle group (51.3%±3.6%) compared to regular plasmid group (42.3%±4.1%) and saline group (30.5%±2.8%) at week 4 (P<0.05 for both). Histology demonstrated increased neoangiogenesis in both treatment groups. Finally, Western blot showed minicircles express >50% higher HIF-1α level than regular plasmid. Conclusion Taken together, this is the first study to demonstrate that minicircles can significantly improve transfection efficiency, duration of transgene expression, and cardiac contractility. Given the serious drawbacks associated with most viral vectors, we believe this novel non-viral vector can be of great value for cardiac gene therapy protocols. PMID

  8. Impact of Vector Dispersal and Host-Plant Fidelity on the Dissemination of an Emerging Plant Pathogen

    PubMed Central

    Johannesen, Jes; Foissac, Xavier; Kehrli, Patrik; Maixner, Michael

    2012-01-01

    Dissemination of vector-transmitted pathogens depend on the survival and dispersal of the vector and the vector's ability to transmit the pathogen, while the host range of vector and pathogen determine the breath of transmission possibilities. In this study, we address how the interaction between dispersal and plant fidelities of a pathogen (stolbur phytoplasma tuf-a) and its vector (Hyalesthes obsoletus: Cixiidae) affect the emergence of the pathogen. Using genetic markers, we analysed the geographic origin and range expansion of both organisms in Western Europe and, specifically, whether the pathogen's dissemination in the northern range is caused by resident vectors widening their host-plant use from field bindweed to stinging nettle, and subsequent host specialisation. We found evidence for common origins of pathogen and vector south of the European Alps. Genetic patterns in vector populations show signals of secondary range expansion in Western Europe leading to dissemination of tuf-a pathogens, which might be newly acquired and of hybrid origin. Hence, the emergence of stolbur tuf-a in the northern range was explained by secondary immigration of vectors carrying stinging nettle-specialised tuf-a, not by widening the host-plant spectrum of resident vectors with pathogen transmission from field bindweed to stinging nettle nor by primary co-migration from the resident vector's historical area of origin. The introduction of tuf-a to stinging nettle in the northern range was therefore independent of vector's host-plant specialisation but the rapid pathogen dissemination depended on the vector's host shift, whereas the general dissemination elsewhere was linked to plant specialisation of the pathogen but not of the vector. PMID:23284774

  9. Impact of vector dispersal and host-plant fidelity on the dissemination of an emerging plant pathogen.

    PubMed

    Johannesen, Jes; Foissac, Xavier; Kehrli, Patrik; Maixner, Michael

    2012-01-01

    Dissemination of vector-transmitted pathogens depend on the survival and dispersal of the vector and the vector's ability to transmit the pathogen, while the host range of vector and pathogen determine the breath of transmission possibilities. In this study, we address how the interaction between dispersal and plant fidelities of a pathogen (stolbur phytoplasma tuf-a) and its vector (Hyalesthes obsoletus: Cixiidae) affect the emergence of the pathogen. Using genetic markers, we analysed the geographic origin and range expansion of both organisms in Western Europe and, specifically, whether the pathogen's dissemination in the northern range is caused by resident vectors widening their host-plant use from field bindweed to stinging nettle, and subsequent host specialisation. We found evidence for common origins of pathogen and vector south of the European Alps. Genetic patterns in vector populations show signals of secondary range expansion in Western Europe leading to dissemination of tuf-a pathogens, which might be newly acquired and of hybrid origin. Hence, the emergence of stolbur tuf-a in the northern range was explained by secondary immigration of vectors carrying stinging nettle-specialised tuf-a, not by widening the host-plant spectrum of resident vectors with pathogen transmission from field bindweed to stinging nettle nor by primary co-migration from the resident vector's historical area of origin. The introduction of tuf-a to stinging nettle in the northern range was therefore independent of vector's host-plant specialisation but the rapid pathogen dissemination depended on the vector's host shift, whereas the general dissemination elsewhere was linked to plant specialisation of the pathogen but not of the vector.

  10. Development and Application of Modern Optimal Controllers for a Membrane Structure Using Vector Second Order Form

    NASA Astrophysics Data System (ADS)

    Ferhat, Ipar

    With increasing advancement in material science and computational power of current computers that allows us to analyze high dimensional systems, very light and large structures are being designed and built for aerospace applications. One example is a reflector of a space telescope that is made of membrane structures. These reflectors are light and foldable which makes the shipment easy and cheaper unlike traditional reflectors made of glass or other heavy materials. However, one of the disadvantages of membranes is that they are very sensitive to external changes, such as thermal load or maneuvering of the space telescope. These effects create vibrations that dramatically affect the performance of the reflector. To overcome vibrations in membranes, in this work, piezoelectric actuators are used to develop distributed controllers for membranes. These actuators generate bending effects to suppress the vibration. The actuators attached to a membrane are relatively thick which makes the system heterogeneous; thus, an analytical solution cannot be obtained to solve the partial differential equation of the system. Therefore, the Finite Element Model is applied to obtain an approximate solution for the membrane actuator system. Another difficulty that arises with very flexible large structures is the dimension of the discretized system. To obtain an accurate result, the system needs to be discretized using smaller segments which makes the dimension of the system very high. This issue will persist as long as the improving technology will allow increasingly complex and large systems to be designed and built. To deal with this difficulty, the analysis of the system and controller development to suppress the vibration are carried out using vector second order form as an alternative to vector first order form. In vector second order form, the number of equations that need to be solved are half of the number equations in vector first order form. Analyzing the system for control

  11. Quantifying the activity of adenoviral E1A CR2 deletion mutants using renilla luciferase bioluminescence and 3'-deoxy-3'-[18F]fluorothymidine positron emission tomography imaging.

    PubMed

    Leyton, Julius; Lockley, Michelle; Aerts, Joeri L; Baird, Sarah K; Aboagye, Eric O; Lemoine, Nicholas R; McNeish, Iain A

    2006-09-15

    The adenoviral E1A CR2 mutant dl922-947 has potent activity in ovarian cancer. We have used Renilla luciferase bioluminescence imaging to monitor viral E1A expression and replication and [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to quantify the activity of dl922-947 in vivo. We created dlCR2 Ren, with the same E1A CR2 deletion as dl922-947 and the luciferase gene from Renilla reniformis downstream of E1. Light emitted from s.c. and i.p. IGROV1 ovarian carcinoma xenografts was measured following treatment with dlCR2 Ren. Mice bearing s.c. IGROV1 xenografts were injected with 2.96 to 3.7 MBq of [18F]FLT 48 and 168 hours following i.t. injection of dl922-947 or control virus Ad LM-X. The presence of Renilla luciferase in dlCR2 Ren did not reduce in vitro nor in vivo potency compared with dl922-947. Light emission correlated closely with E1A expression in vitro and peaked 48 hours after dlCR2 Ren injection in both s.c. and i.p. IGROV1 xenografts. It diminished by 168 hours in s.c. tumors but persisted for at least 2 weeks in i.p. models. Normalized tumor [18F]FLT uptake at 60 minutes (NUV60), fractional retention, and area under radioactivity curve all decreased marginally 48 hours after dl922-947 treatment and significantly at 168 hours compared with controls. There was a close linear correlation between NUV60 and both tumor proliferation (Ki67 labeling index) and thymidine kinase 1 expression. Renilla luciferase bioluminescence and [18F]FLT-PET imaging are capable of quantifying the activity and effectiveness of E1A CR2-deleted adenoviral mutants in ovarian cancer.

  12. Rortex—A new vortex vector definition and vorticity tensor and vector decompositions

    NASA Astrophysics Data System (ADS)

    Liu, Chaoqun; Gao, Yisheng; Tian, Shuling; Dong, Xiangrui

    2018-03-01

    A vortex is intuitively recognized as the rotational/swirling motion of the fluids. However, an unambiguous and universally accepted definition for vortex is yet to be achieved in the field of fluid mechanics, which is probably one of the major obstacles causing considerable confusions and misunderstandings in turbulence research. In our previous work, a new vector quantity that is called vortex vector was proposed to accurately describe the local fluid rotation and clearly display vortical structures. In this paper, the definition of the vortex vector, named Rortex here, is revisited from the mathematical perspective. The existence of the possible rotational axis is proved through real Schur decomposition. Based on real Schur decomposition, a fast algorithm for calculating Rortex is also presented. In addition, new vorticity tensor and vector decompositions are introduced: the vorticity tensor is decomposed to a rigidly rotational part and a non-rotationally anti-symmetric part, and the vorticity vector is decomposed to a rigidly rotational vector which is called the Rortex vector and a non-rotational vector which is called the shear vector. Several cases, including the 2D Couette flow, 2D rigid rotational flow, and 3D boundary layer transition on a flat plate, are studied to demonstrate the justification of the definition of Rortex. It can be observed that Rortex identifies both the precise swirling strength and the rotational axis, and thus it can reasonably represent the local fluid rotation and provide a new powerful tool for vortex dynamics and turbulence research.

  13. Characterization of Reversibly Immortalized Calvarial Mesenchymal Progenitor Cells.

    PubMed

    Shenaq, Deana S; Teven, Chad M; Seitz, Iris A; Rastegar, Farbod; Greives, Matthew R; He, Tong-Chuan; Reid, Russell R

    2015-06-01

    Bone morphogenetic proteins (BMPs) play a sentinel role in osteoblastic differentiation, and their implementation into clinical practice can revolutionize cranial reconstruction. Preliminary data suggest a therapeutic role of adenoviral gene delivery of BMPs in murine calvarial defect healing. Poor transgene expression inherent in direct adenoviral therapy prompted investigation of cell-based strategies. To isolate and immortalize calvarial cells as a potential progenitor source for osseous tissue engineering. Cells were isolated from murine skulls, cultured, and transduced with a retroviral vector bearing the loxP-flanked SV40 large T antigen. Immortalized calvarial cells (iCALs) were evaluated via light microscopy, immunohistochemistry, and flow cytometry to determine whether the immortalization process altered cell morphology or progenitor cell profile. Immortalized calvarial cells were then infected with adenoviral vectors encoding BMP-2 or GFP and assessed for early and late stages of osteogenic differentiation. Immortalization of calvarial cells did not alter cell morphology as demonstrated by phase contrast microscopy. Mesenchymal progenitor cell markers CD166, CD73, CD44, and CD105 were detected at varying levels in both primary cells and iCALs. Significant elevations in alkaline phosphatase activity, osteocalcin mRNA transcription, and matrix mineralization were detected in BMP-2 treated iCALs compared with GFP-treated cells. Gross and histological analyses revealed ectopic bone production from treated cells compared with controls in an in vivo stem cell implantation assay. We have established an immortalized osteoprogenitor cell line from juvenile calvarial cells that retain a progenitor cell phenotype and can successfully undergo osteogenic differentiation upon BMP-2 stimulation. These cells provide a valuable platform to investigate the molecular mechanisms underlying intramembranous bone formation and to screen for factors/small molecules that can

  14. Vector sensor for scanning SQUID microscopy

    NASA Astrophysics Data System (ADS)

    Dang, Vu The; Toji, Masaki; Thanh Huy, Ho; Miyajima, Shigeyuki; Shishido, Hiroaki; Hidaka, Mutsuo; Hayashi, Masahiko; Ishida, Takekazu

    2017-07-01

    We plan to build a novel 3-dimensional (3D) scanning SQUID microscope with high sensitivity and high spatial resolution. In the system, a vector sensor consists of three SQUID sensors and three pick-up coils realized on a single chip. Three pick-up coils are configured in orthogonal with each other to measure the magnetic field vector of X, Y, Z components. We fabricated some SQUID chips with one uniaxial pick-up coil or three vector pick-up coils and carried out fundamental measurements to reveal the basic characteristics. Josephson junctions (JJs) of sensors are designed to have the critical current density J c of 320 A/cm2, and the critical current I c becomes 12.5 μA for the 2.2μm × 2.2μm JJ. We carefully positioned the three pickup coils so as to keep them at the same height at the centers of all three X, Y and Z coils. This can be done by arranging them along single line parallel to a sample surface. With the aid of multilayer technology of Nb-based fabrication, we attempted to reduce an inner diameter of the pickup coils to enhance both sensitivity and spatial resolution. The method for improving a spatial resolution of a local magnetic field image is to employ an XYZ piezo-driven scanner for controlling the positions of the pick-up coils. The fundamental characteristics of our SQUID sensors confirmed the proper operation of our SQUID sensors and found a good agreement with our design parameters.

  15. Application of Genomics for Understanding Plant Virus-Insect Vector Interactions and Insect Vector Control.

    PubMed

    Kaur, Navneet; Hasegawa, Daniel K; Ling, Kai-Shu; Wintermantel, William M

    2016-10-01

    The relationships between plant viruses and their vectors have evolved over the millennia, and yet, studies on viruses began <150 years ago and investigations into the virus and vector interactions even more recently. The advent of next generation sequencing, including rapid genome and transcriptome analysis, methods for evaluation of small RNAs, and the related disciplines of proteomics and metabolomics offer a significant shift in the ability to elucidate molecular mechanisms involved in virus infection and transmission by insect vectors. Genomic technologies offer an unprecedented opportunity to examine the response of insect vectors to the presence of ingested viruses through gene expression changes and altered biochemical pathways. This review focuses on the interactions between viruses and their whitefly or thrips vectors and on potential applications of genomics-driven control of the insect vectors. Recent studies have evaluated gene expression in vectors during feeding on plants infected with begomoviruses, criniviruses, and tospoviruses, which exhibit very different types of virus-vector interactions. These studies demonstrate the advantages of genomics and the potential complementary studies that rapidly advance our understanding of the biology of virus transmission by insect vectors and offer additional opportunities to design novel genetic strategies to manage insect vectors and the viruses they transmit.

  16. Multi-perspective views of students’ difficulties with one-dimensional vector and two-dimensional vector

    NASA Astrophysics Data System (ADS)

    Fauzi, Ahmad; Ratna Kawuri, Kunthi; Pratiwi, Retno

    2017-01-01

    Researchers of students’ conceptual change usually collects data from written tests and interviews. Moreover, reports of conceptual change often simply refer to changes in concepts, such as on a test, without any identification of the learning processes that have taken place. Research has shown that students have difficulties with vectors in university introductory physics courses and high school physics courses. In this study, we intended to explore students’ understanding of one-dimensional and two-dimensional vector in multi perspective views. In this research, we explore students’ understanding through test perspective and interviews perspective. Our research study adopted the mixed-methodology design. The participants of this research were sixty students of third semester of physics education department. The data of this research were collected by testand interviews. In this study, we divided the students’ understanding of one-dimensional vector and two-dimensional vector in two categories, namely vector skills of the addition of one-dimensionaland two-dimensional vector and the relation between vector skills and conceptual understanding. From the investigation, only 44% of students provided correct answer for vector skills of the addition of one-dimensional and two-dimensional vector and only 27% students provided correct answer for the relation between vector skills and conceptual understanding.

  17. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors

    PubMed Central

    Ban, Hiroshi; Nishishita, Naoki; Fusaki, Noemi; Tabata, Toshiaki; Saeki, Koichi; Shikamura, Masayuki; Takada, Nozomi; Inoue, Makoto; Hasegawa, Mamoru; Kawamata, Shin; Nishikawa, Shin-Ichi

    2011-01-01

    After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34+ cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future. PMID:21821793

  18. High-dimensional vector semantics

    NASA Astrophysics Data System (ADS)

    Andrecut, M.

    In this paper we explore the “vector semantics” problem from the perspective of “almost orthogonal” property of high-dimensional random vectors. We show that this intriguing property can be used to “memorize” random vectors by simply adding them, and we provide an efficient probabilistic solution to the set membership problem. Also, we discuss several applications to word context vector embeddings, document sentences similarity, and spam filtering.

  19. Custodial vector model

    NASA Astrophysics Data System (ADS)

    Becciolini, Diego; Franzosi, Diogo Buarque; Foadi, Roshan; Frandsen, Mads T.; Hapola, Tuomas; Sannino, Francesco

    2015-07-01

    We analyze the Large Hadron Collider (LHC) phenomenology of heavy vector resonances with a S U (2 )L×S U (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 and interactions with the standard model fields lead to distinct signatures at the LHC in the diboson, dilepton, and associated Higgs channels.

  20. Helper-Free Foamy Virus Vectors

    PubMed Central

    TROBRIDGE, GRANT D.; RUSSELL, DAVID W.

    2010-01-01

    Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV–LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 105 transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR. OVERVIEW SUMMARY Vectors based on human foamy virus have been developed but low titers and the presence of replication-competent retrovirus (RCR) in vector stocks have prevented their use in preclinical animal experiments. We have developed a transient transfection method that can be used to produce replication-incompetent HFV vector stocks at titers greater than 105/ml, and that does not produce contaminating RCR. The use of CMV-HFV LTR fusion promoters in the helper and vector constructs has circumvented the requirement for the HFV Bel-1 trans-activator protein. Consequently, the potential for generating wild-type HFV by recombination between helper and

  1. Elliptic-symmetry vector optical fields.

    PubMed

    Pan, Yue; Li, Yongnan; Li, Si-Min; Ren, Zhi-Cheng; Kong, Ling-Jun; Tu, Chenghou; Wang, Hui-Tian

    2014-08-11

    We present in principle and demonstrate experimentally a new kind of vector fields: elliptic-symmetry vector optical fields. This is a significant development in vector fields, as this breaks the cylindrical symmetry and enriches the family of vector fields. Due to the presence of an additional degrees of freedom, which is the interval between the foci in the elliptic coordinate system, the elliptic-symmetry vector fields are more flexible than the cylindrical vector fields for controlling the spatial structure of polarization and for engineering the focusing fields. The elliptic-symmetry vector fields can find many specific applications from optical trapping to optical machining and so on.

  2. Chikungunya Virus–Vector Interactions

    PubMed Central

    Coffey, Lark L.; Failloux, Anna-Bella; Weaver, Scott C.

    2014-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever, a severe, debilitating disease that often produces chronic arthralgia. Since 2004, CHIKV has emerged in Africa, Indian Ocean islands, Asia, Europe, and the Americas, causing millions of human infections. Central to understanding CHIKV emergence is knowledge of the natural ecology of transmission and vector infection dynamics. This review presents current understanding of CHIKV infection dynamics in mosquito vectors and its relationship to human disease emergence. The following topics are reviewed: CHIKV infection and vector life history traits including transmission cycles, genetic origins, distribution, emergence and spread, dispersal, vector competence, vector immunity and microbial interactions, and co-infection by CHIKV and other arboviruses. The genetics of vector susceptibility and host range changes, population heterogeneity and selection for the fittest viral genomes, dual host cycling and its impact on CHIKV adaptation, viral bottlenecks and intrahost diversity, and adaptive constraints on CHIKV evolution are also discussed. The potential for CHIKV re-emergence and expansion into new areas and prospects for prevention via vector control are also briefly reviewed. PMID:25421891

  3. Hyperbolic-symmetry vector fields.

    PubMed

    Gao, Xu-Zhen; Pan, Yue; Cai, Meng-Qiang; Li, Yongnan; Tu, Chenghou; Wang, Hui-Tian

    2015-12-14

    We present and construct a new kind of orthogonal coordinate system, hyperbolic coordinate system. We present and design a new kind of local linearly polarized vector fields, which is defined as the hyperbolic-symmetry vector fields because the points with the same polarization form a series of hyperbolae. We experimentally demonstrate the generation of such a kind of hyperbolic-symmetry vector optical fields. In particular, we also study the modified hyperbolic-symmetry vector optical fields with the twofold and fourfold symmetric states of polarization when introducing the mirror symmetry. The tight focusing behaviors of these vector fields are also investigated. In addition, we also fabricate micro-structures on the K9 glass surfaces by several tightly focused (modified) hyperbolic-symmetry vector fields patterns, which demonstrate that the simulated tightly focused fields are in good agreement with the fabricated micro-structures.

  4. [Human diffusion of arthropod disease vectors throughout the world].

    PubMed

    Mouchet, J; Giacomini, T; Julvez, J

    1995-01-01

    The present distribution of animals and plants throughout the world is the result of an evolutionary process involving tectonic, climatic and biotic factors. Humans, since their appearance, have contributed to the spreading of many species including disease vectors and pests. When humans left their native African home, they brought with them ectoparasites such as lice and acarids. During the neolithic era, humans were leading domesticated animals which carried their own parasites into new areas. Dwelling commensals, flea, bugs, triatomids, flies, and cockroaches followed human migrations. In the second millennium, sailboats transported mosquito species which were resistant and reproduced on board, including Culex quinquefasciatus, Aedes aegypti, and Ae. albopictus. Steamers further shortened the length of trips and allowed the transport of anophelines. The opening of the Tamatave-Port-Louis line was immediately followed by the transport of An. gambiae from Madagascar to Mauritius and Reunion, and epidemics of malaria occurred on the two islands which had been free of the disease. Also, An. gambiae was transported from Senegal to Brazil. Old tires destined for recycling carried Ae. albopictus to the USA, Brazil, and then Italy. The pandemic of the plague at the end of the nineteenth century was propagated from harbour to harbour by steamers carrying both infected rats and their fleas Xenopsylla cheopsis. Aircrafts have reduced the travel time so much that in less than two days an insect could reach every point of the world. As soon as the airports had been built on the islands of French Polynesia, they were all colonized by Ae. aegypti. The same phenomenon occurred with midges (Fig. 2). Also, the construction of the airport on a Galapagos Island coincided with the importation of the blackfly Simulium bipunctatum from the continent. In addition, infected malaria mosquitos imported from tropical countries reached Europe and contaminated airport employees and local

  5. pYEMF, a pUC18-derived XcmI T-vector for efficient cloning of PCR products.

    PubMed

    Gu, Jingsong; Ye, Chunjiang

    2011-03-01

    A 1330-bp DNA sequence with two XcmI cassettes was inserted into pUC18 to construct an efficient XcmI T-vector parent plasmid, pYEMF. The large size of the inserted DNA fragment improved T-vector cleavage efficiency, and guaranteed good separation of the molecular components after restriction digestion. The pYEMF-T-vector generated from parent plasmid pYEMF permits blue/white colony screening; cloning efficiency analysis showed that most white colonies (>75%) were putative transformants which carried the cloning product. The sequence analysis and design approach presented here will facilitate applications in the fields of molecular biology and genetic engineering.

  6. The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells

    PubMed Central

    Liu, Xiu-Huai; Xu, Wenqin; Russ, Jill; Eiden, Lee E.; Eiden, Maribeth V.

    2011-01-01

    Background Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. Methodology/Principal Findings Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. Conclusions/Significance These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease. PMID:21464894

  7. A potential food-grade cloning vector for Streptococcus thermophilus that uses cadmium resistance as the selectable marker.

    PubMed

    Wong, Wing Yee; Su, Ping; Allison, Gwen E; Liu, Chun-Qiang; Dunn, Noel W

    2003-10-01

    A potential food-grade cloning vector, pND919, was constructed and transformed into S. thermophilus ST3-1, a plasmid-free strain. The vector contains DNAs from two different food-approved organisms, Streptococcus thermophilus and Lactococcus lactis. The 5.0-kb pND919 is a derivative of the cloning vector pND918 (9.3 kb) and was constructed by deletion of the 4.3-kb region of pND918 which contained DNA from non-food-approved organisms. pND919 carries a heterologous native cadmium resistance selectable marker from L. lactis M71 and expresses the Cd(r) phenotype in S. thermophilus transformants. With the S. thermophilus replicon derived from the shuttle vector pND913, pND919 is able to replicate in the two S. thermophilus industrial strains tested, ST3-1 and ST4-1. Its relatively high retention rate in S. thermophilus further indicates its usefulness as a potential food-grade cloning vector. To our knowledge, this is the first report of a replicative potential food-grade vector for the industrially important organism S. thermophilus.

