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Sample records for lentiviral integration sites

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

    PubMed Central

    Qian, Wei; Wang, Yong; Li, Rui-fu; Zhou, Xin; Liu, Jing; Peng, Dai-zhi

    2017-01-01

    Background Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood. Material/Methods In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software. Results The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions. Conclusions This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases. PMID:28255155

  2. Integration-deficient Lentiviral Vectors: A Slow Coming of Age

    PubMed Central

    Wanisch, Klaus; Yáñez-Muñoz, Rafael J

    2009-01-01

    Lentiviral vectors are very efficient at transducing dividing and quiescent cells, which makes them highly useful tools for genetic analysis and gene therapy. Traditionally this efficiency was considered dependent on provirus integration in the host cell genome; however, recent results have challenged this view. So called integration-deficient lentiviral vectors (IDLVs) can be produced through the use of integrase mutations that specifically prevent proviral integration, resulting in the generation of increased levels of circular vector episomes in transduced cells. These lentiviral episomes lack replication signals and are gradually lost by dilution in dividing cells, but are stable in quiescent cells. Compared to integrating lentivectors, IDLVs have a greatly reduced risk of causing insertional mutagenesis and a lower risk of generating replication-competent recombinants (RCRs). IDLVs can mediate transient gene expression in proliferating cells, stable expression in nondividing cells in vitro and in vivo, specific immune responses, RNA interference, homologous recombination (gene repair, knock-in, and knock-out), site-specific recombination, and transposition. IDLVs can be converted into replicating episomes, suggesting that if a clinically applicable system can be developed they would also become highly appropriate for stable transduction of proliferating tissues in therapeutic applications. PMID:19491821

  3. Differences in Vector Genome Processing and Illegitimate Integration of Non-Integrating Lentiviral Vectors

    PubMed Central

    Shaw, Aaron M.; Joseph, Guiandre L.; Jasti, Aparna C.; Sastry-Dent, Lakshmi; Witting, Scott; Cornetta, Kenneth

    2016-01-01

    A variety of mutations in lentiviral vector expression systems have been shown to generate a non-integrating phenotype. We studied a novel 12 base-pair U3-LTR integrase attachment site deletion (U3-LTR att site) mutant and found similar physical titers to the previously reported integrase catalytic core mutant IN/D116N. Both mutations led to a greater than two log reduction in vector integration; with IN/D116N providing lower illegitimate integration frequency, while the U3-LTR att site mutant provided a higher level of transgene expression. The improved expression of the U3-LTR att site mutant could not be explained solely based on an observed modest increase in integration frequency. In evaluating processing, we noted significant differences in unintegrated vector forms, with the U3-LTR att site mutant leading to a predominance of 1-LTR circles. The mutations also differed in the manner of illegitimate integration. The U3-LTR att site mutant vector demonstrated integrase-mediated integration at the intact U5-LTR att site and non-integrase mediated integration at the mutated U3-LTR att site. Finally, we combined a variety of mutations and modifications and assessed transgene expression and integration frequency to show that combining modifications can improve the potential clinical utility of non-integrating lentiviral vectors. PMID:27682478

  4. A supramolecular assembly mediates lentiviral DNA integration

    PubMed Central

    Serrao, Erik; Locke, Julia; Swuec, Paolo; Jónsson, Stefán R.; Kotecha, Abhay; Cook, Nicola J.; Pye, Valerie E.; Taylor, Ian A.; Andrésdóttir, Valgerdur; Engelman, Alan N.; Costa, Alessandro; Cherepanov, Peter

    2017-01-01

    Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 Å resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors. PMID:28059770

  5. High efficiency restriction enzyme-free linear amplification-mediated polymerase chain reaction approach for tracking lentiviral integration sites does not abrogate retrieval bias.