  8. Design and experimental verification for optical module of optical vector-matrix multiplier.

    PubMed

    Zhu, Weiwei; Zhang, Lei; Lu, Yangyang; Zhou, Ping; Yang, Lin

    2013-06-20

    Optical computing is a new method to implement signal processing functions. The multiplication between a vector and a matrix is an important arithmetic algorithm in the signal processing domain. The optical vector-matrix multiplier (OVMM) is an optoelectronic system to carry out this operation, which consists of an electronic module and an optical module. In this paper, we propose an optical module for OVMM. To eliminate the cross talk and make full use of the optical elements, an elaborately designed structure that involves spherical lenses and cylindrical lenses is utilized in this optical system. The optical design software package ZEMAX is used to optimize the parameters and simulate the whole system. Finally, experimental data is obtained through experiments to evaluate the overall performance of the system. The results of both simulation and experiment indicate that the system constructed can implement the multiplication between a matrix with dimensions of 16 by 16 and a vector with a dimension of 16 successfully.

  9. [Sendai virus vector: vector development and its application to health care and biotechnology].

    PubMed

    Iida, Akihiro

    2007-06-01

    Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome and a member of the paramyxovirus family. We have developed SeV vector which has shown a high efficiently of gene transfer and expression of foreign genes to a wide range of dividing and non-dividing mammalian cells and tissues. One of the characteristics of the vector is that the genome is located exclusively in the cytoplasm of infected cells and does not go through a DNA phase; thus there is no concern about unwanted integration of foreign sequences into chromosomal DNA. Therefore, this new class of "cytoplasmic RNA vector", an RNA vector with cytoplasmic expression, is expected to be a safer and more efficient viral vector than existing vectors for application to human therapy in various fields including gene therapy and vaccination. In this review, I describe development of Sendai virus vector, its application in the field of biotechnology and clinical application aiming to treat for a large number of diseases including cancer, cardiovascular disease, infectious diseases and neurologic disorders.

  10. The analysis of the quality of the frequency control of induction motor carried out on the basis of the processes in the rotor circuit

    NASA Astrophysics Data System (ADS)

    Kodkin, V. L.; Anikin, A. S.; Baldenkov, A. A.

    2018-01-01

    The results of researches of asynchronous electric drives with the frequency control which are carried out for the purpose of establishment of causes and effect relationships between a control method, the implementable standard frequency converter of the Schneider Electric company (ATV-71, ATV-32) and its efficiency are given in article. Tests with asynchronous motors with wound rotor were for the first time carried out. It allowed registering during the experiments the instantaneous values not only the stator currents, but also rotor currents. Authors for the first time applied spectrum analysis of stator and rotor currents, it showed that «sensorless vector» control leads to origin of high-frequency harmonicas with the considerable amplitude and, as a result of they are non-sinusoidal of the created torque and inefficiency of the electric drive. The accelerations that are carried out during the researches to 94, 157 and 251 Rad/s confirmed this feature of vector control that appears incapable to linearize the asynchronous electric drive as it was supposed authors of a method. These results do not contradict theoretical provisions if not to neglect assumptions which usually become in case of an output of the equations of vector control. Unfortunately, the modern researchers do not subject these assumptions to doubts. Continued studies make it possible to create an effective frequency management of asynchronous electric drives required for current technology.

  11. Light scattering of rectangular slot antennas: parallel magnetic vector vs perpendicular electric vector

    NASA Astrophysics Data System (ADS)

    Lee, Dukhyung; Kim, Dai-Sik

    2016-01-01

    We study light scattering off rectangular slot nano antennas on a metal film varying incident polarization and incident angle, to examine which field vector of light is more important: electric vector perpendicular to, versus magnetic vector parallel to the long axis of the rectangle. While vector Babinet’s principle would prefer magnetic field along the long axis for optimizing slot antenna function, convention and intuition most often refer to the electric field perpendicular to it. Here, we demonstrate experimentally that in accordance with vector Babinet’s principle, the incident magnetic vector parallel to the long axis is the dominant component, with the perpendicular incident electric field making a small contribution of the factor of 1/|ε|, the reciprocal of the absolute value of the dielectric constant of the metal, owing to the non-perfectness of metals at optical frequencies.

  12. Extended vector-tensor theories

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kimura, Rampei; Naruko, Atsushi; Yoshida, Daisuke, E-mail: rampei@th.phys.titech.ac.jp, E-mail: naruko@th.phys.titech.ac.jp, E-mail: yoshida@th.phys.titech.ac.jp

    Recently, several extensions of massive vector theory in curved space-time have been proposed in many literatures. In this paper, we consider the most general vector-tensor theories that contain up to two derivatives with respect to metric and vector field. By imposing a degeneracy condition of the Lagrangian in the context of ADM decomposition of space-time to eliminate an unwanted mode, we construct a new class of massive vector theories where five degrees of freedom can propagate, corresponding to three for massive vector modes and two for massless tensor modes. We find that the generalized Proca and the beyond generalized Procamore » theories up to the quartic Lagrangian, which should be included in this formulation, are degenerate theories even in curved space-time. Finally, introducing new metric and vector field transformations, we investigate the properties of thus obtained theories under such transformations.« less

  13. "Analytical" vector-functions I

    NASA Astrophysics Data System (ADS)

    Todorov, Vladimir Todorov

    2017-12-01

    In this note we try to give a new (or different) approach to the investigation of analytical vector functions. More precisely a notion of a power xn; n ∈ ℕ+ of a vector x ∈ ℝ3 is introduced which allows to define an "analytical" function f : ℝ3 → ℝ3. Let furthermore f (ξ )= ∑n =0 ∞ anξn be an analytical function of the real variable ξ. Here we replace the power ξn of the number ξ with the power of a vector x ∈ ℝ3 to obtain a vector "power series" f (x )= ∑n =0 ∞ anxn . We research some properties of the vector series as well as some applications of this idea. Note that an "analytical" vector function does not depend of any basis, which may be used in research into some problems in physics.

  14. Influence of adenovirus and MVA vaccines on the breadth and hierarchy of T cell responses.

    PubMed

    Rollier, Christine S; Hill, Adrian V S; Reyes-Sandoval, Arturo

    2016-08-31

    Viral-vectored vaccines are in clinical development for several infectious diseases where T-cell responses can mediate protection, and responses to sub-dominant epitopes is needed. Little is known about the influence of MVA or adenoviral vectors on the hierarchy of the dominant and sub-dominant T-cell epitopes. We investigated this aspect in mice using a malaria immunogen. Our results demonstrate that the T-cell hierarchy is influenced by the timing of analysis, rather than by the vector after a single immunization, with hierarchy changing over time. Repeated homologous immunization reduced the breadth of responses, while heterologous prime-boost induced the strongest response to the dominant epitope, albeit with only modest response to the sub-dominant epitopes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Validation of SplitVectors Encoding for Quantitative Visualization of Large-Magnitude-Range Vector Fields

    PubMed Central

    Zhao, Henan; Bryant, Garnett W.; Griffin, Wesley; Terrill, Judith E.; Chen, Jian

    2017-01-01

    We designed and evaluated SplitVectors, a new vector field display approach to help scientists perform new discrimination tasks on large-magnitude-range scientific data shown in three-dimensional (3D) visualization environments. SplitVectors uses scientific notation to display vector magnitude, thus improving legibility. We present an empirical study comparing the SplitVectors approach with three other approaches - direct linear representation, logarithmic, and text display commonly used in scientific visualizations. Twenty participants performed three domain analysis tasks: reading numerical values (a discrimination task), finding the ratio between values (a discrimination task), and finding the larger of two vectors (a pattern detection task). Participants used both mono and stereo conditions. Our results suggest the following: (1) SplitVectors improve accuracy by about 10 times compared to linear mapping and by four times to logarithmic in discrimination tasks; (2) SplitVectors have no significant differences from the textual display approach, but reduce cluttering in the scene; (3) SplitVectors and textual display are less sensitive to data scale than linear and logarithmic approaches; (4) using logarithmic can be problematic as participants' confidence was as high as directly reading from the textual display, but their accuracy was poor; and (5) Stereoscopy improved performance, especially in more challenging discrimination tasks. PMID:28113469

  16. Validation of SplitVectors Encoding for Quantitative Visualization of Large-Magnitude-Range Vector Fields.

    PubMed

    Henan Zhao; Bryant, Garnett W; Griffin, Wesley; Terrill, Judith E; Jian Chen

    2017-06-01

    We designed and evaluated SplitVectors, a new vector field display approach to help scientists perform new discrimination tasks on large-magnitude-range scientific data shown in three-dimensional (3D) visualization environments. SplitVectors uses scientific notation to display vector magnitude, thus improving legibility. We present an empirical study comparing the SplitVectors approach with three other approaches - direct linear representation, logarithmic, and text display commonly used in scientific visualizations. Twenty participants performed three domain analysis tasks: reading numerical values (a discrimination task), finding the ratio between values (a discrimination task), and finding the larger of two vectors (a pattern detection task). Participants used both mono and stereo conditions. Our results suggest the following: (1) SplitVectors improve accuracy by about 10 times compared to linear mapping and by four times to logarithmic in discrimination tasks; (2) SplitVectors have no significant differences from the textual display approach, but reduce cluttering in the scene; (3) SplitVectors and textual display are less sensitive to data scale than linear and logarithmic approaches; (4) using logarithmic can be problematic as participants' confidence was as high as directly reading from the textual display, but their accuracy was poor; and (5) Stereoscopy improved performance, especially in more challenging discrimination tasks.

  17. Vector matter waves in two-component Bose-Einstein condensates with spatially modulated nonlinearities

    NASA Astrophysics Data System (ADS)

    Xu, Si-Liu; He, Jun-Rong; Xue, Li; Belić, Milivoj R.

    2018-02-01

    We demonstrate three-dimensional (3D) vector solitary waves in the coupled (3 + 1)-D nonlinear Gross-Pitaevskii equations with variable nonlinearity coefficients. The analysis is carried out in spherical coordinates, providing novel localized solutions that depend on three modal numbers, l, m, and n. Using the similarity transformation (ST) method in 3D, vector solitary waves are built with the help of a combination of harmonic and trapping potentials, including multipole solutions and necklace rings. In general, the solutions found are stable for low values of the modal numbers; for values larger than 2, the solutions are found to be unstable. Variable nonlinearity allows the utilization of soliton management methods.

  18. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

    PubMed

    Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

    2014-11-01

    Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates

  19. The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice.

    PubMed

    Uchida, Kenzo; Nakajima, Hideaki; Hirai, Takayuki; Yayama, Takafumi; Chen, Kebing; Guerrero, Alexander Rodriguez; Johnson, William Eustace; Baba, Hisatoshi

    2012-12-15

    The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS

  20. Equivalent Vectors

    ERIC Educational Resources Information Center

    Levine, Robert

    2004-01-01

    The cross-product is a mathematical operation that is performed between two 3-dimensional vectors. The result is a vector that is orthogonal or perpendicular to both of them. Learning about this for the first time while taking Calculus-III, the class was taught that if AxB = AxC, it does not necessarily follow that B = C. This seemed baffling. The…

  1. The Interaction between Vector Life History and Short Vector Life in Vector-Borne Disease Transmission and Control.

    PubMed

    Brand, Samuel P C; Rock, Kat S; Keeling, Matt J

    2016-04-01

    Epidemiological modelling has a vital role to play in policy planning and prediction for the control of vectors, and hence the subsequent control of vector-borne diseases. To decide between competing policies requires models that can generate accurate predictions, which in turn requires accurate knowledge of vector natural histories. Here we highlight the importance of the distribution of times between life-history events, using short-lived midge species as an example. In particular we focus on the distribution of the extrinsic incubation period (EIP) which determines the time between infection and becoming infectious, and the distribution of the length of the gonotrophic cycle which determines the time between successful bites. We show how different assumptions for these periods can radically change the basic reproductive ratio (R0) of an infection and additionally the impact of vector control on the infection. These findings highlight the need for detailed entomological data, based on laboratory experiments and field data, to correctly construct the next-generation of policy-informing models.

  2. Detecting Vessels Carrying Migrants Using Machine Learning

    NASA Astrophysics Data System (ADS)

    Sfyridis, A.; Cheng, T.; Vespe, M.

    2017-10-01

    Political instability, conflicts and inequalities result into significant flows of people worldwide, moving to different countries in search of a better life, safety or to be reunited with their families. Irregular crossings into Europe via sea routes, despite not being new, have recently increased together with the loss of lives of people in the attempt to reach EU shores. This highlights the need to find ways to improve the understanding of what is happening at sea. This paper, intends to expand the knowledge available on practices among smugglers and contribute to early warning and maritime situational awareness. By identifying smuggling techniques and based on anomaly detection methods, behaviours of interest are modelled and one class support vector machines are used to classify unlabelled data and detect potential smuggling vessels. Nine vessels are identified as potentially carrying irregular migrants and refugees. Though, further inspection of the results highlights possible misclassifications caused by data gaps and limited knowledge on smuggling tactics. Accepted classifications are considered subject to further investigation by the authorities.

  3. An effective method for adenoviral-mediated delivery of small interfering RNA into mesenchymal stem cells.

    PubMed

    Forte, Amalia; Napolitano, Marco A; Cipollaro, Marilena; Giordano, Antonio; Cascino, Antonino; Galderisi, Umberto

    2007-02-01

    Mesenchymal stem cells (MSCs) promise as a main actor of cell-based therapeutic strategies, due to their intrinsic ability to differentiate along different mesenchymal cell lineages, able to repair the diseased or injured tissue in which they are localized. The application of MSCs in therapies requires an in depth knowledge of their biology and of the molecular mechanisms leading to MSC multilineage differentiation. The knockdown of target genes through small interfering RNA (siRNA) carried by adenoviruses (Ad) represents a valid tool for the study of the role of specific molecules in cell biology. Unfortunately, MSCs are poorly transfected by conventional Ad serotype 5 (Ad5) vectors. We set up a method to obtain a very efficient transduction of rat MSCs with low doses of unmodified Ad5, carrying the siRNA targeted against the mRNA coding for Rb2/p130 (Ad-siRNA-Rb2), which plays a fundamental role in cell differentiation. This method allowed a 95% transduction rate of Ad-siRNA in MSC, along with a siRNA-mediated 85% decrease of Rb2/p130 mRNA and a 70% decrease of Rb2/p130 protein 48 h after transduction with 50 multiplicities of infection (MOIs) of Ad5. The effect on Rb2/p130 protein persisted 15 days after transduction. Finally, Ad-siRNA did not compromise the viability of transduced MSCs neither induced any cell cycle modification. The effective Ad-siRNA-Rb2 we constructed, together with the efficient method of delivery in MSCs we set up, will allow an in depth analysis of the role of Rb2/p130 in MSC biology and multilineage differentiation.

  4. Vector systems for prenatal gene therapy: principles of retrovirus vector design and production.

    PubMed

    Howe, Steven J; Chandrashekran, Anil

    2012-01-01

    Vectors derived from the Retroviridae family have several attributes required for successful gene delivery. Retroviral vectors have an adequate payload size for the coding regions of most genes; they are safe to handle and simple to produce. These vectors can be manipulated to target different cell types with low immunogenicity and can permanently insert genetic information into the host cells' genome. Retroviral vectors have been used in gene therapy clinical trials and successfully applied experimentally in vitro, in vivo, and in utero.

  5. Therapeutic vaccines in HBV: lessons from HCV.

    PubMed

    Barnes, Eleanor

    2015-02-01

    Currently, millions of people infected with hepatitis B virus (HBV) are committed to decades of treatment with anti-viral therapy to control viral replication. However, new tools for immunotherapy that include both viral vectors and molecular checkpoint inhibitors are now available. This has led to a resurgence of interest in new strategies to develop immunotherapeutic strategies with the aim of inducing HBeAg seroconversion--an end-point that has been associated with a decrease in the rates of disease progression. Ultimately, a true cure will involve the elimination of covalently closed circular DNA which presents a greater challenge for immunotherapy. In this manuscript, I describe the development of immunotherapeutic strategies for HBV that are approaching or currently in clinical studies, and draw on observations of T cell function in natural infection supported by recent animal studies that may lead to additional rational vaccine strategies using checkpoint inhibitors. I also draw on our recent experience in developing potent vaccines for HCV prophylaxis based on simian adenoviral and MVA vectors used in prime-boost strategies in both healthy volunteers and HCV infected patients. I have shown that the induction of T cell immune responses is markedly attenuated when administered to people with persistent HCV viremia. These studies and recently published animal studies using the woodchuck model suggest that potent vaccines based on DNA or adenoviral vectored vaccination represent a rational way forward. However, combining these with drugs to suppress viral replication, alongside checkpoint inhibitors may be required to induce long-term immune control.

  6. A recombinant rabies virus carrying GFP between N and P affects viral transcription in vitro.

    PubMed

    Luo, Jun; Zhao, Jing; Tian, Qin; Mo, Weiyu; Wang, Yifei; Chen, Hao; Guo, Xiaofeng

    2016-06-01

    Several studies have demonstrated the rabies virus to be a perfect potential vaccine vector to insert foreign genes into the target genome. For this study, a green fluorescent protein (GFP) gene was cloned into the rabies virus (RABV) genome between the N and P gene. CT dinucleotide was inserted as intergenic region. The recombinant high egg passage Flury strain (HEP-Flury) of RABV, carrying GFP (rHEP-NP-GFP), was generated in BHK-21 cells using reverse genetics. According to the viral growth kinetics assay, the addition of GFP between N and P gene has little effect on the viral growth compared to the parental strain HEP-Flury. Quantitative real-time PCR (qPCR) indicated that rHEP-NP-GFP showed different viral gene transcription, especially for G gene, compared to HEP-Flury. The same is true for one other recombinant RABV carrying GFP between G and L gene in NA cells. In addition, parent HEP-Flury showed more expression of innate immune-related molecules in NA cells. Compared to HEP-Flury, Western blotting (WB) indicated that insertion of a foreign gene following N gene enhanced the expression of M and G proteins. According to the qPCR and WB, GFP expression levels of rHEP-NP-GFP were significantly higher than rHEP-GFP. This study indicates HEP-Flury as valid vector to express exogenous genes between N and P.

  7. Reciprocity relationships in vector acoustics and their application to vector field calculations.

    PubMed

    Deal, Thomas J; Smith, Kevin B

    2017-08-01

    The reciprocity equation commonly stated in underwater acoustics relates pressure fields and monopole sources. It is often used to predict the pressure measured by a hydrophone for multiple source locations by placing a source at the hydrophone location and calculating the field everywhere for that source. A similar equation that governs the orthogonal components of the particle velocity field is needed to enable this computational method to be used for acoustic vector sensors. This paper derives a general reciprocity equation that accounts for both monopole and dipole sources. This vector-scalar reciprocity equation can be used to calculate individual components of the received vector field by altering the source type used in the propagation calculation. This enables a propagation model to calculate the received vector field components for an arbitrary number of source locations with a single model run for each vector field component instead of requiring one model run for each source location. Application of the vector-scalar reciprocity principle is demonstrated with analytic solutions for a range-independent environment and with numerical solutions for a range-dependent environment using a parabolic equation model.

  8. Optimization and scale-up of cell culture and purification processes for production of an adenovirus-vectored tuberculosis vaccine candidate.

    PubMed

    Shen, Chun Fang; Jacob, Danielle; Zhu, Tao; Bernier, Alice; Shao, Zhongqi; Yu, Xuefeng; Patel, Mehul; Lanthier, Stephane; Kamen, Amine

    2016-06-17

    Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. The only available TB vaccine is the Bacille Calmette-Guerin (BCG). However, parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A, an adenoviral vector expressing the mycobacterial protein Ag85A, is a new tuberculosis vaccine candidate, and has shown promising results in pre-clinical studies and phase I trial. This adenovirus vectored vaccine is produced using HEK 293 cell culture. Here we report on the optimization of cell culture conditions, scale-up of production and purification of the AdAg85A at different scales. Four commercial serum-free media were evaluated under various conditions for supporting the growth of HEK293 cell and production of AdAg85A. A culturing strategy was employed to take advantages of two culture media with respective strengths in supporting the cell growth and virus production, which enabled to maintain virus productivity at higher cell densities and resulted in more than two folds of increases in culture titer. The production of AdAg85A was successfully scaled up and validated at 60L bioreactor under the optimal conditions. The AdAg85A generated from the 3L and 60L bioreactor runs was purified through several purification steps. More than 98% of total cellular proteins was removed, over 60% of viral particles was recovered after the purification process, and purity of AdAg85A was similar to that of the ATCC VR-1516 Ad5 standard. Vaccination of mice with the purified AdAg85A demonstrated a very good level of Ag85A-specific antibody responses. The optimized production and purification conditions were transferred to a GMP facility for manufacturing of AdAg85A for generation of clinical grade material to support clinical trials. Crown Copyright © 2016. Published by Elsevier Ltd. All rights

  9. Vector quantization

    NASA Technical Reports Server (NTRS)

    Gray, Robert M.

    1989-01-01

    During the past ten years Vector Quantization (VQ) has developed from a theoretical possibility promised by Shannon's source coding theorems into a powerful and competitive technique for speech and image coding and compression at medium to low bit rates. In this survey, the basic ideas behind the design of vector quantizers are sketched and some comments made on the state-of-the-art and current research efforts.

  10. Integrating Transgenic Vector Manipulation with Clinical Interventions to Manage Vector-Borne Diseases.

    PubMed

    Okamoto, Kenichi W; Gould, Fred; Lloyd, Alun L

    2016-03-01

    Many vector-borne diseases lack effective vaccines and medications, and the limitations of traditional vector control have inspired novel approaches based on using genetic engineering to manipulate vector populations and thereby reduce transmission. Yet both the short- and long-term epidemiological effects of these transgenic strategies are highly uncertain. If neither vaccines, medications, nor transgenic strategies can by themselves suffice for managing vector-borne diseases, integrating these approaches becomes key. Here we develop a framework to evaluate how clinical interventions (i.e., vaccination and medication) can be integrated with transgenic vector manipulation strategies to prevent disease invasion and reduce disease incidence. We show that the ability of clinical interventions to accelerate disease suppression can depend on the nature of the transgenic manipulation deployed (e.g., whether vector population reduction or replacement is attempted). We find that making a specific, individual strategy highly effective may not be necessary for attaining public-health objectives, provided suitable combinations can be adopted. However, we show how combining only partially effective antimicrobial drugs or vaccination with transgenic vector manipulations that merely temporarily lower vector competence can amplify disease resurgence following transient suppression. Thus, transgenic vector manipulation that cannot be sustained can have adverse consequences-consequences which ineffective clinical interventions can at best only mitigate, and at worst temporarily exacerbate. This result, which arises from differences between the time scale on which the interventions affect disease dynamics and the time scale of host population dynamics, highlights the importance of accounting for the potential delay in the effects of deploying public health strategies on long-term disease incidence. We find that for systems at the disease-endemic equilibrium, even modest

  11. Integrating Transgenic Vector Manipulation with Clinical Interventions to Manage Vector-Borne Diseases

    PubMed Central

    Okamoto, Kenichi W.; Gould, Fred; Lloyd, Alun L.