    PubMed

    Wu, Chuanfeng; Jares, Alexander; Winkler, Thomas; Xie, Jianjun; Metais, Jean-Yves; Dunbar, Cynthia E

    2013-01-01

    Retroviral vectors are an efficient and widely employed means of introducing an exogenous expression cassette into target cells. These vectors have been shown to integrate semi-randomly into the cellular genome, and can be associated with genotoxicity due to impact on expression of proximate genes. Therefore, efficient and accurate integration site analysis, while quantifying contributions of individual vector-containing clones, is desirable. Linear amplification-mediated polymerase chain reaction (LAM-PCR) is a widely used technique for identifying integrated proviral and host genomic DNA junctions. However, LAM-PCR is subject to selection bias inherent in the reliance of the assay on the presence of a restriction enzyme-cutting site adjacent to a retrievable integration site, and it is further limited by an inability to discriminate prior to sequencing between the flanking genomic DNA of interest and uninformative internal vector DNA. We report a modified restriction enzyme-free LAM-PCR (Re-free LAM-PCR) approach that is less time and labor intensive compared to conventional LAM-PCR, but in contrast to some other nonrestrictive methods, compares in efficiency and sensitivity, excludes retrieval of uninformative internal vector sequences, and allows retrieval of integration sites unbiased by the presence of nearby restriction sites. However, we report that Re-free LAM-PCR remains inaccurate for quantitation of the relative contributions of individual integration site-containing clones in a polyclonal setting, suggesting that bias in LAM-PCR retrieval of integration sites is not wholly explained by restriction enzyme-related factors.

  6. High Efficiency Restriction Enzyme–Free Linear Amplification-Mediated Polymerase Chain Reaction Approach for Tracking Lentiviral Integration Sites Does Not Abrogate Retrieval Bias

    PubMed Central

    Wu, Chuanfeng; Jares, Alexander; Winkler, Thomas; Xie, Jianjun; Metais, Jean-Yves

    2013-01-01

    Abstract Retroviral vectors are an efficient and widely employed means of introducing an exogenous expression cassette into target cells. These vectors have been shown to integrate semi-randomly into the cellular genome, and can be associated with genotoxicity due to impact on expression of proximate genes. Therefore, efficient and accurate integration site analysis, while quantifying contributions of individual vector-containing clones, is desirable. Linear amplification-mediated polymerase chain reaction (LAM-PCR) is a widely used technique for identifying integrated proviral and host genomic DNA junctions. However, LAM-PCR is subject to selection bias inherent in the reliance of the assay on the presence of a restriction enzyme–cutting site adjacent to a retrievable integration site, and it is further limited by an inability to discriminate prior to sequencing between the flanking genomic DNA of interest and uninformative internal vector DNA. We report a modified restriction enzyme–free LAM-PCR (Re-free LAM-PCR) approach that is less time and labor intensive compared to conventional LAM-PCR, but in contrast to some other nonrestrictive methods, compares in efficiency and sensitivity, excludes retrieval of uninformative internal vector sequences, and allows retrieval of integration sites unbiased by the presence of nearby restriction sites. However, we report that Re-free LAM-PCR remains inaccurate for quantitation of the relative contributions of individual integration site–containing clones in a polyclonal setting, suggesting that bias in LAM-PCR retrieval of integration sites is not wholly explained by restriction enzyme–related factors. PMID:22992116

  7. A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

    PubMed

    Ebina, Hirotaka; Kanemura, Yuka; Misawa, Naoko; Sakuma, Tetsushi; Kobayashi, Tomoko; Yamamoto, Takashi; Koyanagi, Yoshio

    2015-01-01

    DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV) provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease system targeting HIV long terminal repeats (LTR). Here, we generated a more efficient strategy that enables the excision of HIV proviral DNA using customized transcription activator-like effector nucleases (TALENs) targeting the same HIV LTR site. A single transfection of TALEN-encoding mRNA, prepared from in vitro transcription, resulted in more than 80% of lentiviral vector DNA being successfully removed from the T cell lines. Furthermore, we developed a lentiviral vector system that takes advantage of the efficient proviral excision with TALENs and permits the simple selection of gene-transduced and excised cells in T cell lines.

  8. Notable reduction in illegitimate integration mediated by a PPT-deleted, nonintegrating lentiviral vector.

    PubMed

    Kantor, Boris; Bayer, Matthew; Ma, Hong; Samulski, Jude; Li, Chengwen; McCown, Thomas; Kafri, Tal

    2011-03-01

    Nonintegrating lentiviral vectors present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling short-term expression of potentially hazardous gene products. However, residual, integrase-independent integration raises a concern that may limit the usefulness of this system. Here we present a novel 3' polypurine tract (PPT)-deleted lentiviral vector that demonstrates impaired integration efficiency and, when packaged into integrase-deficient particles, significantly reduced illegitimate integration. Cells transduced with PPT-deleted vectors exhibited predominantly 1-long terminal repeat (LTR) circles and a low level of linear genomes after reverse transcription (RT). Importantly, the PPT-deleted vector exhibited titers and in vitro and in vivo expression levels matching those of conventional nonintegrating lentiviral vectors. This safer nonintegrating lentiviral vector system will support emerging technologies, such as those based on transient expression of zinc-finger nucleases (ZFNs) for gene editing, as well as reprogramming factors for inducing pluripotency.

  9. Notable Reduction in Illegitimate Integration Mediated by a PPT-deleted, Nonintegrating Lentiviral Vector

    PubMed Central

    Kantor, Boris; Bayer, Matthew; Ma, Hong; Samulski, Jude; Li, Chengwen; McCown, Thomas; Kafri, Tal

    2011-01-01

    Nonintegrating lentiviral vectors present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling short-term expression of potentially hazardous gene products. However, residual, integrase-independent integration raises a concern that may limit the usefulness of this system. Here we present a novel 3′ polypurine tract (PPT)-deleted lentiviral vector that demonstrates impaired integration efficiency and, when packaged into integrase-deficient particles, significantly reduced illegitimate integration. Cells transduced with PPT-deleted vectors exhibited predominantly 1–long terminal repeat (LTR) circles and a low level of linear genomes after reverse transcription (RT). Importantly, the PPT-deleted vector exhibited titers and in vitro and in vivo expression levels matching those of conventional nonintegrating lentiviral vectors. This safer nonintegrating lentiviral vector system will support emerging technologies, such as those based on transient expression of zinc–finger nucleases (ZFNs) for gene editing, as well as reprogramming factors for inducing pluripotency. PMID:21157436

  10. Intrastriatal Delivery of Integration-Deficient Lentiviral Vectors in a Rat Model of Parkinson's Disease.

    PubMed

    Lu-Nguyen, Ngoc B; Broadstock, Martin; Yáñez-Muñoz, Rafael J

    2016-01-01

    Standard integration-proficient lentiviral vectors (IPLVs) are effective at much lower doses than other vector systems and have shown promise in several gene therapy approaches. Their main drawback is the potential risk of insertional mutagenesis. Novel biosafety-enhanced integration-deficient lentiviral vectors (IDLVs) offer a significant improvement and comparable transduction efficacy to their integrating counterparts in some central nervous system applications. We describe here methods for (1) production of IDLVs (and IPLVs), (2) IDLV/IPLV delivery into the striatum of a rat model of Parkinson's disease, and (3) postmortem brain processing.

  11. Towards a Safer, More Randomized Lentiviral Vector Integration Profile Exploring Artificial LEDGF Chimeras

    PubMed Central

    Vranckx, Lenard S.; Demeulemeester, Jonas; Debyser, Zeger

    2016-01-01

    The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells. PMID:27788138

  12. Development of Lentiviral Vectors for Targeted Integration and Protein Delivery.

    PubMed

    Schenkwein, Diana; Ylä-Herttuala, Seppo

    2016-01-01

    The method in this chapter describes the design of human immunodeficiency virus type 1 (HIV-1) integrase (IN)-fusion proteins which we have developed to transport different proteins into the nuclei of lentiviral vector (LV)-transduced cells. The IN-fusion protein cDNA is incorporated into the LV packaging plasmid, which leads to its incorporation into vector particles as part of a large Gag-Pol polyprotein. This specific feature of protein packaging enables also the incorporation of cytotoxic and proapoptotic proteins, such as frequently cutting endonucleases and P53. The vectors can hence be used for various protein transduction needs. An outline of the necessary methods is also given to study the functionality of a chosen IN-fusion protein in a cell culture assay.

  13. Genomic integration occurs in the packaging cell via unexported lentiviral precursors.

    PubMed

    Mosabbir, Abdullah Al; Truong, Kevin

    2016-10-01

    To use HIV-1 based lentivirus components to produce gene integration and the formation of a stable cell line in the packaging cell line without viral infection. A co-transfection of a Human Embryonic Kidney (HEK) 293 packaging cell line with Gag-pol (GP) and a transfer vector, without the envelope vector, produces a stable cell line after 2 weeks of selection. Furthermore, a matrix protein deficient GP in the packaging vector enhances this integration. This supports that, in theory, unexported lentiviral cores produced within the packaging cell can infect itself without requiring the release of any lentiviral particles. If the packaging cell is also the target cell, then gene integration leading to a stable cell line can be accomplished without viral particle infection.