    2016-01-01

    Many vector-borne diseases lack effective vaccines and medications, and the limitations of traditional vector control have inspired novel approaches based on using genetic engineering to manipulate vector populations and thereby reduce transmission. Yet both the short- and long-term epidemiological effects of these transgenic strategies are highly uncertain. If neither vaccines, medications, nor transgenic strategies can by themselves suffice for managing vector-borne diseases, integrating these approaches becomes key. Here we develop a framework to evaluate how clinical interventions (i.e., vaccination and medication) can be integrated with transgenic vector manipulation strategies to prevent disease invasion and reduce disease incidence. We show that the ability of clinical interventions to accelerate disease suppression can depend on the nature of the transgenic manipulation deployed (e.g., whether vector population reduction or replacement is attempted). We find that making a specific, individual strategy highly effective may not be necessary for attaining public-health objectives, provided suitable combinations can be adopted. However, we show how combining only partially effective antimicrobial drugs or vaccination with transgenic vector manipulations that merely temporarily lower vector competence can amplify disease resurgence following transient suppression. Thus, transgenic vector manipulation that cannot be sustained can have adverse consequences—consequences which ineffective clinical interventions can at best only mitigate, and at worst temporarily exacerbate. This result, which arises from differences between the time scale on which the interventions affect disease dynamics and the time scale of host population dynamics, highlights the importance of accounting for the potential delay in the effects of deploying public health strategies on long-term disease incidence. We find that for systems at the disease-endemic equilibrium, even modest

  12. Tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage N15.

    PubMed

    Mardanov, Andrey V; Strakhova, Taisia S; Smagin, Vladimir A; Ravin, Nikolai V

    2007-06-15

    A new Escherichia coli host/vector system has been developed to allow a dual regulation of both the plasmid copy number and gene expression. The new pN15E vectors are low copy number plasmids based on the replicon of temperate phage N15, comprising the repA replicase gene and cB repressor gene, controlling the plasmid copy number. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the PBAD promoter. The host strain also carried phage N15 partition operon, sop, allowing stable inheritance of pN15E vectors in the absence of selection pressure. In the first vector, pN15E4, the same PBAD promoter controls expression of a cloned gene. The second vector, pN15E6, carries the phage T5 promoter with a double lac operator repression module thus allowing independent regulation of promoter activity and copy number. Using the lacZ gene to monitor expression in these vectors, we show that the ratio of induction/repression can be about 7600-fold for pN15E4 and more than 15,000-fold for pN15E6. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products that was demonstrated by the stable maintenance of a gene encoding a restriction endonuclease in pN15E4. The tight control of transcription and the potential to regulate gene activities quantitatively over wide ranges will open up new approaches in the study of gene function in vivo and controlled expression of heterologous genes.

  13. Video Vectorization via Tetrahedral Remeshing.

    PubMed

    Wang, Chuan; Zhu, Jie; Guo, Yanwen; Wang, Wenping

    2017-02-09

    We present a video vectorization method that generates a video in vector representation from an input video in raster representation. A vector-based video representation offers the benefits of vector graphics, such as compactness and scalability. The vector video we generate is represented by a simplified tetrahedral control mesh over the spatial-temporal video volume, with color attributes defined at the mesh vertices. We present novel techniques for simplification and subdivision of a tetrahedral mesh to achieve high simplification ratio while preserving features and ensuring color fidelity. From an input raster video, our method is capable of generating a compact video in vector representation that allows a faithful reconstruction with low reconstruction errors.

  14. Integrating vector control across diseases.

    PubMed

    Golding, Nick; Wilson, Anne L; Moyes, Catherine L; Cano, Jorge; Pigott, David M; Velayudhan, Raman; Brooker, Simon J; Smith, David L; Hay, Simon I; Lindsay, Steve W

    2015-10-01

    Vector-borne diseases cause a significant proportion of the overall burden of disease across the globe, accounting for over 10 % of the burden of infectious diseases. Despite the availability of effective interventions for many of these diseases, a lack of resources prevents their effective control. Many existing vector control interventions are known to be effective against multiple diseases, so combining vector control programmes to simultaneously tackle several diseases could offer more cost-effective and therefore sustainable disease reductions. The highly successful cross-disease integration of vaccine and mass drug administration programmes in low-resource settings acts a precedent for cross-disease vector control. Whilst deliberate implementation of vector control programmes across multiple diseases has yet to be trialled on a large scale, a number of examples of 'accidental' cross-disease vector control suggest the potential of such an approach. Combining contemporary high-resolution global maps of the major vector-borne pathogens enables us to quantify overlap in their distributions and to estimate the populations jointly at risk of multiple diseases. Such an analysis shows that over 80 % of the global population live in regions of the world at risk from one vector-borne disease, and more than half the world's population live in areas where at least two different vector-borne diseases pose a threat to health. Combining information on co-endemicity with an assessment of the overlap of vector control methods effective against these diseases allows us to highlight opportunities for such integration. Malaria, leishmaniasis, lymphatic filariasis, and dengue are prime candidates for combined vector control. All four of these diseases overlap considerably in their distributions and there is a growing body of evidence for the effectiveness of insecticide-treated nets, screens, and curtains for controlling all of their vectors. The real-world effectiveness of cross

  15. Combination Gene Therapy for Liver Metastasis of Colon Carcinoma in vivo

    NASA Astrophysics Data System (ADS)

    Chen, Shu-Hsai; Chen, X. H. Li; Wang, Yibin; Kosai, Ken-Ichiro; Finegold, Milton J.; Rich, Susan S.

    1995-03-01

    The efficacy of combination therapy with a "suicide gene" and a cytokine gene to treat metastatic colon carcinoma in the liver was investigated. Tumor in the liver was generated by intrahepatic injection of a colon carcinoma cell line (MCA-26) in syngeneic BALB/c mice. Recombinant adenoviral vectors containing various control and therapeutic genes were injected directly into the solid tumors, followed by treatment with ganciclovir. While the tumors continued to grow in all animals treated with a control vector or a mouse interleukin 2 vector, those treated with a herpes simplex virus thymidine kinase vector, with or without the coadministration of the mouse interleukin 2 vector, exhibited dramatic necrosis and regression. However, only animals treated with both vectors developed an effective systemic antitumoral immunity against challenges of tumorigenic doses of parental tumor cells inoculated at distant sites. The antitumoral immunity was associated with the presence of MCA-26 tumor-specific cytolytic CD8^+ T lymphocytes. The results suggest that combination suicide and cytokine gene therapy in vivo can be a powerful approach for treatment of metastatic colon carcinoma in the liver.

  16. Bacteriophages carrying antibiotic resistance genes in fecal waste from cattle, pigs, and poultry.

    PubMed

    Colomer-Lluch, Marta; Imamovic, Lejla; Jofre, Juan; Muniesa, Maite

    2011-10-01

    This study evaluates the occurrence of bacteriophages carrying antibiotic resistance genes in animal environments. bla(TEM), bla(CTX-M) (clusters 1 and 9), and mecA were quantified by quantitative PCR in 71 phage DNA samples from pigs, poultry, and cattle fecal wastes. Densities of 3 to 4 log(10) gene copies (GC) of bla(TEM), 2 to 3 log(10) GC of bla(CTX-M), and 1 to 3 log(10) GC of mecA per milliliter or gram of sample were detected, suggesting that bacteriophages can be environmental vectors for the horizontal transfer of antibiotic resistance genes.

  17. Adenoviral gene transfer of Akt enhances myocardial contractility and intracellular calcium handling

    PubMed Central

    Cittadini, A; Monti, MG; Iaccarino, G; Di Rella, F; Tsichlis, PN; Di Gianni, A; Strömer, H; Sorriento, D; Peschle, C; Trimarco, B; Saccà, L; Condorelli, G

    2010-01-01

    The serine-threonine kinase Akt/PKB mediates stimuli from different classes of cardiomyocyte receptors, including the growth hormone/insulin like growth factor and the β-adrenergic receptors. Whereas the growth-promoting and antiapoptotic properties of Akt activation are well established, little is known about the effects of Akt on myocardial contractility, intracellular calcium (Ca2+) handling, oxygen consumption, and β-adrenergic pathway. To this aim, Sprague–Dawley rats were subjected to a wild-type Akt in vivo adenoviral gene transfer using a catheter-based technique combined with aortopulmonary crossclamping. Left ventricular (LV) contractility and intracellular Ca2+ handling were evaluated in an isolated isovolumic buffer-perfused, aequorin-loaded whole heart preparations 10 days after the surgery. The Ca2+–force relationship was obtained under steady-state conditions in tetanized muscles. No significant hypertrophy was detected in adenovirus with wild-type Akt (Ad.Akt) versus controls rats (LV-to-body weight ratio 2.6±0.2 versus 2.7±0.1 mg/g, controls versus Ad.Akt, P, NS). LV contractility, measured as developed pressure, increased by 41% in Ad.Akt. This was accounted for by both more systolic Ca2+ available to the contractile machinery (+19% versus controls) and by enhanced myofilament Ca2+ responsiveness, documented by an increased maximal Ca2+-activated pressure (+19% versus controls) and a shift to the left of the Ca2+–force relationship. Such increased contractility was paralleled by a slight increase of myocardial oxygen consumption (14%), while titrated dose of dobutamine providing similar inotropic effect augmented oxygen consumption by 39% (P<0.01). Phospholamban, calsequestrin, and ryanodine receptor LV mRNA and protein content were not different among the study groups, while sarcoplasmic reticulum Ca2+ ATPase protein levels were significantly increased in Ad.Akt rats. β-Adrenergic receptor density, affinity, kinase-1 levels, and

  18. Application of eco-friendly tools and eco-bio-social strategies to control dengue vectors in urban and peri-urban settings in Thailand

    PubMed Central

    Kittayapong, Pattamaporn; Thongyuan, Suporn; Olanratmanee, Phanthip; Aumchareoun, Worawit; Koyadun, Surachart; Kittayapong, Rungrith; Butraporn, Piyarat

    2012-01-01

    Background Dengue is considered one of the most important vector-borne diseases in Thailand. Its incidence is increasing despite routine implementation of national dengue control programmes. This study, conducted during 2010, aimed to demonstrate an application of integrated, community-based, eco-bio-social strategies in combination with locally-produced eco-friendly vector control tools in the dengue control programme, emphasizing urban and peri-urban settings in eastern Thailand. Methodology Three different community settings were selected and were randomly assigned to intervention and control clusters. Key community leaders and relevant governmental authorities were approached to participate in this intervention programme. Ecohealth volunteers were identified and trained in each study community. They were selected among active community health volunteers and were trained by public health experts to conduct vector control activities in their own communities using environmental management in combination with eco-friendly vector control tools. These trained ecohealth volunteers carried out outreach health education and vector control during household visits. Management of public spaces and public properties, especially solid waste management, was efficiently carried out by local municipalities. Significant reduction in the pupae per person index in the intervention clusters when compared to the control ones was used as a proxy to determine the impact of this programme. Results Our community-based dengue vector control programme demonstrated a significant reduction in the pupae per person index during entomological surveys which were conducted at two-month intervals from May 2010 for the total of six months in the intervention and control clusters. The programme also raised awareness in applying eco-friendly vector control approaches and increased intersectoral and household participation in dengue control activities. Conclusion An eco-friendly dengue vector control

  19. Application of eco-friendly tools and eco-bio-social strategies to control dengue vectors in urban and peri-urban settings in Thailand.

    PubMed

    Kittayapong, Pattamaporn; Thongyuan, Suporn; Olanratmanee, Phanthip; Aumchareoun, Worawit; Koyadun, Surachart; Kittayapong, Rungrith; Butraporn, Piyarat

    2012-12-01

    Dengue is considered one of the most important vector-borne diseases in Thailand. Its incidence is increasing despite routine implementation of national dengue control programmes. This study, conducted during 2010, aimed to demonstrate an application of integrated, community-based, eco-bio-social strategies in combination with locally-produced eco-friendly vector control tools in the dengue control programme, emphasizing urban and peri-urban settings in eastern Thailand. Three different community settings were selected and were randomly assigned to intervention and control clusters. Key community leaders and relevant governmental authorities were approached to participate in this intervention programme. Ecohealth volunteers were identified and trained in each study community. They were selected among active community health volunteers and were trained by public health experts to conduct vector control activities in their own communities using environmental management in combination with eco-friendly vector control tools. These trained ecohealth volunteers carried out outreach health education and vector control during household visits. Management of public spaces and public properties, especially solid waste management, was efficiently carried out by local municipalities. Significant reduction in the pupae per person index in the intervention clusters when compared to the control ones was used as a proxy to determine the impact of this programme. Our community-based dengue vector control programme demonstrated a significant reduction in the pupae per person index during entomological surveys which were conducted at two-month intervals from May 2010 for the total of six months in the intervention and control clusters. The programme also raised awareness in applying eco-friendly vector control approaches and increased intersectoral and household participation in dengue control activities. An eco-friendly dengue vector control programme was successfully implemented in

  20. Justification of Fuzzy Declustering Vector Quantization Modeling in Classification of Genotype-Image Phenotypes

    NASA Astrophysics Data System (ADS)

    Ng, Theam Foo; Pham, Tuan D.; Zhou, Xiaobo

    2010-01-01

    With the fast development of multi-dimensional data compression and pattern classification techniques, vector quantization (VQ) has become a system that allows large reduction of data storage and computational effort. One of the most recent VQ techniques that handle the poor estimation of vector centroids due to biased data from undersampling is to use fuzzy declustering-based vector quantization (FDVQ) technique. Therefore, in this paper, we are motivated to propose a justification of FDVQ based hidden Markov model (HMM) for investigating its effectiveness and efficiency in classification of genotype-image phenotypes. The performance evaluation and comparison of the recognition accuracy between a proposed FDVQ based HMM (FDVQ-HMM) and a well-known LBG (Linde, Buzo, Gray) vector quantization based HMM (LBG-HMM) will be carried out. The experimental results show that the performances of both FDVQ-HMM and LBG-HMM are almost similar. Finally, we have justified the competitiveness of FDVQ-HMM in classification of cellular phenotype image database by using hypotheses t-test. As a result, we have validated that the FDVQ algorithm is a robust and an efficient classification technique in the application of RNAi genome-wide screening image data.

  1. Selection vector filter framework

    NASA Astrophysics Data System (ADS)

    Lukac, Rastislav; Plataniotis, Konstantinos N.; Smolka, Bogdan; Venetsanopoulos, Anastasios N.

    2003-10-01

    We provide a unified framework of nonlinear vector techniques outputting the lowest ranked vector. The proposed framework constitutes a generalized filter class for multichannel signal processing. A new class of nonlinear selection filters are based on the robust order-statistic theory and the minimization of the weighted distance function to other input samples. The proposed method can be designed to perform a variety of filtering operations including previously developed filtering techniques such as vector median, basic vector directional filter, directional distance filter, weighted vector median filters and weighted directional filters. A wide range of filtering operations is guaranteed by the filter structure with two independent weight vectors for angular and distance domains of the vector space. In order to adapt the filter parameters to varying signal and noise statistics, we provide also the generalized optimization algorithms taking the advantage of the weighted median filters and the relationship between standard median filter and vector median filter. Thus, we can deal with both statistical and deterministic aspects of the filter design process. It will be shown that the proposed method holds the required properties such as the capability of modelling the underlying system in the application at hand, the robustness with respect to errors in the model of underlying system, the availability of the training procedure and finally, the simplicity of filter representation, analysis, design and implementation. Simulation studies also indicate that the new filters are computationally attractive and have excellent performance in environments corrupted by bit errors and impulsive noise.

  2. Transduction of satellite cells after prenatal intramuscular administration of lentiviral vectors.

    PubMed

    MacKenzie, Tippi C; Kobinger, Gary P; Louboutin, Jean-Pierre; Radu, Antoneta; Javazon, Elizabeth H; Sena-Esteves, Miguel; Wilson, James M; Flake, Alan W

    2005-01-01

    We have previously reported long-term expression of lacZ in myocytes after in utero intramuscular injection of Mokola and Ebola pseudotyped lentiviral vectors. In further experiments, we have noted that these vectors also transduce small cells at the periphery of the muscle fibers that have the morphology of satellite cells, or muscle stem cells. In this study we performed experiments to further define the morphology and function of these cells. Balb/c mice at 14-15 days gestation were injected intramuscularly with Ebola or Mokola pseudotyped lentiviral vectors carrying CMV-lacZ. Animals were harvested at various time points, muscles were stained with X-gal, and processed for electron microscopy (EM) and immunofluorescence. To determine whether transduced satellite cells were functionally capable of regenerating injured muscles, animals were injected with notexin in the same area 8 weeks after the in utero injection of viral vector. Transmission EM of transduced cells confirmed the ultrastructural appearance of satellite cells. Double immunofluorescence for beta-galactosidase and satellite cell markers demonstrated co-localization of these markers in transduced cells. In the notexin-injured animals, small blue cells were seen at the areas of regeneration that co-localized beta-galactosidase with markers of regenerating satellite cells. Central nucleated blue fibers were seen at late time points, indicating regenerated muscle fibers arising from a transduced satellite cell. This study demonstrates transduction of muscle satellite cells following prenatal viral vector mediated gene transfer. These findings may have important implications for gene therapy strategies directed toward muscular dystrophy.

  3. Understanding Singular Vectors

    ERIC Educational Resources Information Center

    James, David; Botteron, Cynthia

    2013-01-01

    matrix yields a surprisingly simple, heuristical approximation to its singular vectors. There are correspondingly good approximations to the singular values. Such rules of thumb provide an intuitive interpretation of the singular vectors that helps explain why the SVD is so…

  4. Parvovirus-like particles as vaccine vectors.

    PubMed

    Casal, J I; Rueda, P; Hurtado, A

    1999-09-01

    A wide array of systems have been developed to improve "classic" vaccines. The use of small polypeptides able to elicit potent antibody and cytotoxic responses seems to have enormous potential in the design of safer vaccines. While peptide coupling to large soluble proteins such as keyhole limpet hemocyanin is the current method of choice for eliciting antibody responses and insertion in live viruses for cytotoxic T-lymphocyte responses, alternative cheaper and/or safer methods will clearly be required in the future. Virus-like particles constitute very immunogenic molecules that allow for covalent coupling of the epitopes of interest in a simple way. In this article, we detail the methodology employed for the preparation of efficient virus vectors as delivery systems. We used parvovirus as the model for the design of new vaccine vectors. Recently parvovirus-like particles have been engineered to express foreign polypeptides in certain positions, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. We discuss the different alternatives and the necessary steps to carry out this process, placing special emphasis on the flow of decisions that need to be made during the project. Copyright 1999 Academic Press.

  5. Symbolic computer vector analysis

    NASA Technical Reports Server (NTRS)

    Stoutemyer, D. R.

    1977-01-01

    A MACSYMA program is described which performs symbolic vector algebra and vector calculus. The program can combine and simplify symbolic expressions including dot products and cross products, together with the gradient, divergence, curl, and Laplacian operators. The distribution of these operators over sums or products is under user control, as are various other expansions, including expansion into components in any specific orthogonal coordinate system. There is also a capability for deriving the scalar or vector potential of a vector field. Examples include derivation of the partial differential equations describing fluid flow and magnetohydrodynamics, for 12 different classic orthogonal curvilinear coordinate systems.

  6. A replicative plasmid vector allows efficient complementation of pathogenic Leptospira strains.

    PubMed

    Pappas, Christopher J; Benaroudj, Nadia; Picardeau, Mathieu

    2015-05-01

    Leptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. The linear combination of vectors implies the existence of the cross and dot products

    NASA Astrophysics Data System (ADS)

    Pujol, Jose

    2018-07-01

    Given two vectors u and v, their cross product u × v is a vector perpendicular to u and v. The motivation for this property, however, is never addressed. Here we show that the existence of the cross and dot products and the perpendicularity property follow from the concept of linear combination, which does not involve products of vectors. For our proof we consider the plane generated by a linear combination of uand v. When looking for the coefficients in the linear combination required to reach a desired point on the plane, the solution involves the existence of a normal vector n = u × v. Our results have a bearing on the history of vector analysis, as a product similar to the cross product but without the perpendicularity requirement existed at the same time. These competing products originate in the work of two major nineteen-century mathematicians, W. Hamilton, and H. Grassmann. These historical aspects are discussed in some detail here. We also address certain aspects of the teaching of u × v to undergraduate students, which is known to carry some difficulties. This includes the algebraic and geometric denitions of u × v, the rule for the direction of u × v, and the pseudovectorial nature of u × v.

  8. Evidence of man-vector contact in torn long-lasting insecticide-treated nets

    PubMed Central

    2013-01-01

    Background Studies indicate that physical damage to long-lasting insecticide-treated nets (LLINs) occurs at a surprisingly rapid rate following net distribution. To what extent does such damage affect the impact of LLINs? Can vectors pass a compromised LLIN barrier to bite? Do more resistant vectors enter the insecticide-treated nets (ITNs) through holes? Methods The study was carried out in three geo-locations. Two types of LLINs (polyester and polyethylene) with ‘standardized’ physical damage were compared with similarly damaged, but non-insecticidal (control) nets. The proportionate Holes Index (pHI) of each net was 276. Mosquitoes were captured inside the nets, identified taxonomically, and subjected to molecular analysis to estimate Knock-down resistance (Kdr) frequency. Results The most commonly observed species was Anopheles gambiae, accounting for approximately 70% (1,076/1,550) of the total mosquitoes collected both in LLINs and non-insecticidal nets. When compared with controls, number of vectors captured in torn LLINs was significantly reduced. Nonetheless in a night, an average of 5 An. gambiae s.l could enter the damaged LLINs to bite. Similar numbers of resistant mosquitoes were collected in both LLINs and non-insecticidal (control) nets (p > 0.05). Conclusions At a pHI of 276, man-vector contact was observed in torn LLINs. The insecticide at the surface of LLINs could only reduce the number of vectors. Resistant mosquitoes have opportunity to enter both non-insecticidal (control) nets and LLINs to bite. PMID:23941585

  9. Family leader empowerment program using participatory learning process for dengue vector control.

    PubMed

    Pengvanich, Veerapong

    2011-02-01

    Assess the performance of the empowerment program using participatory learning process for the control of Dengue vector The program focuses on using the leaders of families as the main executer of the vector control protocol. This quasi-experimental research utilized the two-group pretest-posttest design. The sample group consisted of 120 family leaders from two communities in Mueang Municipality, Chachoengsao Province. The research was conducted during an 8-week period between April and June 2010. The data were collected and analyzed based on frequency, percentage, mean, paired t-test, and independent t-test. The result was evaluated by comparing the difference between the mean prevalence index of mosquito larvae before and after the process implementation in terms of the container index (CI) and the house index (HI). After spending eight weeks in the empowerment program, the family leader's behavior in the aspect of Dengue vector control has improved. The Container Index and the House Index were found to decrease with p = 0.05 statistical significance. The reduction of CI and HI suggested that the program worked well in the selected communities. The success of the Dengue vector control program depended on cooperation and participation of many groups, especially the families in the community When the family leaders have good attitude and are capable of carrying out the vector control protocol, the risk factor leading to the incidence of Dengue rims infection can be reduced.

  10. GNSS Single Frequency, Single Epoch Reliable Attitude Determination Method with Baseline Vector Constraint.

    PubMed

    Gong, Ang; Zhao, Xiubin; Pang, Chunlei; Duan, Rong; Wang, Yong

    2015-12-02

    For Global Navigation Satellite System (GNSS) single frequency, single epoch attitude determination, this paper proposes a new reliable method with baseline vector constraint. First, prior knowledge of baseline length, heading, and pitch obtained from other navigation equipment or sensors are used to reconstruct objective function rigorously. Then, searching strategy is improved. It substitutes gradually Enlarged ellipsoidal search space for non-ellipsoidal search space to ensure correct ambiguity candidates are within it and make the searching process directly be carried out by least squares ambiguity decorrelation algorithm (LAMBDA) method. For all vector candidates, some ones are further eliminated by derived approximate inequality, which accelerates the searching process. Experimental results show that compared to traditional method with only baseline length constraint, this new method can utilize a priori baseline three-dimensional knowledge to fix ambiguity reliably and achieve a high success rate. Experimental tests also verify it is not very sensitive to baseline vector error and can perform robustly when angular error is not great.

  11. Vectorized Jiles-Atherton hysteresis model

    NASA Astrophysics Data System (ADS)

    Szymański, Grzegorz; Waszak, Michał

    2004-01-01

    This paper deals with vector hysteresis modeling. A vector model consisting of individual Jiles-Atherton components placed along principal axes is proposed. The cross-axis coupling ensures general vector model properties. Minor loops are obtained using scaling method. The model is intended for efficient finite element method computations defined in terms of magnetic vector potential. Numerical efficiency is ensured by differential susceptibility approach.