  14. Mobilization and Mechanism of Transcription of Integrated Self-Inactivating Lentiviral Vectors

    PubMed Central

    Hanawa, Hideki; Persons, Derek A.; Nienhuis, Arthur W.

    2005-01-01

    Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3′ long terminal repeat (LTR) is copied over into the 5′ LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector. PMID:15956585

  15. Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo

    PubMed Central

    Heun, Yvonn; Hildebrand, Staffan; Heidsieck, Alexandra; Gleich, Bernhard; Anton, Martina; Pircher, Joachim; Ribeiro, Andrea; Mykhaylyk, Olga; Eberbeck, Dietmar; Wenzel, Daniela; Pfeifer, Alexander; Woernle, Markus; Krötz, Florian; Pohl, Ulrich; Mannell, Hanna

    2017-01-01

    In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro. Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo, we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy. PMID:28042335

  16. Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo.

    PubMed

    Heun, Yvonn; Hildebrand, Staffan; Heidsieck, Alexandra; Gleich, Bernhard; Anton, Martina; Pircher, Joachim; Ribeiro, Andrea; Mykhaylyk, Olga; Eberbeck, Dietmar; Wenzel, Daniela; Pfeifer, Alexander; Woernle, Markus; Krötz, Florian; Pohl, Ulrich; Mannell, Hanna

    2017-01-01

    In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro. Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo, we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy.

  17. Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells.

    PubMed

    Xu, Zhen; Chen, Feng; Zhang, Lingling; Lu, Jing; Xu, Peng; Liu, Guang; Xie, Xuemin; Mu, Wenli; Wang, Yajun; Liu, Depei

    2016-10-01

    Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.

  18. Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells.

    PubMed

    Wang, Yebo; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

    2016-11-28

    Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs.

  19. Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells

    PubMed Central

    Wang, Yebo; Wang, Yingjia; Chang, Tammy

    2017-01-01

    Abstract Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs. PMID:27899664

  20. Transgenic expression of human glial cell line-derived neurotrophic factor from integration-deficient lentiviral vectors is neuroprotective in a rodent model of Parkinson's disease.

    PubMed

    Lu-Nguyen, Ngoc B; Broadstock, Martin; Schliesser, Maximilian G; Bartholomae, Cynthia C; von Kalle, Christof; Schmidt, Manfred; Yáñez-Muñoz, Rafael J

    2014-07-01

    Standard integration-proficient lentiviral vectors (IPLVs) are effective at much lower doses than other vector systems and have shown promise for gene therapy of Parkinson's disease (PD). Their main drawback is the risk of insertional mutagenesis. The novel biosafety-enhanced integration-deficient lentiviral vectors (IDLVs) may offer a significant enhancement in biosafety, but have not been previously tested in a model of a major disease. We have assessed biosafety and transduction efficiency of IDLVs in a rat model of PD, using IPLVs as a reference. Genomic insertion of lentivectors injected into the lesioned striatum was studied by linear amplification-mediated polymerase chain reaction (PCR), followed by deep sequencing and insertion site analysis, demonstrating lack of significant IDLV integration. Reporter gene expression studies showed efficient, long-lived, and transcriptionally targeted expression from IDLVs injected ahead of lesioning in the rat striatum, although at somewhat lower expression levels than from IPLVs. Transgenic human glial cell line-derived neurotrophic factor (hGDNF) expression from IDLVs was used for a long-term investigation of lentivector-mediated, transcriptionally targeted neuroprotection in this PD rat model. Vectors were injected before striatal lesioning, and the results showed improvements in nigral dopaminergic neuron survival and behavioral tests regardless of lentiviral integration proficiency, although they confirmed lower expression levels of hGDNF from IDLVs. These data demonstrate the effectiveness of IDLVs in a model of a major disease and indicate that these vectors could provide long-term PD treatment at low dose, combining efficacy and biosafety for targeted central nervous system applications.

  1. A novel transgenic mouse model produced from lentiviral germline integration for the study of beta-thalassemia gene therapy.

    PubMed

    Li, Wei; Xie, Shuyang; Guo, Xinbing; Gong, Xiuli; Wang, Shu; Lin, Dan; Zhang, Jingzhi; Ren, Zhaorui; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

    2008-03-01

    beta-thalassemia is one of the most common genetic diseases in the world and requires extensive therapy. Lentiviral-mediated gene therapy has been successfully exploited in the treatment of beta-thalassemia and showed promise in clinical application. Using a human beta-globin transgenic mouse line in a beta-thalassemia diseased model generated with a lentiviral-mediated approach, we investigate the stable therapeutic effect on a common thalassemia syndrome. Human beta-globin gene lentiviral vector was constr ucted, followed by subzonal microinjection into single-cell embryos of beta(IVS-2-654)-thalassemia mice to generate a transgenic line. Human beta-globin gene expression was examined with RT-PCR, Western-blotting and ELISA. The hematologic parameters and tissue pathology were investigated over time in founder mice and their off-spring. Transgenic mice with stable expression of the lentivirus carrying human beta-globin gene were obtained. A marked improvement in red blood cell indices and a dramatic reduction in red blood cell anisocytosis, poikilocytosis and target cells were observed. Nucleated cell proportion was greatly decreased in bone marrow, and splenomegaly with extramedullary hematopoiesis was ameliorated. Iron deposition in liver was also reduced. There was a two-fold increase in the survival rate of the beta(IVS-2-654) mice carrying human beta-globin transgene. Significantly, the germline integration of the lentiviral construct was obtained and stable hematologic phenotype correction was observed over the next two generations of the transgenic mice. The generation of human beta-globin transgenic mice in a beta(IVS-2-654)-thalassemia mouse mediated with lentiviral vectors provides a useful model and offers an attractive means to investigate the transgenic stable therapeutic effect in beta-thalassemia.

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

    PubMed

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

    2010-08-01

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

  3. S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection.

    PubMed

    Verghese, Santhosh Chakkaramakkil; Goloviznina, Natalya A; Skinner, Amy M; Lipps, Hans J; Kurre, Peter

    2014-04-01

    Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting 'anchoring' non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.

  4. Retroviral integration: Site matters

    PubMed Central

    Demeulemeester, Jonas; De Rijck, Jan

    2015-01-01

    Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co‐opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine‐grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success. PMID:26293289

  5. Quantitative shearing linear amplification polymerase chain reaction: an improved method for quantifying lentiviral vector insertion sites in transplanted hematopoietic cell systems.

    PubMed

    Zhou, Sheng; Bonner, Melissa A; Wang, Yong-Dong; Rapp, Samuel; De Ravin, Suk See; Malech, Harry L; Sorrentino, Brian P

    2015-02-01

    In gene therapy trials targeting blood disorders, it is important to detect dominance of transduced hematopoietic stem cell (HSC) clones arising from vector insertion site (VIS) effects. Current methods for VIS analysis often do not have defined levels of quantitative accuracy and therefore can fail to detect early clonal dominance. We have developed a rapid and inexpensive method for measuring clone size based on random shearing of genomic DNA, minimal exponential PCR amplification, and shear site counts as a quantitative endpoint. This quantitative shearing linear amplification PCR (qsLAM PCR) assay utilizes an internal control sample containing 19 lentiviral insertion sites per cell that is mixed with polyclonal samples derived from transduced human CD34+ cells. Samples were analyzed from transplanted pigtail macaques and from a participant in our X-linked severe combined immunodeficiency (XSCID) lentiviral vector trial and yielded controlled and quantitative results in all cases. One case of early clonal dominance was detected in a monkey transplanted with limiting numbers of transduced HSCs, while the clinical samples from the XSCID trial participant showed highly diverse clonal representation. These studies demonstrate that qsLAM PCR is a facile and quantitative assay for measuring clonal repertoires in subjects enrolled in human gene therapy trials using lentiviral-transduced HSCs.

  6. Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors

    PubMed Central

    Hu, Peirong; Li, Yedda; Sands, Mark S; McCown, Thomas; Kafri, Tal

    2015-01-01

    The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~107 infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 108 IU/mL, which upon concentration increased to 1010 IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications. PMID:26229972

  7. Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors.