  12. The Cross Product of Two Vectors Is Not Just Another Vector--A Major Misconception Being Perpetuated in Calculus and Vector Analysis Textbooks.

    ERIC Educational Resources Information Center

    Elk, Seymour B.

    1997-01-01

    Suggests that the cross product of two vectors can be more easily and accurately explained by starting from the perspective of dyadics because then the concept of vector multiplication has a simple geometrical picture that encompasses both the dot and cross products in any number of dimensions in terms of orthogonal unit vector components. (AIM)

  13. A Potential Food-Grade Cloning Vector for Streptococcus thermophilus That Uses Cadmium Resistance as the Selectable Marker

    PubMed Central

    Wong, Wing Yee; Su, Ping; Allison, Gwen E.; Liu, Chun-Qiang; Dunn, Noel W.

    2003-01-01

    A potential food-grade cloning vector, pND919, was constructed and transformed into S. thermophilus ST3-1, a plasmid-free strain. The vector contains DNAs from two different food-approved organisms, Streptococcus thermophilus and Lactococcus lactis. The 5.0-kb pND919 is a derivative of the cloning vector pND918 (9.3 kb) and was constructed by deletion of the 4.3-kb region of pND918 which contained DNA from non-food-approved organisms. pND919 carries a heterologous native cadmium resistance selectable marker from L. lactis M71 and expresses the Cdr phenotype in S. thermophilus transformants. With the S. thermophilus replicon derived from the shuttle vector pND913, pND919 is able to replicate in the two S. thermophilus industrial strains tested, ST3-1 and ST4-1. Its relatively high retention rate in S. thermophilus further indicates its usefulness as a potential food-grade cloning vector. To our knowledge, this is the first report of a replicative potential food-grade vector for the industrially important organism S. thermophilus. PMID:14532023

  14. Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector.

    PubMed

    Chen, Yong; Chen, Qian; Li, Manman; Mao, Qianzhuo; Chen, Hongyan; Wu, Wei; Jia, Dongsheng; Wei, Taiyun

    2017-11-01

    Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors.

  15. Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector

    PubMed Central

    Mao, Qianzhuo; Chen, Hongyan; Wu, Wei

    2017-01-01

    Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors. PMID:29125860

  16. Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

    PubMed Central

    2014-01-01

    Induced pluripotent stem cell (iPSC) reprogramming requires sustained expression of multiple reprogramming factors for a limited period of time (10–30 days). Conventional iPSC reprogramming was achieved using lentiviral or simple retroviral vectors. Retroviral reprogramming has flaws of insertional mutagenesis, uncontrolled silencing, residual expression and re-activation of transgenes, and immunogenicity. To overcome these issues, various technologies were explored, including adenoviral vectors, protein transduction, RNA transfection, minicircle DNA, excisable PiggyBac (PB) transposon, Cre-lox excision system, negative-sense RNA replicon, positive-sense RNA replicon, Epstein-Barr virus-based episomal plasmids, and repeated transfections of plasmids. This review provides summaries of the main vectorologies and factor delivery systems used in current reprogramming protocols. PMID:24625220

  17. GPU Accelerated Vector Median Filter

    NASA Technical Reports Server (NTRS)

    Aras, Rifat; Shen, Yuzhong

    2011-01-01

    Noise reduction is an important step for most image processing tasks. For three channel color images, a widely used technique is vector median filter in which color values of pixels are treated as 3-component vectors. Vector median filters are computationally expensive; for a window size of n x n, each of the n(sup 2) vectors has to be compared with other n(sup 2) - 1 vectors in distances. General purpose computation on graphics processing units (GPUs) is the paradigm of utilizing high-performance many-core GPU architectures for computation tasks that are normally handled by CPUs. In this work. NVIDIA's Compute Unified Device Architecture (CUDA) paradigm is used to accelerate vector median filtering. which has to the best of our knowledge never been done before. The performance of GPU accelerated vector median filter is compared to that of the CPU and MPI-based versions for different image and window sizes, Initial findings of the study showed 100x improvement of performance of vector median filter implementation on GPUs over CPU implementations and further speed-up is expected after more extensive optimizations of the GPU algorithm .

  18. The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector system and selection cassettes.

    PubMed

    Bińka, Agnieszka; Orczyk, Wacław; Nadolska-Orczyk, Anna

    2012-02-01

    The influence of two binary vector systems, pGreen and pCAMBIA, on the Agrobacterium-mediated transformation ability of wheat and triticale was studied. Both vectors carried selection cassettes with bar or nptII driven by different promoters. Two cultivars of wheat, Kontesa and Torka, and one cultivar of triticale, Wanad, were tested. The transformation rates for the wheat cultivars ranged from 0.00 to 3.58% and from 0.00 to 6.79% for triticale. The best values for wheat were 3.58% for Kontesa and 3.14% for Torka, and these were obtained after transformation with the pGreen vector carrying the nptII selection gene under the control of 35S promoter. In the case of the bar selection system, the best transformation rates were, respectively, 1.46 and 1.79%. Such rates were obtained when the 35S::bar cassette was carried by the pCAMBIA vector; they were significantly lower with the pGreen vector. The triticale cultivar Wanad had its highest transformation rate after transformation with nptII driven by 35S in pCAMBIA. The bar selection system for the same triticale cultivar was better when the gene was driven by nos and the selection cassette was carried by pGreen. The integration of the transgenes was confirmed with at least three pairs of specific starters amplifying the fragments of nptII, bar, or gus. The expression of selection genes, measured by reverse transcriptase polymerase chain reaction (RT-PCR) in relation to the actin gene, was low, ranging from 0.00 to 0.63 for nptII and from 0.00 to 0.33 for bar. The highest relative transcript accumulation was observed for nptII driven by 35S and expressed in Kontesa that had been transformed with pGreen.

  19. An Efficient Wait-Free Vector

    DOE PAGES

    Feldman, Steven; Valera-Leon, Carlos; Dechev, Damian

    2016-03-01

    The vector is a fundamental data structure, which provides constant-time access to a dynamically-resizable range of elements. Currently, there exist no wait-free vectors. The only non-blocking version supports only a subset of the sequential vector API and exhibits significant synchronization overhead caused by supporting opposing operations. Since many applications operate in phases of execution, wherein each phase only a subset of operations are used, this overhead is unnecessary for the majority of the application. To address the limitations of the non-blocking version, we present a new design that is wait-free, supports more of the operations provided by the sequential vector,more » and provides alternative implementations of key operations. These alternatives allow the developer to balance the performance and functionality of the vector as requirements change throughout execution. Compared to the known non-blocking version and the concurrent vector found in Intel’s TBB library, our design outperforms or provides comparable performance in the majority of tested scenarios. Over all tested scenarios, the presented design performs an average of 4.97 times more operations per second than the non-blocking vector and 1.54 more than the TBB vector. In a scenario designed to simulate the filling of a vector, performance improvement increases to 13.38 and 1.16 times. This work presents the first ABA-free non-blocking vector. Finally, unlike the other non-blocking approach, all operations are wait-free and bounds-checked and elements are stored contiguously in memory.« less

  20. A gene cassette for adapting Escherichia coli strains as hosts for att-Int-mediated rearrangement and pL expression vectors.

    PubMed

    Balakrishnan, R; Bolten, B; Backman, K C

    1994-01-28

    A cassette of genes from bacteriophage lambda, when carried on a derivative of bacteriophage Mu, renders strains of Escherichia coli (and in principle other Mu-sensitive bacteria) capable of supporting lambda-based expression vectors, such as rearrangement vectors and pL vectors. The gene cassette contains a temperature-sensitive allele of the repressor gene, cIts857, and a shortened leftward operon comprising, oLpL, N, xis and int. Transfection and lysogenization of this cassette into various host bacteria is mediated by phage Mu functions. Examples of regulated expression of the gene encoding T4 DNA ligase are presented.

  1. Larvicidal activity of few select indigenous plants of North East India against disease vector mosquitoes (Diptera: Culicidae).

    PubMed

    Dohutia, C; Bhattacharyya, D R; Sharma, S K; Mohapatra, P K; Bhattacharjee, K; Gogoi, K; Gogoi, P; Mahanta, J; Prakash, A

    2015-03-01

    Mosquitoes are the vectors of several life threatening diseases like dengue, malaria, Japanese encephalitis and lymphatic filariasis, which are widely present in the north-eastern states of India. Investigations on five local plants of north-east India, selected on the basis of their use by indigenous communities as fish poison, were carried out to study their mosquito larvicidal potential against Anopheles stephensi (malaria vector), Stegomyia aegypti (dengue vector) and Culex quinquefasciatus (lymphatic filariasis vector) mosquitoes. Crude Petroleum ether extracts of the roots of three plants viz. Derris elliptica, Linostoma decandrum and Croton tiglium were found to have remarkable larvicidal activity; D. elliptica extract was the most effective and with LC50 value of 0.307 μg/ml its activity was superior to propoxur, the standard synthetic larvicide. Half-life of larvicidal activity of D. elliptica and L. decandrum extracts ranged from 2-4 days.

  2. Effects of Climate and Climate Change on Vectors and Vector-Borne Diseases: Ticks Are Different.

    PubMed

    Ogden, Nick H; Lindsay, L Robbin

    2016-08-01

    There has been considerable debate as to whether global risk from vector-borne diseases will be impacted by climate change. This has focussed on important mosquito-borne diseases that are transmitted by the vectors from infected to uninfected humans. However, this debate has mostly ignored the biological diversity of vectors and vector-borne diseases. Here, we review how climate and climate change may impact those most divergent of arthropod disease vector groups: multivoltine insects and hard-bodied (ixodid) ticks. We contrast features of the life cycles and behaviour of these arthropods, and how weather, climate, and climate change may have very different impacts on the spatiotemporal occurrence and abundance of vectors, and the pathogens they transmit. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  3. Integral transformation solution of free-space cylindrical vector beams and prediction of modified Bessel-Gaussian vector beams.

    PubMed

    Li, Chun-Fang

    2007-12-15

    A unified description of free-space cylindrical vector beams is presented that is an integral transformation solution to the vector Helmholtz equation and the transversality condition. In the paraxial condition, this solution not only includes the known J(1) Bessel-Gaussian vector beam and the axisymmetric Laguerre-Gaussian vector beam that were obtained by solving the paraxial wave equations but also predicts two kinds of vector beam, called a modified Bessel-Gaussian vector beam.

  4. Reduced Order Model Basis Vector Generation: Generates Basis Vectors fro ROMs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Arrighi, Bill

    2016-03-03

    libROM is a library that implements order reduction via singular value decomposition (SVD) of sampled state vectors. It implements 2 parallel, incremental SVD algorithms and one serial, non-incremental algorithm. It also provides a mechanism for adaptive sampling of basis vectors.

  5. Generation of arbitrary vector fields based on a pair of orthogonal elliptically polarized base vectors.

    PubMed

    Xu, Danfeng; Gu, Bing; Rui, Guanghao; Zhan, Qiwen; Cui, Yiping

    2016-02-22

    We present an arbitrary vector field with hybrid polarization based on the combination of a pair of orthogonal elliptically polarized base vectors on the Poincaré sphere. It is shown that the created vector field is only dependent on the latitude angle 2χ but is independent on the longitude angle 2ψ on the Poincaré sphere. By adjusting the latitude angle 2χ, which is related to two identical waveplates in a common path interferometric arrangement, one could obtain arbitrary type of vector fields. Experimentally, we demonstrate the generation of such kind of vector fields and confirm the distribution of state of polarization by the measurement of Stokes parameters. Besides, we investigate the tight focusing properties of these vector fields. It is found that the additional degree of freedom 2χ provided by arbitrary vector field with hybrid polarization allows one to control the spatial structure of polarization and to engineer the focusing field.

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

    PubMed

    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 (P<0.01). There was no significant difference in the expression level (P>0.05) 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.

  7. Reducing vector-borne disease by empowering farmers in integrated vector management.

    PubMed

    van den Berg, Henk; von Hildebrand, Alexander; Ragunathan, Vaithilingam; Das, Pradeep K

    2007-07-01

    Irrigated agriculture exposes rural people to health risks associated with vector-borne diseases and pesticides used in agriculture and for public health protection. Most developing countries lack collaboration between the agricultural and health sectors to jointly address these problems. We present an evaluation of a project that uses the "farmer field school" method to teach farmers how to manage vector-borne diseases and how to improve rice yields. Teaching farmers about these two concepts together is known as "integrated pest and vector management". An intersectoral project targeting rice irrigation systems in Sri Lanka. Project partners developed a new curriculum for the field school that included a component on vector-borne diseases. Rice farmers in intervention villages who graduated from the field school took vector-control actions as well as improving environmental sanitation and their personal protection measures against disease transmission. They also reduced their use of agricultural pesticides, especially insecticides. The intervention motivated and enabled rural people to take part in vector-management activities and to reduce several environmental health risks. There is scope for expanding the curriculum to include information on the harmful effects of pesticides on human health and to address other public health concerns. Benefits of this approach for community-based health programmes have not yet been optimally assessed. Also, the institutional basis of the integrated management approach needs to be broadened so that people from a wider range of organizations take part. A monitoring and evaluation system needs to be established to measure the performance of integrated management initiatives.

  8. Tensor calculus: unlearning vector calculus

    NASA Astrophysics Data System (ADS)

    Lee, Wha-Suck; Engelbrecht, Johann; Moller, Rita

    2018-02-01

    Tensor calculus is critical in the study of the vector calculus of the surface of a body. Indeed, tensor calculus is a natural step-up for vector calculus. This paper presents some pitfalls of a traditional course in vector calculus in transitioning to tensor calculus. We show how a deeper emphasis on traditional topics such as the Jacobian can serve as a bridge for vector calculus into tensor calculus.

  9. HIV-1 vaccine strategies utilizing viral vectors including antigen- displayed inoviral vectors.

    PubMed

    Hassapis, Kyriakos A; Kostrikis, Leondios G

    2013-12-01

    Antigen-presenting viral vectors have been extensively used as vehicles for the presentation of antigens to the immune system in numerous vaccine strategies. Particularly in HIV vaccine development efforts, two main viral vectors have been used as antigen carriers: (a) live attenuated vectors and (b) virus-like particles (VLPs); the former, although highly effective in animal studies, cannot be clinically tested in humans due to safety concerns and the latter have failed to induce broadly neutralizing anti-HIV antibodies. For more than two decades, Inoviruses (non-lytic bacterial phages) have also been utilized as antigen carriers in several vaccine studies. Inoviral vectors are important antigen-carriers in vaccine development due to their ability to present an antigen on their outer architecture in many copies and to their natural high immunogenicity. Numerous fundamental studies have been conducted, which have established the unique properties of antigen-displayed inoviral vectors in HIV vaccine efforts. The recent isolation of new, potent anti-HIV broadly neutralizing monoclonal antibodies provides a new momentum in this emerging technology.

  10. Test of understanding of vectors: A reliable multiple-choice vector concept test

    NASA Astrophysics Data System (ADS)

    Barniol, Pablo; Zavala, Genaro

    2014-06-01

    In this article we discuss the findings of our research on students' understanding of vector concepts in problems without physical context. First, we develop a complete taxonomy of the most frequent errors made by university students when learning vector concepts. This study is based on the results of several test administrations of open-ended problems in which a total of 2067 students participated. Using this taxonomy, we then designed a 20-item multiple-choice test [Test of understanding of vectors (TUV)] and administered it in English to 423 students who were completing the required sequence of introductory physics courses at a large private Mexican university. We evaluated the test's content validity, reliability, and discriminatory power. The results indicate that the TUV is a reliable assessment tool. We also conducted a detailed analysis of the students' understanding of the vector concepts evaluated in the test. The TUV is included in the Supplemental Material as a resource for other researchers studying vector learning, as well as instructors teaching the material.

  11. Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Puntel, Mariana; Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689; Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048

    2013-05-01

    Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, andmore » can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation

  12. Vector generator scan converter

    DOEpatents

    Moore, James M.; Leighton, James F.

    1990-01-01

    High printing speeds for graphics data are achieved with a laser printer by transmitting compressed graphics data from a main processor over an I/O (input/output) channel to a vector generator scan converter which reconstructs a full graphics image for input to the laser printer through a raster data input port. The vector generator scan converter includes a microprocessor with associated microcode memory containing a microcode instruction set, a working memory for storing compressed data, vector generator hardward for drawing a full graphic image from vector parameters calculated by the microprocessor, image buffer memory for storing the reconstructed graphics image and an output scanner for reading the graphics image data and inputting the data to the printer. The vector generator scan converter eliminates the bottleneck created by the I/O channel for transmitting graphics data from the main processor to the laser printer, and increases printer speed up to thirty fold.

  13. Vector generator scan converter

    DOEpatents

    Moore, J.M.; Leighton, J.F.

    1988-02-05

    High printing speeds for graphics data are achieved with a laser printer by transmitting compressed graphics data from a main processor over an I/O channel to a vector generator scan converter which reconstructs a full graphics image for input to the laser printer through a raster data input port. The vector generator scan converter includes a microprocessor with associated microcode memory containing a microcode instruction set, a working memory for storing compressed data, vector generator hardware for drawing a full graphic image from vector parameters calculated by the microprocessor, image buffer memory for storing the reconstructed graphics image and an output scanner for reading the graphics image data and inputting the data to the printer. The vector generator scan converter eliminates the bottleneck created by the I/O channel for transmitting graphics data from the main processor to the laser printer, and increases printer speed up to thirty fold. 7 figs.

  14. A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

    PubMed

    Fonseca, Jairo Andres; Cabrera-Mora, Monica; Kashentseva, Elena A; Villegas, John Paul; Fernandez, Alejandra; Van Pelt, Amelia; Dmitriev, Igor P; Curiel, David T; Moreno, Alberto

    2016-01-01

    A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective

  15. Fast higher-order MR image reconstruction using singular-vector separation.

    PubMed

    Wilm, Bertram J; Barmet, Christoph; Pruessmann, Klaas P

    2012-07-01

    Medical resonance imaging (MRI) conventionally relies on spatially linear gradient fields for image encoding. However, in practice various sources of nonlinear fields can perturb the encoding process and give rise to artifacts unless they are suitably addressed at the reconstruction level. Accounting for field perturbations that are neither linear in space nor constant over time, i.e., dynamic higher-order fields, is particularly challenging. It was previously shown to be feasible with conjugate-gradient iteration. However, so far this approach has been relatively slow due to the need to carry out explicit matrix-vector multiplications in each cycle. In this work, it is proposed to accelerate higher-order reconstruction by expanding the encoding matrix such that fast Fourier transform can be employed for more efficient matrix-vector computation. The underlying principle is to represent the perturbing terms as sums of separable functions of space and time. Compact representations with this property are found by singular-vector analysis of the perturbing matrix. Guidelines for balancing the accuracy and speed of the resulting algorithm are derived by error propagation analysis. The proposed technique is demonstrated for the case of higher-order field perturbations due to eddy currents caused by diffusion weighting. In this example, image reconstruction was accelerated by two orders of magnitude.

  16. Effects of Cucumber mosaic virus infection on vector and non-vector herbivores of squash.

    PubMed

    Mauck, Kerry E; De Moraes, Consuelo M; Mescher, Mark C

    2010-11-01

    Plant chemicals mediating interactions with insect herbivores seem a likely target for manipulation by insectvectored plant pathogens. Yet, little is currently known about the chemical ecology of insect-vectored diseases or their effects on the ecology of vector and nonvector insects. We recently reported that a widespread plant pathogen, Cucumber mosaic virus (CMV), greatly reduces the quality of host-plants (squash) for aphid vectors, but that aphids are nevertheless attracted to the odors of infected plants-which exhibit elevated emissions of a volatile blend otherwise similar to the odor of healthy plants. This finding suggests that exaggerating existing host-location cues can be a viable vector attraction strategy for pathogens that otherwise reduce host quality for vectors. Here we report additional data regarding the effects of CMV infection on plant interactions with a common nonvector herbivore, the squash bug, Anasa tristis, which is a pest in this system. We found that adult A. tristis females preferred to oviposit on healthy plants in the field, and that healthy plants supported higher populations of nymphs. Collectively, our recent findings suggest that CMV-induced changes in host plant chemistry influence the behavior of both vector and non-vector herbivores, with significant implications both for disease spread and for broader community-level interactions.

  17. Learning with LOGO: Logo and Vectors.

    ERIC Educational Resources Information Center

    Lough, Tom; Tipps, Steve

    1986-01-01

    This is the first of a two-part series on the general concept of vector space. Provides tool procedures to allow investigation of vector properties, vector addition and subtraction, and X and Y components. Lists several sources of additional vector ideas. (JM)

  18. [What makes an insect a vector?].

    PubMed

    Kampen, Helge

    2009-01-01

    Blood-feeding insects transmit numerous viruses, bacteria, protozoans and helminths to vertebrates. The developmental cycles of the microorganisms in their vectors and the mechanisms of transmission are generally extremely complex and the result of a long-lasting coevolution of vector and vectored pathogen based on mutual adaptation. The conditions necessary for an insect to become a vector are multiple but require an innate vector competence as a genetic basis. Next to the vector competence plenty of entomological, ecological and pathogen-related factors are decisive, given the availability of infection sources. The various modes of pathogen transmission by vectors are connected to the developmental routes of the microorganisms in their vectors. In particular, pathogens transmitted by saliva encounter a lot of cellular and acellular barriers during their migration from the insect's midgut through the hemocele into the salivary fluid, including components of the insect's immune system. With regard to intracellular development, receptor-mediated invasion mechanisms are of relevance. As an environmental factor, the temperature has a paramount impact on the vectorial roles of hematophagous insects. Not only has it a considerable influence on the duration of a pathogen's development in its vector (extrinsic incubation period) but it can render putatively vector-incompetent insects to vectors ("leaky gut" phenomenon). Equally crucial are behavioural aspects of both the insect and the pathogen such as blood host preferences, seasonal appearance and circadian biting activity on the vector's side and diurnal/nocturnal periodicity on the pathogen's side which facilitate a contact in the first place.

  19. Integrated vector management: a critical strategy for combating vector-borne diseases in South Sudan.

    PubMed

    Chanda, Emmanuel; Govere, John M; Macdonald, Michael B; Lako, Richard L; Haque, Ubydul; Baba, Samson P; Mnzava, Abraham

    2013-10-25

    Integrated vector management (IVM) based vector control is encouraged by the World Health Organization (WHO). However, operational experience with the IVM strategy has mostly come from countries with relatively well-established health systems and with malaria control focused programmes. Little is known about deployment of IVM for combating multiple vector-borne diseases in post-emergency settings, where delivery structures are less developed or absent. This manuscript reports on the feasibility of operational IVM for combating vector-borne diseases in South Sudan. A methodical review of published and unpublished documents on vector-borne diseases for South Sudan was conducted via systematic literature search of online electronic databases, Google Scholar, PubMed and WHO, using a combination of search terms. Additional, non-peer reviewed literature was examined for information related to the subject. South Sudan is among the heartlands of vector-borne diseases in the world, characterized by enormous infrastructure, human and financial resource constraints and a weak health system against an increasing number of refugees, returnees and internally displaced people. The presence of a multiplicity of vector-borne diseases in this post-conflict situation presents a unique opportunity to explore the potential of a rational IVM strategy for multiple disease control and optimize limited resource utilization, while maximizing the benefits and providing a model for countries in a similar situation. The potential of integrating vector-borne disease control is enormous in South Sudan. However, strengthened coordination, intersectoral collaboration and institutional and technical capacity for entomological monitoring and evaluation, including enforcement of appropriate legislation are crucial.