    PubMed

    Hu, Peirong; Li, Yedda; Sands, Mark S; McCown, Thomas; Kafri, Tal

    2015-01-01

    The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~10(7) infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 10(8) IU/mL, which upon concentration increased to 10(10) IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Everson, Elizabeth M; Olzsko, Miles E; Leap, David J; Hocum, Jonah D; Trobridge, Grant D

    2016-01-01

    Hematopoietic stem cell (HSC) gene therapy using retroviral vectors has immense potential, but vector-mediated genotoxicity limits use in the clinic. Lentiviral vectors are less genotoxic than gammaretroviral vectors and have become the vector of choice in clinical trials. Foamy retroviral vectors have a promising integration profile and are less prone to read-through transcription than gammaretroviral or lentiviral vectors. Here, we directly compared the safety and efficacy of foamy vectors to lentiviral vectors in human CD34+ repopulating cells in immunodeficient mice. To increase their genotoxic potential, foamy and lentiviral vectors with identical transgene cassettes with a known genotoxic spleen focus forming virus promoter were used. Both vectors resulted in efficient marking in vivo and a total of 825 foamy and 460 lentiviral vector unique integration sites were recovered in repopulating cells 19 weeks after transplantation. Foamy vector proviruses were observed less often near RefSeq gene and proto-oncogene transcription start sites than lentiviral vectors. The foamy vector group were also more polyclonal with fewer dominant clones (two out of six mice) than the lentiviral vector group (eight out of eight mice), and only lentiviral vectors had integrants near known proto-oncogenes in dominant clones. Our data further support the relative safety of foamy vectors for HSC gene therapy. PMID:27579335

  11. Histone Deacetylase Inhibition Activates Transgene Expression from Integration-Defective Lentiviral Vectors in Dividing and Non-Dividing Cells

    PubMed Central

    Pelascini, Laetitia P.L.; Janssen, Josephine M.

    2013-01-01

    Abstract Integration-defective lentiviral vectors (IDLVs) are being increasingly deployed in both basic and preclinical gene transfer settings. Often, however, the IDLV transgene expression profile is muted when compared to that of their integration-proficient counterparts. We hypothesized that the episomal nature of IDLVs turns them into preferential targets for epigenetic silencing involving chromatin-remodeling histone deacetylation. Therefore, vectors carrying an array of cis-acting elements and transcriptional unit components were assembled with the aid of packaging constructs encoding either the wild-type or the class I mutant D116N integrase moieties. The transduction levels and transgene-product yields provided by each vector class were assessed in the presence and absence of the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A. To investigate the role of the target cell replication status, we performed experiments in growth-arrested human mesenchymal stem cells and in post-mitotic syncytial myotubes. We found that IDLVs are acutely affected by HDACs regardless of their genetic makeup or target cell replication rate. Interestingly, the magnitude of IDLV transgene expression rescue due to HDAC inhibition varied in a vector backbone– and cell type–dependent manner. Finally, investigation of histone modifications by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) revealed a paucity of euchromatin marks distributed along IDLV genomes when compared to those measured on isogenic integration-competent vector templates. These findings support the view that IDLVs constitute preferential targets for epigenetic silencing involving histone deacetylation, which contributes to dampening their full transcriptional potential. Our data provide leads on how to most optimally titrate and deploy these promising episomal gene delivery vehicles. PMID:23140481

  12. Development of a Non-integrating Rev-dependent Lentiviral Vector Carrying Diphtheria Toxin A Chain and Human TRAF6 to Target HIV Reservoirs

    PubMed Central

    Wang, Zhirui; Tang, Zhongwei; Zheng, Yanfang; Yu, Dongyang; Spear, Mark; Iyer, Subashini R.; Bishop, Barney; Wu, Yuntao

    2010-01-01

    Persistence of HIV despite highly active antiretroviral therapy (HAART) is a lasting challenge to virus eradication. To develop a strategy complementary to HAART, we constructed a series of Rev-dependent lentiviral vectors carrying diphtheria toxin A chain (DT-A) and its attenuated mutants, as well as human TRAF6. Expression of these suicide genes following delivery through viral particles is dependent on Rev, which exists only in infected cells. Among these toxins, DT-A has been known to trigger cell death with as little as a single molecule, whereas two of the attenuated mutants in this study, DT-A(176) and DT-A(ΔN), were well-tolerated by cells at low levels. TRAF6 induced apoptosis only with persistent overexpression. Thus, these suicide genes, which induce cell death at different expression levels, offer a balance between efficacy and safety. To minimize possible mutagenesis introduced by retroviral integration in non-target cells, we further developed a non-integrating Rev-dependent (NIRD) lentiviral vector to deliver these genes. In addition, we constructed a DT-A-resistant human cell line by introducing a human elongation factor 2 (EF-2) mutant into HEK293T cells. This allowed us to manufacture the first high-titer NIRD lentiviral particles carrying DT-A to target HIV-positive cells. PMID:20410930

  13. Biosafety Features of Lentiviral Vectors

    PubMed Central

    Schambach, Axel; Zychlinski, Daniela; Ehrnstroem, Birgitta

    2013-01-01

    Abstract Over the past decades, lentiviral vectors have evolved as a benchmark tool for stable gene transfer into cells with a high replicative potential. Their relatively flexible genome and ability to transduce many forms of nondividing cells, combined with the potential for cell-specific pseudotyping, provides a rich resource for numerous applications in experimental platforms and therapeutic settings. Here, we give an overview of important biosafety features of lentiviral vectors, with detailed discussion of (i) the principles of the lentiviral split-genome design used for the construction of packaging cells; (ii) the relevance of modifications introduced into the lentiviral long terminal repeat (deletion of enhancer/promoter sequences and introduction of insulators); (iii) the basic features of mRNA processing, including the Rev/Rev-responsive element (RRE) interaction and the modifications of the 3′ untranslated region of lentiviral vectors with various post-transcriptional regulatory elements affecting transcriptional termination, polyadenylation, and differentiation-specific degradation of mRNA; and (iv) the characteristic integration pattern with the associated risk of transcriptional interference with cellular genes. We conclude with considerations regarding the importance of cell targeting via envelope modifications. Along this course, we address canonical biosafety issues encountered with any type of viral vector: the risks of shedding, mobilization, germline transmission, immunogenicity, and insertional mutagenesis. PMID:23311447

  14. Biosafety features of lentiviral vectors.

    PubMed

    Schambach, Axel; Zychlinski, Daniela; Ehrnstroem, Birgitta; Baum, Christopher

    2013-02-01

    Over the past decades, lentiviral vectors have evolved as a benchmark tool for stable gene transfer into cells with a high replicative potential. Their relatively flexible genome and ability to transduce many forms of nondividing cells, combined with the potential for cell-specific pseudotyping, provides a rich resource for numerous applications in experimental platforms and therapeutic settings. Here, we give an overview of important biosafety features of lentiviral vectors, with detailed discussion of (i) the principles of the lentiviral split-genome design used for the construction of packaging cells; (ii) the relevance of modifications introduced into the lentiviral long terminal repeat (deletion of enhancer/promoter sequences and introduction of insulators); (iii) the basic features of mRNA processing, including the Rev/Rev-responsive element (RRE) interaction and the modifications of the 3' untranslated region of lentiviral vectors with various post-transcriptional regulatory elements affecting transcriptional termination, polyadenylation, and differentiation-specific degradation of mRNA; and (iv) the characteristic integration pattern with the associated risk of transcriptional interference with cellular genes. We conclude with considerations regarding the importance of cell targeting via envelope modifications. Along this course, we address canonical biosafety issues encountered with any type of viral vector: the risks of shedding, mobilization, germline transmission, immunogenicity, and insertional mutagenesis.

  15. Lentivector integration sites in ependymal cells from a model of metachromatic leukodystrophy: non-B DNA as a new factor influencing integration.

    PubMed

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

    2014-08-26

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

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

    PubMed Central

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

    2014-01-01

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

  17. VISPA: a computational pipeline for the identification and analysis of genomic vector integration sites.

    PubMed

    Calabria, Andrea; Leo, Simone; Benedicenti, Fabrizio; Cesana, Daniela; Spinozzi, Giulio; Orsini, Massimilano; Merella, Stefania; Stupka, Elia; Zanetti, Gianluigi; Montini, Eugenio

    2014-01-01

    The analysis of the genomic distribution of viral vector genomic integration sites is a key step in hematopoietic stem cell-based gene therapy applications, allowing to assess both the safety and the efficacy of the treatment and to study the basic aspects of hematopoiesis and stem cell biology. Identifying vector integration sites requires ad-hoc bioinformatics tools with stringent requirements in terms of computational efficiency, flexibility, and usability. We developed VISPA (Vector Integration Site Parallel Analysis), a pipeline for automated integration site identification and annotation based on a distributed environment with a simple Galaxy web interface. VISPA was successfully used for the bioinformatics analysis of the follow-up of two lentiviral vector-based hematopoietic stem-cell gene therapy clinical trials. Our pipeline provides a reliable and efficient tool to assess the safety and efficacy of integrating vectors in clinical settings.