  20. Integrated vector management: a critical strategy for combating vector-borne diseases in South Sudan

    PubMed Central

    2013-01-01

    Background Integrated vector management (IVM) based vector control is encouraged by the World Health Organization (WHO). However, operational experience with the IVM strategy has mostly come from countries with relatively well-established health systems and with malaria control focused programmes. Little is known about deployment of IVM for combating multiple vector-borne diseases in post-emergency settings, where delivery structures are less developed or absent. This manuscript reports on the feasibility of operational IVM for combating vector-borne diseases in South Sudan. Case description A methodical review of published and unpublished documents on vector-borne diseases for South Sudan was conducted via systematic literature search of online electronic databases, Google Scholar, PubMed and WHO, using a combination of search terms. Additional, non-peer reviewed literature was examined for information related to the subject. Discussion South Sudan is among the heartlands of vector-borne diseases in the world, characterized by enormous infrastructure, human and financial resource constraints and a weak health system against an increasing number of refugees, returnees and internally displaced people. The presence of a multiplicity of vector-borne diseases in this post-conflict situation presents a unique opportunity to explore the potential of a rational IVM strategy for multiple disease control and optimize limited resource utilization, while maximizing the benefits and providing a model for countries in a similar situation. Conclusion The potential of integrating vector-borne disease control is enormous in South Sudan. However, strengthened coordination, intersectoral collaboration and institutional and technical capacity for entomological monitoring and evaluation, including enforcement of appropriate legislation are crucial. PMID:24156749

  1. Major vectors and vector-borne diseases in small ruminants in Ethiopia: A systematic review.

    PubMed

    Asmare, Kassahun; Abayneh, Takele; Sibhat, Berhanu; Shiferaw, Dessie; Szonyi, Barbara; Krontveit, Randi I; Skjerve, Eystein; Wieland, Barbara

    2017-06-01

    Vector-borne diseases are among major health constraints of small ruminant in Ethiopia. While various studies on single vector-borne diseases or presence of vectors have been conducted, no summarized evidence is available on the occurrence of these diseases and the related vectors. This systematic literature review provides a comprehensive summary on major vectors and vector-borne diseases in small ruminants in Ethiopia. Search for published and unpublished literature was conducted between 8th of January and 25th of June 2015. The search was both manual and electronic. The databases used in electronic search were PubMed, Web of Science, CAB Direct and AJOL. For most of the vector-borne diseases, the summary was limited to narrative synthesis due to lack of sufficient data. Meta-analysis was computed for trypanosomosis and dermatophilosis while meta-regression and sensitivity analysis was done only for trypanososmosis due to lack of sufficient reports on dermatophilosis. Owing emphasis to their vector role, ticks and flies were summarized narratively at genera/species level. In line with inclusion criteria, out of 106 initially identified research reports 43 peer-reviewed articles passed the quality assessment. Data on 7 vector-borne diseases were extracted at species and region level from each source. Accordingly, the pooled prevalence estimate of trypanosomosis was 3.7% with 95% confidence interval (CI) 2.8, 4.9), while that of dermatophilosis was 3.1% (95% CI: 1.6, 6.0). The in-between study variance noted for trypanosomosis was statistically significant (p<0.05). Among the three covariates considered for meta-regression, only one (species) fitted the final model significantly (p<0.05) and explained 65.44% of the between studies variance (R 2 ). The prevalence in sheep (5.5%) increased nearly by 34% compared to goats (2.9%). The parasitic presence in blood was documented for babesiosis (3.7% in goats); and anaplasmosis (3.9% in sheep). Serological evidence was

  2. The Weighted Burgers Vector: a new quantity for constraining dislocation densities and types using electron backscatter diffraction on 2D sections through crystalline materials.

    PubMed

    Wheeler, J; Mariani, E; Piazolo, S; Prior, D J; Trimby, P; Drury, M R

    2009-03-01

    The Weighted Burgers Vector (WBV) is defined here as the sum, over all types of dislocations, of [(density of intersections of dislocation lines with a map) x (Burgers vector)]. Here we show that it can be calculated, for any crystal system, solely from orientation gradients in a map view, unlike the full dislocation density tensor, which requires gradients in the third dimension. No assumption is made about gradients in the third dimension and they may be non-zero. The only assumption involved is that elastic strains are small so the lattice distortion is entirely due to dislocations. Orientation gradients can be estimated from gridded orientation measurements obtained by EBSD mapping, so the WBV can be calculated as a vector field on an EBSD map. The magnitude of the WBV gives a lower bound on the magnitude of the dislocation density tensor when that magnitude is defined in a coordinate invariant way. The direction of the WBV can constrain the types of Burgers vectors of geometrically necessary dislocations present in the microstructure, most clearly when it is broken down in terms of lattice vectors. The WBV has three advantages over other measures of local lattice distortion: it is a vector and hence carries more information than a scalar quantity, it has an explicit mathematical link to the individual Burgers vectors of dislocations and, since it is derived via tensor calculus, it is not dependent on the map coordinate system. If a sub-grain wall is included in the WBV calculation, the magnitude of the WBV becomes dependent on the step size but its direction still carries information on the Burgers vectors in the wall. The net Burgers vector content of dislocations intersecting an area of a map can be simply calculated by an integration round the edge of that area, a method which is fast and complements point-by-point WBV calculations.

  3. Vectoring of parallel synthetic jets

    NASA Astrophysics Data System (ADS)

    Berk, Tim; Ganapathisubramani, Bharathram; Gomit, Guillaume

    2015-11-01

    A pair of parallel synthetic jets can be vectored by applying a phase difference between the two driving signals. The resulting jet can be merged or bifurcated and either vectored towards the actuator leading in phase or the actuator lagging in phase. In the present study, the influence of phase difference and Strouhal number on the vectoring behaviour is examined experimentally. Phase-locked vorticity fields, measured using Particle Image Velocimetry (PIV), are used to track vortex pairs. The physical mechanisms that explain the diversity in vectoring behaviour are observed based on the vortex trajectories. For a fixed phase difference, the vectoring behaviour is shown to be primarily influenced by pinch-off time of vortex rings generated by the synthetic jets. Beyond a certain formation number, the pinch-off timescale becomes invariant. In this region, the vectoring behaviour is determined by the distance between subsequent vortex rings. We acknowledge the financial support from the European Research Council (ERC grant agreement no. 277472).

  4. Analysis of the Three-Dimensional Vector FAÇADE Model Created from Photogrammetric Data

    NASA Astrophysics Data System (ADS)

    Kamnev, I. S.; Seredovich, V. A.

    2017-12-01

    The results of the accuracy assessment analysis for creation of a three-dimensional vector model of building façade are described. In the framework of the analysis, analytical comparison of three-dimensional vector façade models created by photogrammetric and terrestrial laser scanning data has been done. The three-dimensional model built from TLS point clouds was taken as the reference one. In the course of the experiment, the three-dimensional model to be analyzed was superimposed on the reference one, the coordinates were measured and deviations between the same model points were determined. The accuracy estimation of the three-dimensional model obtained by using non-metric digital camera images was carried out. Identified façade surface areas with the maximum deviations were revealed.

  5. Stability of Lentiviral Vector-Mediated Transgene Expression in the Brain in the Presence of Systemic Antivector Immune Responses

    PubMed Central

    ABORDO-ADESIDA, EVELYN; FOLLENZI, ANTONIA; BARCIA, CARLOS; SCIASCIA, SANDRA; CASTRO, MARIA G.; NALDINI, LUIGI; LOWENSTEIN, PEDRO R.

    2009-01-01

    Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose—response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 102, and maximum expression at 106, transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune

  6. Vector and axial-vector decomposition of Einstein's gravitational action

    NASA Astrophysics Data System (ADS)

    Soh, Kwang S.

    1991-08-01

    Vector and axial-vector gravitational fields are introduced to express the Einstein action in the manner of electromagnetism. Their conformal scaling properties are examined, and the resemblance between the general coordinate and electromagnetic gauge transformation is elucidated. The chiral formulation of the gravitational action is constructed. I am deeply grateful to Professor S. Hawking, and Professor G. Lloyd for warm hospitality at DAMTP, and Darwin College, University of Cambridge, respectively. I also appreciate much help received from Dr. Q.-H. Park.

  7. Rapid Assembly of Customized TALENs into Multiple Delivery Systems

    PubMed Central

    Zhang, Zhengxing; Zhang, Siliang; Huang, Xin; Orwig, Kyle E.; Sheng, Yi

    2013-01-01

    Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway® Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse Ddx3 subfamily genes, and demonstrated that our modified TALEN assembly approach efficiently generates accurate TALEN moieties that effectively introduce mutations into target genes. We generated “user friendly” TALEN Entry vectors containing TALEN expression cassettes with fluorescent reporter genes that can be efficiently transferred via Gateway (LR) recombination into different delivery systems. We demonstrated that the TALEN Entry vectors can be easily transferred to an adenoviral delivery system to expand application to cells that are difficult to transfect. Since TALENs work in pairs, we also generated a TALEN Entry vector set that combines a TALEN pair into one PiggyBac transposon-based destination vector. The approach described here can also be modified for construction of TALE transcriptional activators, repressors or other functional domains. PMID:24244669

  8. Effects of internal yaw-vectoring devices on the static performance of a pitch-vectoring nonaxisymmetric convergent-divergent nozzle

    NASA Technical Reports Server (NTRS)

    Asbury, Scott C.

    1993-01-01

    An investigation was conducted in the static test facility of the Langley 16-Foot Transonic Tunnel to evaluate the internal performance of a nonaxisymmetric convergent divergent nozzle designed to have simultaneous pitch and yaw thrust vectoring capability. This concept utilized divergent flap deflection for thrust vectoring in the pitch plane and flow-turning deflectors installed within the divergent flaps for yaw thrust vectoring. Modifications consisting of reducing the sidewall length and deflecting the sidewall outboard were investigated as means to increase yaw-vectoring performance. This investigation studied the effects of multiaxis (pitch and yaw) thrust vectoring on nozzle internal performance characteristics. All tests were conducted with no external flow, and nozzle pressure ratio was varied from 2.0 to approximately 13.0. The results indicate that this nozzle concept can successfully generate multiaxis thrust vectoring. Deflection of the divergent flaps produced resultant pitch vector angles that, although dependent on nozzle pressure ratio, were nearly equal to the geometric pitch vector angle. Losses in resultant thrust due to pitch vectoring were small or negligible. The yaw deflectors produced resultant yaw vector angles up to 21 degrees that were controllable by varying yaw deflector rotation. However, yaw deflector rotation resulted in significant losses in thrust ratios and, in some cases, nozzle discharge coefficient. Either of the sidewall modifications generally reduced these losses and increased maximum resultant yaw vector angle. During multiaxis (simultaneous pitch and yaw) thrust vectoring, little or no cross coupling between the thrust vectoring processes was observed.

  9. Vectors a Fortran 90 module for 3-dimensional vector and dyadic arithmetic

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brock, B.C.

    1998-02-01

    A major advance contained in the new Fortran 90 language standard is the ability to define new data types and the operators associated with them. Writing computer code to implement computations with real and complex three-dimensional vectors and dyadics is greatly simplified if the equations can be implemented directly, without the need to code the vector arithmetic explicitly. The Fortran 90 module described here defines new data types for real and complex 3-dimensional vectors and dyadics, along with the common operations needed to work with these objects. Routines to allow convenient initialization and output of the new types are alsomore » included. In keeping with the philosophy of data abstraction, the details of the implementation of the data types are maintained private, and the functions and operators are made generic to simplify the combining of real, complex, single- and double-precision vectors and dyadics.« less

  10. Nonviral vectors for cancer gene therapy: prospects for integrating vectors and combination therapies.

    PubMed

    Ohlfest, John R; Freese, Andrew B; Largaespada, David A

    2005-12-01

    Gene therapy has the potential to improve the clinical outcome of many cancers by transferring therapeutic genes into tumor cells or normal host tissue. Gene transfer into tumor cells or tumor-associated stroma is being employed to induce tumor cell death, stimulate anti-tumor immune response, inhibit angiogenesis, and control tumor cell growth. Viral vectors have been used to achieve this proof of principle in animal models and, in select cases, in human clinical trials. Nevertheless, there has been considerable interest in developing nonviral vectors for cancer gene therapy. Nonviral vectors are simpler, more amenable to large-scale manufacture, and potentially safer for clinical use. Nonviral vectors were once limited by low gene transfer efficiency and transient or steadily declining gene expression. However, recent improvements in plasmid-based vectors and delivery methods are showing promise in circumventing these obstacles. This article reviews the current status of nonviral cancer gene therapy, with an emphasis on combination strategies, long-term gene transfer using transposons and bacteriophage integrases, and future directions.

  11. pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

    PubMed Central

    Kugeratski, Fernanda G; Batista, Michel; Inoue, Alexandre Haruo; Ramos, Bruno Dias; Krieger, Marco Aurelio; Marchini/, Fabricio K

    2015-01-01

    The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzi genes and is freely available for scientific community. PMID:26200713

  12. Prediction of Spirometric Forced Expiratory Volume (FEV1) Data Using Support Vector Regression

    NASA Astrophysics Data System (ADS)

    Kavitha, A.; Sujatha, C. M.; Ramakrishnan, S.

    2010-01-01

    In this work, prediction of forced expiratory volume in 1 second (FEV1) in pulmonary function test is carried out using the spirometer and support vector regression analysis. Pulmonary function data are measured with flow volume spirometer from volunteers (N=175) using a standard data acquisition protocol. The acquired data are then used to predict FEV1. Support vector machines with polynomial kernel function with four different orders were employed to predict the values of FEV1. The performance is evaluated by computing the average prediction accuracy for normal and abnormal cases. Results show that support vector machines are capable of predicting FEV1 in both normal and abnormal cases and the average prediction accuracy for normal subjects was higher than that of abnormal subjects. Accuracy in prediction was found to be high for a regularization constant of C=10. Since FEV1 is the most significant parameter in the analysis of spirometric data, it appears that this method of assessment is useful in diagnosing the pulmonary abnormalities with incomplete data and data with poor recording.

  13. Project Physics Programmed Instruction, Vectors 1.

    ERIC Educational Resources Information Center

    Harvard Univ., Cambridge, MA. Harvard Project Physics.

    This programmed instruction booklet is an interim version of instructional materials being developed by Harvard Project Physics. It is the first in a series of three booklets on vectors and covers the definitions of vectors and scalars, drawing vector quantities to scale, and negative vectors. For others in this series, see SE 015 550 and SE 015…

  14. Insecticide resistance in disease vectors from Mayotte: an opportunity for integrated vector management

    PubMed Central

    2014-01-01

    Background Mayotte, a small island in the Indian Ocean, has been affected for many years by vector-borne diseases. Malaria, Bancroftian filariasis, dengue, chikungunya and Rift Valley fever have circulated or still circulate on the island. They are all transmitted by Culicidae mosquitoes. To limit the impact of these diseases on human health, vector control has been implemented for more than 60 years on Mayotte. In this study, we assessed the resistance levels of four major vector species (Anopheles gambiae, Culex pipiens quinquefasciatus, Aedes aegypti and Aedes albopictus) to two types of insecticides: i) the locally currently-used insecticides (organophosphates, pyrethroids) and ii) alternative molecules that are promising for vector control and come from different insecticide families (bacterial toxins or insect growth regulators). When some resistance was found to one of these insecticides, we characterized the mechanisms involved. Methods Larval and adult bioassays were used to evaluate the level of resistance. When resistance was found, we tested for the presence of metabolic resistance through detoxifying enzyme activity assays, or for target-site mutations through molecular identification of known resistance alleles. Results Resistance to currently-used insecticides varied greatly between the four vector species. While no resistance to any insecticides was found in the two Aedes species, bioassays confirmed multiple resistance in Cx. p. quinquefasciatus (temephos: ~ 20 fold and deltamethrin: only 10% mortality after 24 hours). In An. gambiae, resistance was scarce: only a moderate resistance to temephos was found (~5 fold). This resistance appears to be due only to carboxyl-esterase overexpression and not to target modification. Finally, and comfortingly, none of the four species showed resistance to any of the new insecticides. Conclusions The low resistance observed in Mayotte’s main disease vectors is particularly interesting, because it leaves a

  15. Insecticide resistance in disease vectors from Mayotte: an opportunity for integrated vector management.

    PubMed

    Pocquet, Nicolas; Darriet, Frédéric; Zumbo, Betty; Milesi, Pascal; Thiria, Julien; Bernard, Vincent; Toty, Céline; Labbé, Pierrick; Chandre, Fabrice

    2014-07-01

    Mayotte, a small island in the Indian Ocean, has been affected for many years by vector-borne diseases. Malaria, Bancroftian filariasis, dengue, chikungunya and Rift Valley fever have circulated or still circulate on the island. They are all transmitted by Culicidae mosquitoes. To limit the impact of these diseases on human health, vector control has been implemented for more than 60 years on Mayotte. In this study, we assessed the resistance levels of four major vector species (Anopheles gambiae, Culex pipiens quinquefasciatus, Aedes aegypti and Aedes albopictus) to two types of insecticides: i) the locally currently-used insecticides (organophosphates, pyrethroids) and ii) alternative molecules that are promising for vector control and come from different insecticide families (bacterial toxins or insect growth regulators). When some resistance was found to one of these insecticides, we characterized the mechanisms involved. Larval and adult bioassays were used to evaluate the level of resistance. When resistance was found, we tested for the presence of metabolic resistance through detoxifying enzyme activity assays, or for target-site mutations through molecular identification of known resistance alleles. Resistance to currently-used insecticides varied greatly between the four vector species. While no resistance to any insecticides was found in the two Aedes species, bioassays confirmed multiple resistance in Cx. p. quinquefasciatus (temephos: ~ 20 fold and deltamethrin: only 10% mortality after 24 hours). In An. gambiae, resistance was scarce: only a moderate resistance to temephos was found (~5 fold). This resistance appears to be due only to carboxyl-esterase overexpression and not to target modification. Finally, and comfortingly, none of the four species showed resistance to any of the new insecticides. The low resistance observed in Mayotte's main disease vectors is particularly interesting, because it leaves a range of tools useable by vector control

  16. Rate determination from vector observations

    NASA Technical Reports Server (NTRS)

    Weiss, Jerold L.

    1993-01-01

    Vector observations are a common class of attitude data provided by a wide variety of attitude sensors. Attitude determination from vector observations is a well-understood process and numerous algorithms such as the TRIAD algorithm exist. These algorithms require measurement of the line of site (LOS) vector to reference objects and knowledge of the LOS directions in some predetermined reference frame. Once attitude is determined, it is a simple matter to synthesize vehicle rate using some form of lead-lag filter, and then, use it for vehicle stabilization. Many situations arise, however, in which rate knowledge is required but knowledge of the nominal LOS directions are not available. This paper presents two methods for determining spacecraft angular rates from vector observations without a priori knowledge of the vector directions. The first approach uses an extended Kalman filter with a spacecraft dynamic model and a kinematic model representing the motion of the observed LOS vectors. The second approach uses a 'differential' TRIAD algorithm to compute the incremental direction cosine matrix, from which vehicle rate is then derived.

  17. Will integrated surveillance systems for vectors and vector-borne diseases be the future of controlling vector-borne diseases? A practical example from China.

    PubMed

    Wu, Y; Ling, F; Hou, J; Guo, S; Wang, J; Gong, Z

    2016-07-01

    Vector-borne diseases are one of the world's major public health threats and annually responsible for 30-50% of deaths reported to the national notifiable disease system in China. To control vector-borne diseases, a unified, effective and economic surveillance system is urgently needed; all of the current surveillance systems in China waste resources and/or information. Here, we review some current surveillance systems and present a concept for an integrated surveillance system combining existing vector and vector-borne disease monitoring systems. The integrated surveillance system has been tested in pilot programmes in China and led to a 21·6% cost saving in rodent-borne disease surveillance. We share some experiences gained from these programmes.

  18. Tensor Calculus: Unlearning Vector Calculus

    ERIC Educational Resources Information Center

    Lee, Wha-Suck; Engelbrecht, Johann; Moller, Rita

    2018-01-01

    Tensor calculus is critical in the study of the vector calculus of the surface of a body. Indeed, tensor calculus is a natural step-up for vector calculus. This paper presents some pitfalls of a traditional course in vector calculus in transitioning to tensor calculus. We show how a deeper emphasis on traditional topics such as the Jacobian can…

  19. Project Physics Programmed Instruction, Vectors 2.

    ERIC Educational Resources Information Center

    Harvard Univ., Cambridge, MA. Harvard Project Physics.

    This is the second of a series of three programmed instruction booklets on vectors developed by Harvard Project Physics. It covers adding two or more vectors together, and finding a third vector that could be added to two given vectors to make a sum of zero. For other booklets in this series, see SE 015 549 and SE 015 551. (DT)

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

    PubMed

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

    2015-01-01

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

  1. An adaptive vector quantization scheme

    NASA Technical Reports Server (NTRS)

    Cheung, K.-M.

    1990-01-01

    Vector quantization is known to be an effective compression scheme to achieve a low bit rate so as to minimize communication channel bandwidth and also to reduce digital memory storage while maintaining the necessary fidelity of the data. However, the large number of computations required in vector quantizers has been a handicap in using vector quantization for low-rate source coding. An adaptive vector quantization algorithm is introduced that is inherently suitable for simple hardware implementation because it has a simple architecture. It allows fast encoding and decoding because it requires only addition and subtraction operations.

  2. Segmentation of discrete vector fields.

    PubMed

    Li, Hongyu; Chen, Wenbin; Shen, I-Fan

    2006-01-01

    In this paper, we propose an approach for 2D discrete vector field segmentation based on the Green function and normalized cut. The method is inspired by discrete Hodge Decomposition such that a discrete vector field can be broken down into three simpler components, namely, curl-free, divergence-free, and harmonic components. We show that the Green Function Method (GFM) can be used to approximate the curl-free and the divergence-free components to achieve our goal of the vector field segmentation. The final segmentation curves that represent the boundaries of the influence region of singularities are obtained from the optimal vector field segmentations. These curves are composed of piecewise smooth contours or streamlines. Our method is applicable to both linear and nonlinear discrete vector fields. Experiments show that the segmentations obtained using our approach essentially agree with human perceptual judgement.

  3. Vector Acoustics, Vector Sensors, and 3D Underwater Imaging

    NASA Astrophysics Data System (ADS)

    Lindwall, D.

    2007-12-01

    Vector acoustic data has two more dimensions of information than pressure data and may allow for 3D underwater imaging with much less data than with hydrophone data. The vector acoustic sensors measures the particle motions due to passing sound waves and, in conjunction with a collocated hydrophone, the direction of travel of the sound waves. When using a controlled source with known source and sensor locations, the reflection points of the sound field can be determined with a simple trigonometric calculation. I demonstrate this concept with an experiment that used an accelerometer based vector acoustic sensor in a water tank with a short-pulse source and passive scattering targets. The sensor consists of a three-axis accelerometer and a matched hydrophone. The sound source was a standard transducer driven by a short 7 kHz pulse. The sensor was suspended in a fixed location and the hydrophone was moved about the tank by a robotic arm to insonify the tank from many locations. Several floats were placed in the tank as acoustic targets at diagonal ranges of approximately one meter. The accelerometer data show the direct source wave as well as the target scattered waves and reflections from the nearby water surface, tank bottom and sides. Without resorting to the usual methods of seismic imaging, which in this case is only two dimensional and relied entirely on the use of a synthetic source aperture, the two targets, the tank walls, the tank bottom, and the water surface were imaged. A directional ambiguity inherent to vector sensors is removed by using collocated hydrophone data. Although this experiment was in a very simple environment, it suggests that 3-D seismic surveys may be achieved with vector sensors using the same logistics as a 2-D survey that uses conventional hydrophones. This work was supported by the Office of Naval Research, program element 61153N.

  4. Dengue Vectors and their Spatial Distribution

    PubMed Central

    Higa, Yukiko

    2011-01-01

    The distribution of dengue vectors, Ae. aegypti and Ae. albopictus, is affected by climatic factors. In addition, since their life cycles are well adapted to the human environment, environmental changes resulting from human activity such as urbanization exert a great impact on vector distribution. The different responses of Ae. aegypti and Ae albopictus to various environments result in a difference in spatial distribution along north-south and urban-rural gradients, and between the indoors and outdoors. In the north-south gradient, climate associated with survival is an important factor in spatial distribution. In the urban-rural gradient, different distribution reflects a difference in adult niches and is modified by geographic and human factors. The direct response of the two species to the environment around houses is related to different spatial distribution indoors and outdoors. Dengue viruses circulate mainly between human and vector mosquitoes, and the vector presence is a limiting factor of transmission. Therefore, spatial distribution of dengue vectors is a significant concern in the epidemiology of the disease. Current technologies such as GIS, satellite imagery and statistical models allow researchers to predict the spatial distribution of vectors in the changing environment. Although it is difficult to confirm the actual effect of environmental and climate changes on vector abundance and vector-borne diseases, environmental changes caused by humans and human behavioral changes due to climate change can be expected to exert an impact on dengue vectors. Longitudinal monitoring of dengue vectors and viruses is therefore necessary. PMID:22500133

  5. TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

    PubMed Central

    Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

    2012-01-01

    Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

  6. The Vector, Signal, and Image Processing Library (VSIPL): an Open Standard for Astronomical Data Processing

    NASA Astrophysics Data System (ADS)

    Kepner, J. V.; Janka, R. S.; Lebak, J.; Richards, M. A.

    1999-12-01

    The Vector/Signal/Image Processing Library (VSIPL) is a DARPA initiated effort made up of industry, government and academic representatives who have defined an industry standard API for vector, signal, and image processing primitives for real-time signal processing on high performance systems. VSIPL supports a wide range of data types (int, float, complex, ...) and layouts (vectors, matrices and tensors) and is ideal for astronomical data processing. The VSIPL API is intended to serve as an open, vendor-neutral, industry standard interface. The object-based VSIPL API abstracts the memory architecture of the underlying machine by using the concept of memory blocks and views. Early experiments with VSIPL code conversions have been carried out by the High Performance Computing Program team at the UCSD. Commercially, several major vendors of signal processors are actively developing implementations. VSIPL has also been explicitly required as part of a recent Rome Labs teraflop procurement. This poster presents the VSIPL API, its functionality and the status of various implementations.

  7. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

    PubMed

    Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

    2009-12-30

    Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements

  8. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

    PubMed Central

    2009-01-01

    Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T

  9. [Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].

    PubMed

    He, Zuoping; Luo, Peifang; Hu, Feihuan; Weng, Yunceng; Wang, Wenjing; Li, Chengyao

    2016-04-01

    To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis. Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis. The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells. The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.

  10. Non-coaxial superposition of vector vortex beams.

    PubMed

    Aadhi, A; Vaity, Pravin; Chithrabhanu, P; Reddy, Salla Gangi; Prabakar, Shashi; Singh, R P

    2016-02-10

    Vector vortex beams are classified into four types depending upon spatial variation in their polarization vector. We have generated all four of these types of vector vortex beams by using a modified polarization Sagnac interferometer with a vortex lens. Further, we have studied the non-coaxial superposition of two vector vortex beams. It is observed that the superposition of two vector vortex beams with same polarization singularity leads to a beam with another kind of polarization singularity in their interaction region. The results may be of importance in ultrahigh security of the polarization-encrypted data that utilizes vector vortex beams and multiple optical trapping with non-coaxial superposition of vector vortex beams. We verified our experimental results with theory.

  11. Fractal vector optical fields.

    PubMed

    Pan, Yue; Gao, Xu-Zhen; Cai, Meng-Qiang; Zhang, Guan-Lin; Li, Yongnan; Tu, Chenghou; Wang, Hui-Tian

    2016-07-15

    We introduce the concept of a fractal, which provides an alternative approach for flexibly engineering the optical fields and their focal fields. We propose, design, and create a new family of optical fields-fractal vector optical fields, which build a bridge between the fractal and vector optical fields. The fractal vector optical fields have polarization states exhibiting fractal geometry, and may also involve the phase and/or amplitude simultaneously. The results reveal that the focal fields exhibit self-similarity, and the hierarchy of the fractal has the "weeding" role. The fractal can be used to engineer the focal field.

  12. Reversible vector ratchets for skyrmion systems

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ma, Xiu; Reichhardt, Cynthia Jane Olson; Reichhardt, Charles

    In this paper, we show that ac driven skyrmions interacting with an asymmetric substrate provide a realization of a 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 clockwise or counterclockwise relative to the substrate asymmetry direction. Up to a full 360° rotation is possible for varied ac amplitudes or skyrmion densities. In contrast to overdamped systems, in which ratchet motion is alwaysmore » parallel to the substrate asymmetry direction, vector ratchets allow the ratchet motion to be in any direction relative to the substrate asymmetry. It is also possible to obtain a reversal in the direction of rotation of the vector ratchet, permitting the creation of a reversible vector ratchet. We examine vector ratchets for ac drives applied parallel or perpendicular to the substrate asymmetry direction, and show that reverse ratchet motion can be produced by collective effects. No reversals occur for an isolated skyrmion on an asymmetric substrate. Finally, since a vector ratchet can produce motion in any direction, it could represent a method for controlling skyrmion motion for spintronic applications.« less

  13. Reversible vector ratchets for skyrmion systems

    NASA Astrophysics Data System (ADS)

    Ma, X.; Reichhardt, C. J. Olson; Reichhardt, C.

    2017-03-01

    We show that ac driven skyrmions interacting with an asymmetric substrate provide a realization of a 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 clockwise or counterclockwise relative to the substrate asymmetry direction. Up to a full 360∘ rotation is possible for varied ac amplitudes or skyrmion densities. In contrast to overdamped systems, in which ratchet motion is always parallel to the substrate asymmetry direction, vector ratchets allow the ratchet motion to be in any direction relative to the substrate asymmetry. It is also possible to obtain a reversal in the direction of rotation of the vector ratchet, permitting the creation of a reversible vector ratchet. We examine vector ratchets for ac drives applied parallel or perpendicular to the substrate asymmetry direction, and show that reverse ratchet motion can be produced by collective effects. No reversals occur for an isolated skyrmion on an asymmetric substrate. Since a vector ratchet can produce motion in any direction, it could represent a method for controlling skyrmion motion for spintronic applications.

  14. Reversible vector ratchets for skyrmion systems

    DOE PAGES

    Ma, Xiu; Reichhardt, Cynthia Jane Olson; Reichhardt, Charles

    2017-03-03

    In this paper, we show that ac driven skyrmions interacting with an asymmetric substrate provide a realization of a 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 clockwise or counterclockwise relative to the substrate asymmetry direction. Up to a full 360° rotation is possible for varied ac amplitudes or skyrmion densities. In contrast to overdamped systems, in which ratchet motion is alwaysmore » parallel to the substrate asymmetry direction, vector ratchets allow the ratchet motion to be in any direction relative to the substrate asymmetry. It is also possible to obtain a reversal in the direction of rotation of the vector ratchet, permitting the creation of a reversible vector ratchet. We examine vector ratchets for ac drives applied parallel or perpendicular to the substrate asymmetry direction, and show that reverse ratchet motion can be produced by collective effects. No reversals occur for an isolated skyrmion on an asymmetric substrate. Finally, since a vector ratchet can produce motion in any direction, it could represent a method for controlling skyrmion motion for spintronic applications.« less

  15. Correlated Topic Vector for Scene Classification.

    PubMed

    Wei, Pengxu; Qin, Fei; Wan, Fang; Zhu, Yi; Jiao, Jianbin; Ye, Qixiang

    2017-07-01

    Scene images usually involve semantic correlations, particularly when considering large-scale image data sets. This paper proposes a novel generative image representation, correlated topic vector, to model such semantic correlations. Oriented from the correlated topic model, correlated topic vector intends to naturally utilize the correlations among topics, which are seldom considered in the conventional feature encoding, e.g., Fisher vector, but do exist in scene images. It is expected that the involvement of correlations can increase the discriminative capability of the learned generative model and consequently improve the recognition accuracy. Incorporated with the Fisher kernel method, correlated topic vector inherits the advantages of Fisher vector. The contributions to the topics of visual words have been further employed by incorporating the Fisher kernel framework to indicate the differences among scenes. Combined with the deep convolutional neural network (CNN) features and Gibbs sampling solution, correlated topic vector shows great potential when processing large-scale and complex scene image data sets. Experiments on two scene image data sets demonstrate that correlated topic vector improves significantly the deep CNN features, and outperforms existing Fisher kernel-based features.

  16. Focusing properties of cylindrical vector vortex beams

    NASA Astrophysics Data System (ADS)

    Xiaoqiang, Zhang; Ruishan, Chen; Anting, Wang

    2018-05-01

    In this paper, following Richards and Wolf vectorial diffraction theory, the focusing properties of cylindrical vector vortex beams (CVVB) are investigated, and a diffractive optical element (DOE) is designed to spatially modulate the amplitude of the CVVB. Simulated results show that the CVVB focused by an objective also carry orbital angular momentum (OAM), and the optical fields near the focal region can be modulated by changing the topological charge of the CVVB. We numerically simulate the focus properties of radially and azimuthally polarized beams with topological charge equal to 0, 1, 2 and 10 respectively. As a result, a dark channel with a length about 20 λ can be obtained. These new properties have the potential applications such as particle acceleration, optical trapping and material processing.

  17. Test of Understanding of Vectors: A Reliable Multiple-Choice Vector Concept Test

    ERIC Educational Resources Information Center

    Barniol, Pablo; Zavala, Genaro

    2014-01-01

    In this article we discuss the findings of our research on students' understanding of vector concepts in problems without physical context. First, we develop a complete taxonomy of the most frequent errors made by university students when learning vector concepts. This study is based on the results of several test administrations of open-ended…

  18. Stimulated scattering of electromagnetic waves carrying orbital angular momentum in quantum plasmas.

    PubMed

    Shukla, P K; Eliasson, B; Stenflo, L

    2012-07-01

    We investigate stimulated scattering instabilities of coherent circularly polarized electromagnetic (CPEM) waves carrying orbital angular momentum (OAM) in dense quantum plasmas with degenerate electrons and nondegenerate ions. For this purpose, we employ the coupled equations for the CPEM wave vector potential and the driven (by the ponderomotive force of the CPEM waves) equations for the electron and ion plasma oscillations. The electrons are significantly affected by the quantum forces (viz., the quantum statistical pressure, the quantum Bohm potential, as well as the electron exchange and electron correlations due to electron spin), which are included in the framework of the quantum hydrodynamical description of the electrons. Furthermore, our investigation of the stimulated Brillouin instability of coherent CPEM waves uses the generalized ion momentum equation that includes strong ion coupling effects. The nonlinear equations for the coupled CPEM and quantum plasma waves are then analyzed to obtain nonlinear dispersion relations which exhibit stimulated Raman, stimulated Brillouin, and modulational instabilities of CPEM waves carrying OAM. The present results are useful for understanding the origin of scattered light off low-frequency density fluctuations in high-energy density plasmas where quantum effects are eminent.

  19. Evidence for carry-over effects of predator exposure on pathogen transmission potential

    PubMed Central

    Roux, Olivier; Vantaux, Amélie; Roche, Benjamin; Yameogo, Koudraogo B.; Dabiré, Kounbobr R.; Diabaté, Abdoulaye; Simard, Frederic; Lefèvre, Thierry

    2015-01-01

    Accumulating evidence indicates that species interactions such as competition and predation can indirectly alter interactions with other community members, including parasites. For example, presence of predators can induce behavioural defences in the prey, resulting in a change in susceptibility to parasites. Such predator-induced phenotypic changes may be especially pervasive in prey with discrete larval and adult stages, for which exposure to predators during larval development can have strong carry-over effects on adult phenotypes. To the best of our knowledge, no study to date has examined possible carry-over effects of predator exposure on pathogen transmission. We addressed this question using a natural food web consisting of the human malaria parasite Plasmodium falciparum, the mosquito vector Anopheles coluzzii and a backswimmer, an aquatic predator of mosquito larvae. Although predator exposure did not significantly alter mosquito susceptibility to P. falciparum, it incurred strong fitness costs on other key mosquito life-history traits, including larval development, adult size, fecundity and longevity. Using an epidemiological model, we show that larval predator exposure should overall significantly decrease malaria transmission. These results highlight the importance of taking into account the effect of environmental stressors on disease ecology and epidemiology. PMID:26674956

  20. Extraction of inland Nypa fruticans (Nipa Palm) using Support Vector Machine

    NASA Astrophysics Data System (ADS)

    Alberto, R. T.; Serrano, S. C.; Damian, G. B.; Camaso, E. E.; Biagtan, A. R.; Panuyas, N. Z.; Quibuyen, J. S.

    2017-09-01

    Mangroves are considered as one of the major habitats in coastal ecosystem, providing a lot of economic and ecological services in human society. Nypa fruticans (Nipa palm) is one of the important species of mangroves because of its versatility and uniqueness as halophytic palm. However, nipas are not only adaptable in saline areas, they can also managed to thrive away from the coastline depending on the favorable soil types available in the area. Because of this, mapping of this species are not limited alone in the near shore areas, but in areas where this species are present as well. The extraction process of Nypa fruticans were carried out using the available LiDAR data. Support Vector Machine (SVM) classification process was used to extract nipas in inland areas. The SVM classification process in mapping Nypa fruticans produced high accuracy of 95+%. The Support Vector Machine classification process to extract inland nipas was proven to be effective by utilizing different terrain derivatives from LiDAR data.

  1. Generation of a neurodegenerative disease mouse model using lentiviral vectors carrying an enhanced synapsin I promoter.

    PubMed

    Matsuzaki, Yasunori; Oue, Miho; Hirai, Hirokazu

    2014-02-15

    Certain inherited progressive neurodegenerative disorders, such as spinocerebellar ataxia (SCA), affect neurons in large areas of the central nervous system (CNS). The selective expression of disease-causing and therapeutic genes in susceptible regions and cell types is critical for the generation of animal models and development of gene therapies for these diseases. Previous studies have demonstrated the advantages of the short synapsin I (SynI) promoter (0.5 kb) as a neuron-specific promoter for robust transgene expression. However, the short SynI promoter has also shown some promoter activity in glia and a lack of transgene expression in significant areas of the CNS. New methods: To improve the SynI promoter, we used a SynI promoter that is twice as long (1.0 kb) as the short SynI promoter and incorporated a minimal CMV (minCMV) sequence. We observed that the 1.0 kb rat SynI promoter with minCMV [rSynI(1.0)-minCMV] exhibited robust promoter strength, excellent neuronal specificity and wide-ranging transgene expression throughout the CNS. Comparison with existing methods: Compared with the two previously reported short (0.5 kb) promoters, the new promoter was superior with respect to neuronal specificity and more efficiently transduced neurons. Moreover, transgenic mice expressing the mutant protein ATXN1[Q98], which causes SCA type 1 (SCA1), under the control of the rSynI(1.0)-minCMV promoter showed robust transgene expression specifically in neurons throughout the CNS and exhibited progressive ataxia. rSynI(1.0)-minCMV drives robust and neuron-specific transgene expression throughout the CNS and is therefore useful for viral vector-mediated neuron-specific gene delivery and generation of neuron-specific transgenic animals. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Vector-Vector Scattering on the Lattice

    NASA Astrophysics Data System (ADS)

    Romero-López, Fernando; Urbach, Carsten; Rusetsky, Akaki

    2018-03-01

    In this work we present an extension of the LüScher formalism to include the interaction of particles with spin, focusing on the scattering of two vector particles. The derived formalism will be applied to Scalar QED in the Higgs Phase, where the U(1) gauge boson acquires mass.

  3. A generalized nonlocal vector calculus

    NASA Astrophysics Data System (ADS)

    Alali, Bacim; Liu, Kuo; Gunzburger, Max

    2015-10-01

    A nonlocal vector calculus was introduced in Du et al. (Math Model Meth Appl Sci 23:493-540, 2013) that has proved useful for the analysis of the peridynamics model of nonlocal mechanics and nonlocal diffusion models. A formulation is developed that provides a more general setting for the nonlocal vector calculus that is independent of particular nonlocal models. It is shown that general nonlocal calculus operators are integral operators with specific integral kernels. General nonlocal calculus properties are developed, including nonlocal integration by parts formula and Green's identities. The nonlocal vector calculus introduced in Du et al. (Math Model Meth Appl Sci 23:493-540, 2013) is shown to be recoverable from the general formulation as a special example. This special nonlocal vector calculus is used to reformulate the peridynamics equation of motion in terms of the nonlocal gradient operator and its adjoint. A new example of nonlocal vector calculus operators is introduced, which shows the potential use of the general formulation for general nonlocal models.

  4. Potential of Gene Therapy for the Treatment of Pituitary Tumors

    PubMed Central

    Goya, R G.; Sarkar, D.K.; Brown, O.A.; Hereñú, C.B.

    2010-01-01

    Pituitary adenomas constitute the most frequent neuroendocrine pathology, comprising up to 15% of primary intracranial tumors. Current therapies for pituitary tumors include surgery and radiotherapy, as well as pharmacological approaches for some types. Although all of these approaches have shown a significant degree of success, they are not devoid of unwanted side effects, and in most cases do not offer a permanent cure. Gene therapy—the transfer of genetic material for therapeutic purposes—has undergone an explosive development in the last few years. Within this context, the development of gene therapy approaches for the treatment of pituitary tumors emerges as a promising area of research. We begin by presenting a brief account of the genesis of prolactinomas, with particular emphasis on how estradiol induces prolactinomas in animals. In so doing, we discuss the role of each of the recently discovered growth inhibitory and growth stimulatory substances and their interactions in estrogen action. We also evaluate the cell-cell communication that may govern these growth factor interactions and subsequently promote the growth and survival of prolactinomas. Current research efforts to implement gene therapy in pituitary tumors include the treatment of experimental prolactinomas or somatomammotropic tumors with adenoviral vector-mediated transfer of the suicide gene for the herpes simplex type 1 (HSV1) thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. In some cases, the suicide transgene has been placed under the control of pituitary cell-type specific promoters, like the human prolactin or human growth hormone promoters. Also, regulatable adenoviral vector systems are being assessed in gene therapy approaches for experimental pituitary tumors. In a different type of approach, an adenoviral vector, encoding the human retinoblastoma suppressor oncogene, has been successfully used to rescue the phenotype of spontaneous pituitary

  5. Poynting-vector filter

    DOEpatents

    Carrigan, Charles R [Tracy, CA

    2011-08-02

    A determination is made of frequency components associated with a particular bearing or location resulting from sources emitting electromagnetic-wave energy for which a Poynting-Vector can be defined. The broadband frequency components associated with a specific direction or location of interest are isolated from other components in the power spectrum that are not associated with the direction or location of interest. The collection of pointing vectors can be used to characterize the source.

  6. [Effects of plant viruses on vector and non-vector herbivorous arthropods and their natural enemies: a mini review].

    PubMed

    He, Xiao-Chan; Xu, Hong-Xing; Zhou, Xiao-Jun; Zheng, Xu-Song; Sun, Yu-Jian; Yang, Ya-Jun; Tian, Jun-Ce; Lü, Zhong-Xian

    2014-05-01

    Plant viruses transmitted by arthropods, as an important biotic factor, may not only directly affect the yield and quality of host plants, and development, physiological characteristics and ecological performances of their vector arthropods, but also directly or indirectly affect the non-vector herbivorous arthropods and their natural enemies in the same ecosystem, thereby causing influences to the whole agro-ecosystem. This paper reviewed the progress on the effects of plant viruses on herbivorous arthropods, including vector and non-vector, and their natural enemies, and on their ecological mechanisms to provide a reference for optimizing the management of vector and non-vector arthropod populations and sustainable control of plant viruses in agro-ecosystem.

  7. Improved exercise capacity and reduced systemic inflammation after adenoviral-mediated SERCA-2a gene transfer.

    PubMed

    Gupta, Dipin; Palma, Jon; Molina, Ezequiel; Gaughan, John P; Long, Walter; Houser, Steven; Macha, Mahender

    2008-04-01

    We hypothesized that sarcoplasmic reticulum Ca2+ ATPase pump (SERCA-2a) gene delivery would have beneficial effects upon exercise capacity and markers of inflammation in the setting of heart failure. A pressure-overload model of experimental heart failure was used in rats. Following a decrease in fractional shortening of >or=25%, animals underwent intracoronary adenoviral-mediated gene transfection using SERCA-2a. Heart failure animals were randomized to receive the SERCA-2a gene, the beta galactosidase (control) gene, or followed without any further intervention. Exercise and hemodynamic testing were performed, and myocardial and systemic markers of inflammation were assayed after 7 and 21 d. Animals receiving Ad.SERCA-2a showed an increase in exercise tolerance (499.0 +/- 14.9 versus 312.8 +/- 10.5 s, P < 0.0001) relative to Ad.Gal group. Groups treated with Ad.SERCA-2a had significantly decreased serum levels of the inflammatory markers interleukin-1, interleukin-6, and tumor necrosis factor-alpha compared with Ad.Gal-treated animals. Serum levels of atrial natriuretic peptide were decreased in animals receiving Ad.SERCA-2a compared with animals receiving Ad.Gal at day 7 (0.35 +/- 0.03 versus 0.52 +/- 0.11 pg/mL, P = 0.001). Myocardial levels of the proapoptotic protein bax were reduced in Ad.SERCA-2a -treated animals compared with those receiving Ad.Gal at day 7 (protein level/actin: 0.24 +/- 0.05 versus 0.33 +/- 0.04, P = 0.04) and day 21 (protein level/actin: 0.61 +/- 0.04 versus 0.69 +/- 0.01, P = 0.001). Genetic modulation of heart failure using the SERCA-2a gene was associated with improvement in cardiac function and exercise capacity as well as improvements in heart-failure associated inflammatory markers.

  8. Virus-Vectored Influenza Virus Vaccines

    PubMed Central

    Tripp, Ralph A.; Tompkins, S. Mark

    2014-01-01

    Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. PMID:25105278

  9. Tetrahedral DNA Nanoparticle Vector for Intracellular Delivery of Targeted Peptide Nucleic Acid Antisense Agents to Restore Antibiotic Sensitivity in Cefotaxime-Resistant Escherichia coli.

    PubMed

    Readman, John Benedict; Dickson, George; Coldham, Nick G

    2017-06-01

    The bacterial cell wall presents a barrier to the uptake of unmodified synthetic antisense oligonucleotides, such as peptide nucleic acids, and so is one of the greatest obstacles to the development of their use as therapeutic anti-bacterial agents. Cell-penetrating peptides have been covalently attached to antisense agents, to facilitate penetration of the bacterial cell wall and deliver their cargo into the cytoplasm. Although they are an effective vector for antisense oligonucleotides, they are not specific for bacterial cells and can exhibit growth inhibitory properties at higher doses. Using a bacterial cell growth assay in the presence of cefotaxime (CTX 16 mg/L), we have developed and evaluated a self-assembling non-toxic DNA tetrahedron nanoparticle vector incorporating a targeted anti-bla CTX-M-group 1 antisense peptide nucleic acid (PNA4) in its structure for penetration of the bacterial cell wall. A dose-dependent CTX potentiating effect was observed when PNA4 (0-40 μM) was incorporated into the structure of a DNA tetrahedron vector. The minimum inhibitory concentration (to CTX) of an Escherichia coli field isolate harboring a plasmid carrying bla CTX-M-3 was reduced from 35 to 16 mg/L in the presence of PNA4 carried by the DNA tetrahedron vector (40 μM), contrasting with no reduction in MIC in the presence of PNA4 alone. No growth inhibitory effects of the DNA tetrahedron vector alone were observed.

  10. The poultry red mite (Dermanyssus gallinae): a potential vector of pathogenic agents.

    PubMed

    Valiente Moro, Claire; De Luna, Carlos J; Tod, Alexander; Guy, Jonathan H; Sparagano, Olivier A E; Zenner, Lionel

    2009-06-01

    The poultry red mite, D. gallinae has been involved in the transmission of many pathogenic agents, responsible for serious diseases both in animals and humans. Nowadays, few effective methods are available to control the ectoparasite in poultry farms. Consequently, this is an emerging problem which must be taken into account to maintain good health in commercial egg production. This paper addresses the vector capacity of the ectoparasite with special emphasis on salmonellae, pathogenic agents responsible for many of the most important outbreaks of food-borne diseases worlwide. It has been experimentally shown that D. gallinae could act as a biological vector of S. enteritidis and natural carriage of these bacteria by the mite on poultry premises has also been reported. It was also found that D. gallinae carried other pathogens such as E. coli, Shigella sp., and Staphylococcus, thus increasing the list of pathogenic agents potentially transmitted by the mite.

  11. Vector SIMP dark matter

    NASA Astrophysics Data System (ADS)

    Choi, Soo-Min; Hochberg, Yonit; Kuflik, Eric; Lee, Hyun Min; Mambrini, Yann; Murayama, Hitoshi; Pierre, Mathias

    2017-10-01

    Strongly Interacting Massive Particles (SIMPs) have recently been proposed as light thermal dark matter relics. Here we consider an explicit realization of the SIMP mechanism in the form of vector SIMPs arising from an SU(2) X hidden gauge theory, where the accidental custodial symmetry protects the stability of the dark matter. We propose several ways of equilibrating the dark and visible sectors in this setup. In particular, we show that a light dark Higgs portal can maintain thermal equilibrium between the two sectors, as can a massive dark vector portal with its generalized Chern-Simons couplings to the vector SIMPs, all while remaining consistent with experimental constraints.

  12. A Model Framework to Estimate Impact and Cost of Genetics-Based Sterile Insect Methods for Dengue Vector Control

    PubMed Central

    Alphey, Nina; Alphey, Luke; Bonsall, Michael B.

    2011-01-01

    Vector-borne diseases impose enormous health and economic burdens and additional methods to control vector populations are clearly needed. The Sterile Insect Technique (SIT) has been successful against agricultural pests, but is not in large-scale use for suppressing or eliminating mosquito populations. Genetic RIDL technology (Release of Insects carrying a Dominant Lethal) is a proposed modification that involves releasing insects that are homozygous for a repressible dominant lethal genetic construct rather than being sterilized by irradiation, and could potentially overcome some technical difficulties with the conventional SIT technology. Using the arboviral disease dengue as an example, we combine vector population dynamics and epidemiological models to explore the effect of a program of RIDL releases on disease transmission. We use these to derive a preliminary estimate of the potential cost-effectiveness of vector control by applying estimates of the costs of SIT. We predict that this genetic control strategy could eliminate dengue rapidly from a human community, and at lower expense (approximately US$ 2∼30 per case averted) than the direct and indirect costs of disease (mean US$ 86–190 per case of dengue). The theoretical framework has wider potential use; by appropriately adapting or replacing each component of the framework (entomological, epidemiological, vector control bio-economics and health economics), it could be applied to other vector-borne diseases or vector control strategies and extended to include other health interventions. PMID:21998654

  13. Poly ICLC increases the potency of a replication-defective human adenovirus vectored foot-and-mouth disease vaccine

    USDA-ARS?s Scientific Manuscript database

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have previously demonstrated that a replication-defective human adenovirus 5 vector carrying the FMDV capsid coding region of serotype A24 Cruzeiro (Ad5-CI-A24-2B) protects swine and cattle against FM...

  14. Camel as a transboundary vector for emerging exotic Salmonella serovars.

    PubMed

    Ghoneim, Nahed H; Abdel-Moein, Khaled A; Zaher, Hala

    2017-05-01

    The current study was conducted to shed light on the role of imported camels as a transboundary vector for emerging exotic Salmonella serovars. Fecal samples were collected from 206 camels directly after slaughtering including 25 local camels and 181 imported ones as well as stool specimens were obtained from 50 slaughterhouse workers at the same abattoir. The obtained samples were cultured while Salmonella serovars were identified through Gram's stain films, biochemical tests and serotyping with antisera kit. Moreover, the obtained Salmonella serovars were examined by PCR for the presence of invA and stn genes. The overall prevalence of Salmonella serovars among the examined camels was 8.3%. Stn gene was detected in the vast majority of exotic strains (11/14) 78.6% including emerging serovars such as Salmonella Saintpaul, S. Chester, S. Typhimurium whereas only one isolate from local camels carried stn gene (1/3) 33.3%. On the other hand, none of the examined humans yielded positive result. Our findings highlight the potential role of imported camels as a transboundary vector for exotic emerging Salomenella serovars.

  15. Attitude Determination Using Two Vector Measurements

    NASA Technical Reports Server (NTRS)

    Markley, F. Landis

    1998-01-01

    Many spacecraft attitude determination methods use exactly two vector measurements. The two vectors are typically the unit vector to the Sun and the Earth's magnetic field vector for coarse "sun-mag" attitude determination or unit vectors to two stars tracked by two star trackers for fine attitude determination. TRIAD, the earliest published algorithm for determining spacecraft attitude from two vector measurements, has been widely used in both ground-based and onboard attitude determination. Later attitude determination methods have been based on Wahba's optimality criterion for n arbitrarily weighted observations. The solution of Wahba's problem is somewhat difficult in the general case, but there is a simple closed-form solution in the two-observation case. This solution reduces to the TRIAD solution for certain choices of measurement weights. This paper presents and compares these algorithms as well as sub-optimal algorithms proposed by Bar-Itzhack, Harman, and Reynolds. Some new results will be presented, but the paper is primarily a review and tutorial.

  16. [New strategy for RNA vectorization in mammalian cells. Use of a peptide vector].

    PubMed

    Vidal, P; Morris, M C; Chaloin, L; Heitz, F; Divita, G

    1997-04-01

    A major barrier for gene delivery is the low permeability of nucleic acids to cellular membranes. The development of antisenses and gene therapy has focused mainly on improving methods of oligonucleotide or gene delivery to the cell. In this report we described a new strategy for RNA cell delivery, based on a short single peptide. This peptide vector is derived from both the fusion domain of the gp41 protein of HIV and the nuclear localization sequence of the SV40 large T antigen. This peptide vector localizes rapidly to the cytoplasm then to the nucleus of human fibroblasts (HS-68) within a few minutes and exhibits a high affinity for a single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase (in a 100 nM range). The peptide/RNA complex formation involves mainly electrostatic interactions between the basic residues of the peptide and the charges on the phosphate group of the RNA. In the presence of the peptide-vector fluorescently-labelled mRNA is delivered into the cytoplasm of mammalian cells (HS68 human fibroblasts) in less than 1 h with a relatively high efficiency (80%). This new concept based on a peptide-derived vector offers several advantages compared to other compounds commonly used in gene delivery. This vector is highly soluble and exhibits no cytotoxicity at the concentrations used for optimal gene delivery. This result clearly supports the fact that this peptide vector is a powerful tool and that it can be used widely, as much for laboratory research as for new applications and development in gene and/or antisense therapy.

  17. Adenoviral Gene Transfer of Hepatic Stimulator Substance Confers Resistance Against Hepatic Ischemia–Reperfusion Injury by Improving Mitochondrial Function

    PubMed Central

    Jiang, Shu-Jun; Li, Wen

    2013-01-01

    Abstract Hepatic stimulator substance (HSS) has been suggested to protect liver cells from various toxins. However, the precise role of HSS in hepatic ischemia–reperfusion (I/R) injury remains unknown. This study aims to elucidate whether overexpression of HSS could attenuate hepatic ischemia–reperfusion injury and its possible mechanisms. Both in vivo hepatic I/R injury in mice and in vitro hypoxia–reoxygenation (H/R) in a cell model were used to evaluate the effect of HSS protection after adenoviral gene transfer. Moreover, a possible mitochondrial mechanism of HSS protection was investigated. Efficient transfer of the HSS gene into liver inhibited hepatic I/R injury in mice, as evidenced by improvement in liver function tests, the preservation of hepatic morphology, and a reduction in hepatocyte apoptosis. HSS overexpression also inhibited H/R-induced cell death, as detected by cell viability and cell apoptosis assays. The underlying mechanism of this hepatic protection might involve the attenuation of mitochondrial dysfunction and mitochondrial-dependent cell apoptosis, as shown by the good preservation of mitochondrial ultrastructure, mitochondrial membrane potential, and the inhibition of cytochrome c leakage and caspase activity. Moreover, the suppression of H/R-induced mitochondrial ROS production and the maintenance of mitochondrial respiratory chain complex activities may participate in this mechanism. This new function of HSS expands the possibility of its application for the prevention of I/R injury, such as hepatic resection and liver transplantation in clinical practice. PMID:23461564

  18. Viral vector-based influenza vaccines.

    PubMed

    de Vries, Rory D; Rimmelzwaan, Guus F

    2016-11-01

    Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors.

  19. Project Physics Programmed Instruction, Vectors 3.

    ERIC Educational Resources Information Center

    Harvard Univ., Cambridge, MA. Harvard Project Physics.

    This is the third of a series of three programmed instruction booklets on vectors developed by Harvard Project Physics. Separating vectors into components and obtaining a vector from its components are the topics covered. For other booklets in this series, see SE 015 549 and SE 015 550. (DT)

  20. Vectors of Crimean Congo Hemorrhagic Fever Virus in Iran

    PubMed Central

    Telmadarraiy, Zakkyeh; Chinikar, Sadegh; Vatandoost, Hassan; Faghihi, Faezeh; Hosseini-Chegeni, Asadollah

    2015-01-01

    Background: Ticks are important vectors and reservoirs of Crimean Congo Hemorrhagic Fever (CCHF) virus. Human beings may be infected whenever the normal life cycle of the infected ticks on non-human vertebrate hosts is interrupted by the undesirable presence of humans in the cycle. A total of 26 species of Argasid and Ixodid ticks have been recorded in Iran; including nine Hyalomma, two Rhipicephalus, two Dermacentor, five Haemaphysalis, two Boophilus, one Ixodes and two Argas as well as three Ornithodoros species as blood sucking ectoparasites of livestock and poultries. The present paper reviews tick vectors of CCHF virus in Iran, focusing on the role of ticks in different provinces of Iran using reverse transcription polymerase chain reaction (RT-PCR) assay. Methods: During ten years study, 1054 tick specimens; including two species of Argasidae and 17 species of Ixodidae were examined for their infection to CCHF virus genome. The output of all studies as well as related publications were discussed in the current paper. Results: The results show that Rhipicephalus sanguineus, Hyalomma marginatum, H. anatolicum, H. asiaticum and H. dromedarii were known as the most frequent species which were positive for CCHF virus. Conclusion: The status of ticks which were positive for CCHF virus revealed that unlike the most common idea that Hyalomma species are the most important vectors of CCHF virus, other ticks including Rhipicephalus, Haemaphysalis and Dermacentor can be reservoir of this virus; thus, considering geographical distribution, type of host and environmental conditions, different tick control measurements should be carried out in areas with high incidence of CCHF disease. PMID:26623426

  1. VectorBase: an updated bioinformatics resource for invertebrate vectors and other organisms related with human diseases

    PubMed Central

    Giraldo-Calderón, Gloria I.; Emrich, Scott J.; MacCallum, Robert M.; Maslen, Gareth; Dialynas, Emmanuel; Topalis, Pantelis; Ho, Nicholas; Gesing, Sandra; Madey, Gregory; Collins, Frank H.; Lawson, Daniel

    2015-01-01

    VectorBase is a National Institute of Allergy and Infectious Diseases supported Bioinformatics Resource Center (BRC) for invertebrate vectors of human pathogens. Now in its 11th year, VectorBase currently hosts the genomes of 35 organisms including a number of non-vectors for comparative analysis. Hosted data range from genome assemblies with annotated gene features, transcript and protein expression data to population genetics including variation and insecticide-resistance phenotypes. Here we describe improvements to our resource and the set of tools available for interrogating and accessing BRC data including the integration of Web Apollo to facilitate community annotation and providing Galaxy to support user-based workflows. VectorBase also actively supports our community through hands-on workshops and online tutorials. All information and data are freely available from our website at https://www.vectorbase.org/. PMID:25510499

  2. Clifford support vector machines for classification, regression, and recurrence.

    PubMed

    Bayro-Corrochano, Eduardo Jose; Arana-Daniel, Nancy

    2010-11-01

    This paper introduces the Clifford support vector machines (CSVM) as a generalization of the real and complex-valued support vector machines using the Clifford geometric algebra. In this framework, we handle the design of kernels involving the Clifford or geometric product. In this approach, one redefines the optimization variables as multivectors. This allows us to have a multivector as output. Therefore, we can represent multiple classes according to the dimension of the geometric algebra in which we work. We show that one can apply CSVM for classification and regression and also to build a recurrent CSVM. The CSVM is an attractive approach for the multiple input multiple output processing of high-dimensional geometric entities. We carried out comparisons between CSVM and the current approaches to solve multiclass classification and regression. We also study the performance of the recurrent CSVM with experiments involving time series. The authors believe that this paper can be of great use for researchers and practitioners interested in multiclass hypercomplex computing, particularly for applications in complex and quaternion signal and image processing, satellite control, neurocomputation, pattern recognition, computer vision, augmented virtual reality, robotics, and humanoids.

  3. Production of non viral DNA vectors.

    PubMed

    Schleef, Martin; Blaesen, Markus; Schmeer, Marco; Baier, Ruth; Marie, Corinne; Dickson, George; Scherman, Daniel

    2010-12-01

    After some decades of research, development and first clinical approaches to use DNA vectors in gene therapy, cell therapy and DNA vaccination, the requirements for the pharmaceutical manufacturing of gene vectors has improved significantly step by step. Even the expression level and specificity of non viral DNA vectors were significantly modified and followed the success of viral vectors. The strict separation of "viral" and "non viral" gene transfer are historic borders between scientist and we will show that both fields together are able to allow the next step towards successful prevention and therapy. Here we summarize the features of producing and modifying these non-viral gene vectors to ensure the required quality to modify cells and to treat human and animals.

  4. A Subdivision-Based Representation for Vector Image Editing.

    PubMed

    Liao, Zicheng; Hoppe, Hugues; Forsyth, David; Yu, Yizhou

    2012-11-01

    Vector graphics has been employed in a wide variety of applications due to its scalability and editability. Editability is a high priority for artists and designers who wish to produce vector-based graphical content with user interaction. In this paper, we introduce a new vector image representation based on piecewise smooth subdivision surfaces, which is a simple, unified and flexible framework that supports a variety of operations, including shape editing, color editing, image stylization, and vector image processing. These operations effectively create novel vector graphics by reusing and altering existing image vectorization results. Because image vectorization yields an abstraction of the original raster image, controlling the level of detail of this abstraction is highly desirable. To this end, we design a feature-oriented vector image pyramid that offers multiple levels of abstraction simultaneously. Our new vector image representation can be rasterized efficiently using GPU-accelerated subdivision. Experiments indicate that our vector image representation achieves high visual quality and better supports editing operations than existing representations.

  5. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  6. [Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

    PubMed

    Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

    2015-09-01

    For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

  7. Increasing the Efficacy of Oncolytic Adenovirus Vectors

    PubMed Central

    Toth, Karoly; Wold, William S. M.

    2010-01-01

    Oncolytic adenovirus (Ad) vectors present a new modality to treat cancer. These vectors attack tumors via replicating in and killing cancer cells. Upon completion of the vector replication cycle, the infected tumor cell lyses and releases progeny virions that are capable of infecting neighboring tumor cells. Repeated cycles of vector replication and cell lysis can destroy the tumor. Numerous Ad vectors have been generated and tested, some of them reaching human clinical trials. In 2005, the first oncolytic Ad was approved for the treatment of head-and-neck cancer by the Chinese FDA. Oncolytic Ads have been proven to be safe, with no serious adverse effects reported even when high doses of the vector were injected intravenously. The vectors demonstrated modest anti-tumor effect when applied as a single agent; their efficacy improved when they were combined with another modality. The efficacy of oncolytic Ads can be improved using various approaches, including vector design, delivery techniques, and ancillary treatment, which will be discussed in this review. PMID:21994711

  8. Bacteriophage-based vectors for site-specific insertion of DNA in the chromosome of Corynebacteria.

    PubMed

    Oram, Mark; Woolston, Joelle E; Jacobson, Andrew D; Holmes, Randall K; Oram, Diana M

    2007-04-15

    In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as beta. beta-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally encoded genes, is regulated by the DtxR protein in response to Fe(2+) levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the beta-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae DeltadtxR strain. Additionally, strains of beta-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for beta, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species.

  9. Bacteriophage-based Vectors for Site-specific Insertion of DNA in the Chromosome of Corynebacteria

    PubMed Central

    Oram, Mark; Woolston, Joelle E.; Jacobson, Andrew D.; Holmes, Randall K.; Oram, Diana M.

    2007-01-01

    In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as β. β-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally-encoded genes, is regulated by the DtxR protein in response to Fe2+ levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the β-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae ΔdtxR strain. Additionally, strains of β-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for β, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species. PMID:17275217

  10. Using rabies virus vaccine strain SRV9 as viral vector to express exogenous gene.

    PubMed

    Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Li, Ling; Qi, Yinglin; Liang, Meng; Zhao, Yongkun; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Jin, Ningyi; Yang, Songtao; Xia, Xianzhu

    2015-04-01

    Rabies virus (RABV) can cause a fatal neurological disease in human and animals, and vaccines were generally applied for the immunoprophylaxis of rabies. Here, a recombinant viral vector carrying the exogenous gene expression component between phosphoprotein (P) and matrix protein (M) genes of RABV was constructed based on the vaccine strain SRV9 used in China. To develop a reverse genetic system, the full-length cDNA plasmids of SRV9 were constructed using the eukaryotic expression vector pCI or pcDNA3.1(+). However, recovery efficiency based on the pcDNA3.1 vector was significantly higher than that of the pCI vector. The exogenous gene expression component PE-PS-BsiWI-PmeI or PS-BsiWI-PmeI-PE was introduced in different locations between the P and M genes of SRV9. When the enhanced green fluorescent protein (eGFP) was used as a reporter gene, both locations could rescue recombinant RABV (rRABV) expressing eGFP with high efficiency. Characterization of rRABV expressing eGFP in vitro revealed that its growth was similar to that of the parental virus. Animal experiments showed that rRABV expressing eGFP could replicate and express eGFP in the brains of suckling mice. Furthermore, rRABV of SRV9 was nonpathogenic for 3-week-old mice and could be cleared from the central nervous system at 5 days post-inoculation. Our results showed that the recombinant SRV9 virus could be used as a useful viral vector for exogenous gene expression.

  11. Production of lentiviral vectors

    PubMed Central

    Merten, Otto-Wilhelm; Hebben, Matthias; Bovolenta, Chiara

    2016-01-01

    Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented. PMID:27110581

  12. Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells.

    PubMed

    Higashimoto, Yuji; Elliott, W Mark; Behzad, Ali R; Sedgwick, Edward G; Takei, Tatsuo; Hogg, James C; Hayashi, Shizu

    2002-07-15

    Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.

  13. Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

    PubMed

    Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

    2010-01-01

    The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

  14. Vector SIMP dark matter

    DOE PAGES

    Choi, Soo -Min; Hochberg, Yonit; Kuflik, Eric; ...

    2017-10-24

    Strongly Interacting Massive Particles (SIMPs) have recently been proposed as light thermal dark matter relics. Here we consider an explicit realization of the SIMP mechanism in the form of vector SIMPs arising from an SU(2) X hidden gauge theory, where the accidental custodial symmetry protects the stability of the dark matter. We propose several ways of equilibrating the dark and visible sectors in this setup. In particular, we show that a light dark Higgs portal can maintain thermal equilibrium between the two sectors, as can a massive dark vector portal with its generalized Chern-Simons couplings to the vector SIMPs, allmore » while remaining consistent with experimental constraints.« less

  15. Vector SIMP dark matter

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Choi, Soo -Min; Hochberg, Yonit; Kuflik, Eric

    Strongly Interacting Massive Particles (SIMPs) have recently been proposed as light thermal dark matter relics. Here we consider an explicit realization of the SIMP mechanism in the form of vector SIMPs arising from an SU(2) X hidden gauge theory, where the accidental custodial symmetry protects the stability of the dark matter. We propose several ways of equilibrating the dark and visible sectors in this setup. In particular, we show that a light dark Higgs portal can maintain thermal equilibrium between the two sectors, as can a massive dark vector portal with its generalized Chern-Simons couplings to the vector SIMPs, allmore » while remaining consistent with experimental constraints.« less

  16. Multiscale vector fields for image pattern recognition

    NASA Technical Reports Server (NTRS)

    Low, Kah-Chan; Coggins, James M.

    1990-01-01

    A uniform processing framework for low-level vision computing in which a bank of spatial filters maps the image intensity structure at each pixel into an abstract feature space is proposed. Some properties of the filters and the feature space are described. Local orientation is measured by a vector sum in the feature space as follows: each filter's preferred orientation along with the strength of the filter's output determine the orientation and the length of a vector in the feature space; the vectors for all filters are summed to yield a resultant vector for a particular pixel and scale. The orientation of the resultant vector indicates the local orientation, and the magnitude of the vector indicates the strength of the local orientation preference. Limitations of the vector sum method are discussed. Investigations show that the processing framework provides a useful, redundant representation of image structure across orientation and scale.

  17. Coffee, its roasted form, and their residues cause birth failure and shorten lifespan in dengue vectors.

    PubMed

    Dieng, Hamady; Ellias, Salbiah Binti; Satho, Tomomitsu; Ahmad, Abu Hassan; Abang, Fatimah; Ghani, Idris Abd; Noor, Sabina; Ahmad, Hamdan; Zuharah, Wan Fatma; Morales Vargas, Ronald E; Morales, Noppawan P; Hipolito, Cirilo N; Attrapadung, Siriluck; Noweg, Gabriel Tonga

    2017-06-01

    In dengue mosquitoes, successful embryonic development and long lifespan are key determinants for the persistence of both virus and vector. Therefore, targeting the egg stage and vector lifespan would be expected to have greater impacts than larvicides or adulticides, both strategies that have lost effectiveness due to the development of resistance. Therefore, there is now a pressing need to find novel chemical means of vector control. Coffee contains many chemicals, and its waste, which has become a growing environmental concern, is as rich in toxicants as the green coffee beans; these chemicals do not have a history of resistance in insects, but some are lost in the roasting process. We examined whether exposure to coffee during embryonic development could alter larval eclosion and lifespan of dengue vectors. A series of bioassays with different coffee forms and their residues indicated that larval eclosion responses of Aedes albopictus and Ae. aegypti were appreciably lower when embryonic maturation occurred in environments containing coffee, especially roasted coffee crude extract (RCC). In addition, the lifespan of adults derived from eggs that hatched successfully in a coffee milieu was reduced, but this effect was less pronounced with roasted and green coffee extracts (RCU and GCU, respectively). Taken together, these findings suggested that coffee and its residues have embryocidal activities with impacts that are carried over onto the adult lifespan of dengue vectors. These effects may significantly reduce the vectorial capacity of these insects. Reutilizing coffee waste in vector control may also represent a realistic solution to the issues associated with its pollution.

  18. Integrated optic vector-matrix multiplier

    DOEpatents

    Watts, Michael R [Albuquerque, NM

    2011-09-27

    A vector-matrix multiplier is disclosed which uses N different wavelengths of light that are modulated with amplitudes representing elements of an N.times.1 vector and combined to form an input wavelength-division multiplexed (WDM) light stream. The input WDM light stream is split into N streamlets from which each wavelength of the light is individually coupled out and modulated for a second time using an input signal representing elements of an M.times.N matrix, and is then coupled into an output waveguide for each streamlet to form an output WDM light stream which is detected to generate a product of the vector and matrix. The vector-matrix multiplier can be formed as an integrated optical circuit using either waveguide amplitude modulators or ring resonator amplitude modulators.

  19. Viral vector-based influenza vaccines

    PubMed Central

    de Vries, Rory D.; Rimmelzwaan, Guus F.

    2016-01-01

    ABSTRACT Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors. PMID:27455345

  20. Pathogenic landscapes: Interactions between land, people, disease vectors, and their animal hosts

    PubMed Central

    2010-01-01

    Background Landscape attributes influence spatial variations in disease risk or incidence. We present a review of the key findings from eight case studies that we conducted in Europe and West Africa on the impact of land changes on emerging or re-emerging vector-borne diseases and/or zoonoses. The case studies concern West Nile virus transmission in Senegal, tick-borne encephalitis incidence in Latvia, sandfly abundance in the French Pyrenees, Rift Valley Fever in the Ferlo (Senegal), West Nile Fever and the risk of malaria re-emergence in the Camargue, and rodent-borne Puumala hantavirus and Lyme borreliosis in Belgium. Results We identified general principles governing landscape epidemiology in these diverse disease systems and geographic regions. We formulated ten propositions that are related to landscape attributes, spatial patterns and habitat connectivity, pathways of pathogen transmission between vectors and hosts, scale issues, land use and ownership, and human behaviour associated with transmission cycles. Conclusions A static view of the "pathogenecity" of landscapes overlays maps of the spatial distribution of vectors and their habitats, animal hosts carrying specific pathogens and their habitat, and susceptible human hosts and their land use. A more dynamic view emphasizing the spatial and temporal interactions between these agents at multiple scales is more appropriate. We also highlight the complementarity of the modelling approaches used in our case studies. Integrated analyses at the landscape scale allows a better understanding of interactions between changes in ecosystems and climate, land use and human behaviour, and the ecology of vectors and animal hosts of infectious agents. PMID:20979609

  1. 4 × 20 Gbit/s mode division multiplexing over free space using vector modes and a q-plate mode (de)multiplexer

    NASA Astrophysics Data System (ADS)

    Milione, Giovanni; Lavery, Martin P. J.; Huang, Hao; Ren, Yongxiong; Xie, Guodong; Nguyen, Thien An; Karimi, Ebrahim; Marrucci, Lorenzo; Nolan, Daniel A.; Alfano, Robert R.; Willner, Alan E.

    2015-05-01

    Vector modes are spatial modes that have spatially inhomogeneous states of polarization, such as, radial and azimuthal polarization. They can produce smaller spot sizes and stronger longitudinal polarization components upon focusing. As a result, they are used for many applications, including optical trapping and nanoscale imaging. In this work, vector modes are used to increase the information capacity of free space optical communication via the method of optical communication referred to as mode division multiplexing. A mode (de)multiplexer for vector modes based on a liquid crystal technology referred to as a q-plate is introduced. As a proof of principle, using the mode (de)multiplexer four vector modes each carrying a 20 Gbit/s quadrature phase shift keying signal on a single wavelength channel (~1550nm), comprising an aggregate 80 Gbit/s, were transmitted ~1m over the lab table with <-16.4 dB (<2%) mode crosstalk. Bit error rates for all vector modes were measured at the forward error correction threshold with power penalties < 3.41dB.

  2. Multiclass Reduced-Set Support Vector Machines

    NASA Technical Reports Server (NTRS)

    Tang, Benyang; Mazzoni, Dominic

    2006-01-01

    There are well-established methods for reducing the number of support vectors in a trained binary support vector machine, often with minimal impact on accuracy. We show how reduced-set methods can be applied to multiclass SVMs made up of several binary SVMs, with significantly better results than reducing each binary SVM independently. Our approach is based on Burges' approach that constructs each reduced-set vector as the pre-image of a vector in kernel space, but we extend this by recomputing the SVM weights and bias optimally using the original SVM objective function. This leads to greater accuracy for a binary reduced-set SVM, and also allows vectors to be 'shared' between multiple binary SVMs for greater multiclass accuracy with fewer reduced-set vectors. We also propose computing pre-images using differential evolution, which we have found to be more robust than gradient descent alone. We show experimental results on a variety of problems and find that this new approach is consistently better than previous multiclass reduced-set methods, sometimes with a dramatic difference.

  3. Spatial orientation of semicircular canals and afferent sensitivity vectors in pigeons

    NASA Technical Reports Server (NTRS)

    Dickman, J. D.

    1996-01-01

    Rotational head motion in vertebrates is detected by the semicircular canal system, whose innervating primary afferent fibers carry information about movement in specific head planes. The semicircular canals have been qualitatively examined over a number of years, and the canal planes have been quantitatively characterized in several animal species. The present study first determined the geometric relationship between individual semicircular canals and between the canals and the stereotactic head planes in pigeons. Stereotactic measurements of multiple points along the circumference of the bony canals were taken, and the measured points fitted with a three-dimensional planar surface. Direction normals to the plane's surface were calculated and used to define angles between semicircular canal pairs. Because of the unusual shape of the anterior semicircular canals in pigeons, two planes, a major and a minor, were fitted to the canal's course. Calculated angle values for all canals indicated that the horizontal and posterior semicircular canals are nearly orthogonal, but the anterior canals have substantial deviations from orthogonality with other canal planes. Next, the responses of the afferent fibers that innervate each of the semicircular canals to 0.5 Hz sinusoidal rotation about an earth-vertical axis were obtained. The head orientation relative to the rotation axis was systematically varied so that directions of maximum sensitivity for each canal afferent could be determined. These sensitivity vectors were then compared with the canal plane direction normals. The afferents that innervated specific semicircular canals formed homogeneous clusters of sensitivity vectors in different head planes. The horizontal and posterior afferents had average sensitivity vectors that were largely co-incident with the innervated canal plane direction normals. Anterior canal afferents, however, appeared to synthesize contributions from the major and minor plane components of the

  4. Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model

    PubMed Central

    Ahmed, Atique U; Rolle, Cleo E; Tyler, Matthew A; Han, Yu; Sengupta, Sadhak; Wainwright, Derek A; Balyasnikova, Irina V; Ulasov, Ilya V; Lesniak, Maciej S

    2010-01-01

    Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow–derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response. PMID:20588259

  5. Tissue Distribution of the Ehrlichia muris-Like Agent in a Tick Vector

    PubMed Central

    Lynn, Geoffrey E.; Oliver, Jonathan D.; Nelson, Curtis M.; Felsheim, Roderick F.; Kurtti, Timothy J.; Munderloh, Ulrike G.

    2015-01-01

    Human pathogens transmitted by ticks undergo complex life cycles alternating between the arthropod vector and a mammalian host. While the latter has been investigated to a greater extent, examination of the biological interactions between microbes and the ticks that carry them presents an equally important opportunity for disruption of the disease cycle. In this study, we used in situ hybridization to demonstrate infection by the Ehrlichia muris-like organism, a newly recognized human pathogen, of Ixodes scapularis ticks, a primary vector for several important human disease agents. This allowed us to assess whole sectioned ticks for the patterns of tissue invasion, and demonstrate generalized dissemination of ehrlichiae in a variety of cell types and organs within ticks infected naturally via blood feeding. Electron microscopy was used to confirm these results. Here we describe a strong ehrlichial affinity for epithelial cells, neuronal cells of the synganglion, salivary glands, and male accessory glands. PMID:25781930

  6. Decays of the vector glueball

    NASA Astrophysics Data System (ADS)

    Giacosa, Francesco; Sammet, Julia; Janowski, Stanislaus

    2017-06-01

    We calculate two- and three-body decays of the (lightest) vector glueball into (pseudo)scalar, (axial-)vector, as well as pseudovector and excited vector mesons in the framework of a model of QCD. While absolute values of widths cannot be predicted because the corresponding coupling constants are unknown, some interesting branching ratios can be evaluated by setting the mass of the yet hypothetical vector glueball to 3.8 GeV as predicted by quenched lattice QCD. We find that the decay mode ω π π should be one of the largest (both through the decay chain O →b1π →ω π π and through the direct coupling O →ω π π ). Similarly, the (direct and indirect) decay into π K K*(892 ) is sizable. Moreover, the decays into ρ π and K*(892 )K are, although subleading, possible and could play a role in explaining the ρ π puzzle of the charmonium state ψ (2 S ) thanks to a (small) mixing with the vector glueball. The vector glueball can be directly formed at the ongoing BESIII experiment as well as at the future PANDA experiment at the FAIR facility. If the width is sufficiently small (≲100 MeV ) it should not escape future detection. It should be stressed that the employed model is based on some inputs and simplifying assumptions: the value of glueball mass (at present, the quenched lattice value is used), the lack of mixing of the glueball with other quarkonium states, and the use of few interaction terms. It then represents a first step toward the identification of the main decay channels of the vector glueball, but shall be improved when corresponding experimental candidates and/or new lattice results will be available.

  7. Image Coding Based on Address Vector Quantization.

    NASA Astrophysics Data System (ADS)

    Feng, Yushu

    Image coding is finding increased application in teleconferencing, archiving, and remote sensing. This thesis investigates the potential of Vector Quantization (VQ), a relatively new source coding technique, for compression of monochromatic and color images. Extensions of the Vector Quantization technique to the Address Vector Quantization method have been investigated. In Vector Quantization, the image data to be encoded are first processed to yield a set of vectors. A codeword from the codebook which best matches the input image vector is then selected. Compression is achieved by replacing the image vector with the index of the code-word which produced the best match, the index is sent to the channel. Reconstruction of the image is done by using a table lookup technique, where the label is simply used as an address for a table containing the representative vectors. A code-book of representative vectors (codewords) is generated using an iterative clustering algorithm such as K-means, or the generalized Lloyd algorithm. A review of different Vector Quantization techniques are given in chapter 1. Chapter 2 gives an overview of codebook design methods including the Kohonen neural network to design codebook. During the encoding process, the correlation of the address is considered and Address Vector Quantization is developed for color image and monochrome image coding. Address VQ which includes static and dynamic processes is introduced in chapter 3. In order to overcome the problems in Hierarchical VQ, Multi-layer Address Vector Quantization is proposed in chapter 4. This approach gives the same performance as that of the normal VQ scheme but the bit rate is about 1/2 to 1/3 as that of the normal VQ method. In chapter 5, a Dynamic Finite State VQ based on a probability transition matrix to select the best subcodebook to encode the image is developed. In chapter 6, a new adaptive vector quantization scheme, suitable for color video coding, called "A Self -Organizing

  8. Baculovirus-mediated vascular endothelial growth factor-D(ΔNΔC) gene transfer induces angiogenesis in rabbit skeletal muscle.

    PubMed

    Heikura, Tommi; Nieminen, Tiina; Roschier, Miia M; Karvinen, Henna; Kaikkonen, Minna U; Mähönen, Anssi J; Lesch, Hanna P; Rissanen, Tuomas T; Laitinen, Olli H; Airenne, Kari J; Ylä-Herttuala, Seppo

    2012-01-01

    Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding β-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene. Copyright © 2012 John Wiley & Sons, Ltd.

  9. Polynomial interpretation of multipole vectors

    NASA Astrophysics Data System (ADS)

    Katz, Gabriel; Weeks, Jeff

    2004-09-01

    Copi, Huterer, Starkman, and Schwarz introduced multipole vectors in a tensor context and used them to demonstrate that the first-year Wilkinson microwave anisotropy probe (WMAP) quadrupole and octopole planes align at roughly the 99.9% confidence level. In the present article, the language of polynomials provides a new and independent derivation of the multipole vector concept. Bézout’s theorem supports an elementary proof that the multipole vectors exist and are unique (up to rescaling). The constructive nature of the proof leads to a fast, practical algorithm for computing multipole vectors. We illustrate the algorithm by finding exact solutions for some simple toy examples and numerical solutions for the first-year WMAP quadrupole and octopole. We then apply our algorithm to Monte Carlo skies to independently reconfirm the estimate that the WMAP quadrupole and octopole planes align at the 99.9% level.

  10. Vector curvaton with varying kinetic function

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dimopoulos, Konstantinos; Karciauskas, Mindaugas; Wagstaff, Jacques M.

    2010-01-15

    A new model realization of the vector curvaton paradigm is presented and analyzed. The model consists of a single massive Abelian vector field, with a Maxwell-type kinetic term. By assuming that the kinetic function and the mass of the vector field are appropriately varying during inflation, it is shown that a scale-invariant spectrum of superhorizon perturbations can be generated. These perturbations can contribute to the curvature perturbation of the Universe. If the vector field remains light at the end of inflation it is found that it can generate substantial statistical anisotropy in the spectrum and bispectrum of the curvature perturbation.more » In this case the non-Gaussianity in the curvature perturbation is predominantly anisotropic, which will be a testable prediction in the near future. If, on the other hand, the vector field is heavy at the end of inflation then it is demonstrated that particle production is approximately isotropic and the vector field alone can give rise to the curvature perturbation, without directly involving any fundamental scalar field. The parameter space for both possibilities is shown to be substantial. Finally, toy models are presented which show that the desired variation of the mass and kinetic function of the vector field can be realistically obtained, without unnatural tunings, in the context of supergravity or superstrings.« less

  11. HSV Recombinant Vectors for Gene Therapy

    PubMed Central

    Manservigi, Roberto; Argnani, Rafaela; Marconi, Peggy

    2010-01-01

    The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges. PMID:20835362

  12. SSM/I and ECMWF Wind Vector Comparison

    NASA Technical Reports Server (NTRS)

    Wentz, Frank J.; Ashcroft, Peter D.

    1996-01-01

    Wentz was the first to convincingly show that satellite microwave radiometers have the potential to measure the oceanic wind vector. The most compelling evidence for this conclusion was the monthly wind vector maps derived solely from a statistical analysis of Special Sensor Microwave Imager (SSM/I) observations. In a qualitative sense, these maps clearly showed the general circulation over the world's oceans. In this report we take a closer look at the SSM/I monthly wind vector maps and compare them to European Center for Medium-Range Weather Forecasts (ECMWF) wind fields. This investigation leads both to an empirical comparison of SSM/I calculated wind vectors with ECMWF wind vectors, and to an examination of possible reasons that the SSM/I calculated wind vector direction would be inherently more reliable at some locations than others.

  13. The maximum vector-angular margin classifier and its fast training on large datasets using a core vector machine.

    PubMed

    Hu, Wenjun; Chung, Fu-Lai; Wang, Shitong

    2012-03-01

    Although pattern classification has been extensively studied in the past decades, how to effectively solve the corresponding training on large datasets is a problem that still requires particular attention. Many kernelized classification methods, such as SVM and SVDD, can be formulated as the corresponding quadratic programming (QP) problems, but computing the associated kernel matrices requires O(n2)(or even up to O(n3)) computational complexity, where n is the size of the training patterns, which heavily limits the applicability of these methods for large datasets. In this paper, a new classification method called the maximum vector-angular margin classifier (MAMC) is first proposed based on the vector-angular margin to find an optimal vector c in the pattern feature space, and all the testing patterns can be classified in terms of the maximum vector-angular margin ρ, between the vector c and all the training data points. Accordingly, it is proved that the kernelized MAMC can be equivalently formulated as the kernelized Minimum Enclosing Ball (MEB), which leads to a distinctive merit of MAMC, i.e., it has the flexibility of controlling the sum of support vectors like v-SVC and may be extended to a maximum vector-angular margin core vector machine (MAMCVM) by connecting the core vector machine (CVM) method with MAMC such that the corresponding fast training on large datasets can be effectively achieved. Experimental results on artificial and real datasets are provided to validate the power of the proposed methods. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Vector Graph Assisted Pedestrian Dead Reckoning Using an Unconstrained Smartphone

    PubMed Central

    Qian, Jiuchao; Pei, Ling; Ma, Jiabin; Ying, Rendong; Liu, Peilin

    2015-01-01

    The paper presents a hybrid indoor positioning solution based on a pedestrian dead reckoning (PDR) approach using built-in sensors on a smartphone. To address the challenges of flexible and complex contexts of carrying a phone while walking, a robust step detection algorithm based on motion-awareness has been proposed. Given the fact that step length is influenced by different motion states, an adaptive step length estimation algorithm based on motion recognition is developed. Heading estimation is carried out by an attitude acquisition algorithm, which contains a two-phase filter to mitigate the distortion of magnetic anomalies. In order to estimate the heading for an unconstrained smartphone, principal component analysis (PCA) of acceleration is applied to determine the offset between the orientation of smartphone and the actual heading of a pedestrian. Moreover, a particle filter with vector graph assisted particle weighting is introduced to correct the deviation in step length and heading estimation. Extensive field tests, including four contexts of carrying a phone, have been conducted in an office building to verify the performance of the proposed algorithm. Test results show that the proposed algorithm can achieve sub-meter mean error in all contexts. PMID:25738763

  15. Evidence for carry-over effects of predator exposure on pathogen transmission potential.

    PubMed

    Roux, Olivier; Vantaux, Amélie; Roche, Benjamin; Yameogo, Koudraogo B; Dabiré, Kounbobr R; Diabaté, Abdoulaye; Simard, Frederic; Lefèvre, Thierry

    2015-12-22

    Accumulating evidence indicates that species interactions such as competition and predation can indirectly alter interactions with other community members, including parasites. For example, presence of predators can induce behavioural defences in the prey, resulting in a change in susceptibility to parasites. Such predator-induced phenotypic changes may be especially pervasive in prey with discrete larval and adult stages, for which exposure to predators during larval development can have strong carry-over effects on adult phenotypes. To the best of our knowledge, no study to date has examined possible carry-over effects of predator exposure on pathogen transmission. We addressed this question using a natural food web consisting of the human malaria parasite Plasmodium falciparum, the mosquito vector Anopheles coluzzii and a backswimmer, an aquatic predator of mosquito larvae. Although predator exposure did not significantly alter mosquito susceptibility to P. falciparum, it incurred strong fitness costs on other key mosquito life-history traits, including larval development, adult size, fecundity and longevity. Using an epidemiological model, we show that larval predator exposure should overall significantly decrease malaria transmission. These results highlight the importance of taking into account the effect of environmental stressors on disease ecology and epidemiology. © 2015 The Author(s).

  16. [Lutzomyia antunesi as suspected vector of cutaneous leishmaniasis in the Orinoquian region of Colombia].

    PubMed

    Vásquez-Trujillo, Adolfo; Santamaría-Herreño, Erika; González-Reina, Angélica E; Buitrago-Alvarez, Luz S; Góngora-Orjuela, Agustín; Cabrera-Quintero, Olga L

    2008-01-01

    Identifying probable cutaneous leishmaniasis vectors in a rural area a few kilometres from the city of Villavicencio, taking the relative abundance of sand-flies and their natural infection with Leishmania spphaving into account. A CDC trap was used for sampling sand-flies in and around 15 dwellings. Pools of three females from the most abundant Lutzomyia species were used for identifying Leishmania spp. by PCR, with OL1 and OL2 primers. 1 304 sand-flies from nine species were captured, of which L. antunesi (75,6 %) and L. walkeri (19,2 %) were the most abundant. These was a low abundance of L. panamensis and L. gomezi anthropophilic species (<2,4 %). PCR detected Leishmania spp. infection in two L. antunesi groups (total=123 processed females). Due to the fact that L. antunesi was the most abundant species and was found to have Leishmania infection, it may be considered to be the main suspected cutaneous leishmaniasis vector in the rural area being studied. It is recommended that detailed studies of this species' biology (including biting and resting behaviour) should be carried out, aimed at furthering vector control measures.

  17. How random is a random vector?

    NASA Astrophysics Data System (ADS)

    Eliazar, Iddo

    2015-12-01

    Over 80 years ago Samuel Wilks proposed that the "generalized variance" of a random vector is the determinant of its covariance matrix. To date, the notion and use of the generalized variance is confined only to very specific niches in statistics. In this paper we establish that the "Wilks standard deviation" -the square root of the generalized variance-is indeed the standard deviation of a random vector. We further establish that the "uncorrelation index" -a derivative of the Wilks standard deviation-is a measure of the overall correlation between the components of a random vector. Both the Wilks standard deviation and the uncorrelation index are, respectively, special cases of two general notions that we introduce: "randomness measures" and "independence indices" of random vectors. In turn, these general notions give rise to "randomness diagrams"-tangible planar visualizations that answer the question: How random is a random vector? The notion of "independence indices" yields a novel measure of correlation for Lévy laws. In general, the concepts and results presented in this paper are applicable to any field of science and engineering with random-vectors empirical data.

  18. Improved distribution of small molecules and viral vectors in the murine brain using a hollow fiber catheter

    PubMed Central

    Seunguk, Oh; Odland, Rick; Wilson, Scott R.; Kroeger, Kurt M.; Liu, Chunyan; Lowenstein, Pedro R.; Castro, Maria G.; Hall, Walter A.; Ohlfest, John R.

    2008-01-01

    Object A hollow fiber catheter was developed to improve the distribution of drugs administered via direct infusion into the central nervous system (CNS). It is a porous catheter that significantly increases the surface area of brain tissue into which a drug is infused. Methods Dye was infused into the mouse brain through convection-enhanced delivery (CED) using a 28-gauge needle compared with a 3-mm-long hollow fiber catheter. To determine whether a hollow fiber catheter could increase the distribution of gene therapy vectors, a recombinant adenovirus expressing the firefly luciferase reporter was injected into the mouse striatum. Gene expression was monitored using in vivo bioluminescent imaging. To assess the distribution of gene transfer, an adenovirus expressing green fluorescent protein was injected into the striatum using a hollow fiber catheter or a needle. Results Hollow fiber catheter—mediated infusion increased the volume of brain tissue labeled with dye by 2.7 times relative to needle-mediated infusion. In vivo imaging revealed that catheter-mediated infusion of adenovirus resulted in gene expression that was 10 times greater than that mediated by a needle. The catheter appreciably increased the area of brain transduced with adenovirus relative to a needle, affecting a significant portion of the injected hemisphere. Conclusions The miniature hollow fiber catheter used in this study significantly increased the distribution of dye and adenoviral-mediated gene transfer in the mouse brain compared with the levels reached using a 28-gauge needle. Compared with standard single-port clinical catheters, the hollow fiber catheter has the advantage of millions of nanoscale pores to increase surface area and bulk flow in the CNS. Extending the scale of the hollow fiber catheter for the large mammalian brain shows promise in increasing the distribution and efficacy of gene therapy and drug therapy using CED. PMID:17886557

  19. Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33K and L4-22K proteins.

    PubMed

    Biasiotto, Roberta; Akusjärvi, Göran

    2015-01-28

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins.

  20. Projection correlation between two random vectors.

    PubMed

    Zhu, Liping; Xu, Kai; Li, Runze; Zhong, Wei

    2017-12-01

    We propose the use of projection correlation to characterize dependence between two random vectors. Projection correlation has several appealing properties. It equals zero if and only if the two random vectors are independent, it is not sensitive to the dimensions of the two random vectors, it is invariant with respect to the group of orthogonal transformations, and its estimation is free of tuning parameters and does not require moment conditions on the random vectors. We show that the sample estimate of the projection correction is [Formula: see text]-consistent if the two random vectors are independent and root-[Formula: see text]-consistent otherwise. Monte Carlo simulation studies indicate that the projection correlation has higher power than the distance correlation and the ranks of distances in tests of independence, especially when the dimensions are relatively large or the moment conditions required by the distance correlation are violated.