  18. HIF-1α Dependent Wound Healing Angiogenesis In Vivo Can Be Controlled by Site-Specific Lentiviral Magnetic Targeting of SHP-2.

    PubMed

    Heun, Yvonn; Pogoda, Kristin; Anton, Martina; Pircher, Joachim; Pfeifer, Alexander; Woernle, Markus; Ribeiro, Andrea; Kameritsch, Petra; Mykhaylyk, Olga; Plank, Christian; Kroetz, Florian; Pohl, Ulrich; Mannell, Hanna

    2017-07-05

    Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1α (HIF-1α), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and non-specific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanoparticle-lentivirus (MNP-LV) complexes allowing site-directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1α dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1α from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1α DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-1α activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  19. Integrating risks at contaminated sites

    SciTech Connect

    MacDonell, M.; Habegger, L.; Nieves, L.; Schreiber, Z.; Travis, C.

    2000-02-17

    The U.S. Department of Energy (DOE) is responsible for a number of large sites across the country that were radioactively and chemically contaminated by past nuclear research, development, and production activities. Multiple risk assessments are being conducted for these sites to evaluate current conditions and determine what measures are needed to protect human health and the environment from today through the long term. Integrating the risks associated with multiple contaminants in different environmental media across extensive areas, over time periods that extend beyond 1,000 years, and for a number of different impact categories--from human health and ecological to social and economic--represents a considerable challenge. A central element of these integrated analyses is the ability to reflect key interrelationships among environmental resources and human communities that may be adversely affected by the actions or inactions being considered for a given site. Complicating the already difficult task of integrating many kinds of risk is the importance of reflecting the diverse values and preferences brought to bear by the multiple parties interested in the risk analysis process and outcome. An initial conceptual framework has been developed to provide an organized structure to this risk integration, with the aim of supporting effective environmental management decisions. This paper highlights key issues associated with comprehensive risk integration and offers suggestions developed from preliminary work at a complex DOE site.

  20. Surface-engineering of lentiviral vectors.

    PubMed

    Verhoeyen, Els; Cosset, François-Loïc

    2004-02-01

    Vectors derived from retroviridae offer particularly flexible properties in gene transfer applications given the numerous possible associations of various viral surface glycoproteins (determining cell tropism) with different types of retroviral cores (determining genome replication and integration). Lentiviral vectors should be preferred gene delivery vehicles over vectors derived from onco-retroviruses such as murine leukemia viruses (MLVs) that cannot transduce non-proliferating target cells. Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors, their interaction with the host immune system and their host range. There are however important gene transfer restrictions to some non-proliferative tissues or cell types and recent studies have shown that progenitor hematopoietic stem cells in G(0), non-activated primary blood lymphocytes or monocytes were not transducible by lentiviral vectors. Moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer applications in vivo. Several innovative approaches have been explored to overcome such problems that have given rise to novel concepts in the field and have provided promising results in preliminary evaluations in vivo. Here we review the different approaches explored to upgrade lentiviral vectors, aiming at developing vectors suitable for in vivo gene delivery.

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

    PubMed

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

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

  2. Comparison of Lentiviral and Sleeping Beauty Mediated αβ T Cell Receptor Gene Transfer

    PubMed Central

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm’s tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies. PMID:23840834

  3. Hanford site integrated pest management plan

    SciTech Connect

    Giddings, R.F.

    1996-04-09

    The Hanford Site Integrated Pest Management Plan (HSIPMP) defines the Integrated Pest Management (IPM) decision process and subsequent strategies by which pest problems are to be solved at all Hanford Site properties per DOE-RL Site Infrastructure Division memo (WHC 9505090). The HSIPMP defines the roles that contractor organizations play in supporting the IPM process. In short the IPM process anticipates and prevents pest activity and infestation by combining several strategies to achieve long-term pest control solutions.

  4. [Transfection of HL-60 cells by Venus lentiviral vector].

    PubMed

    Li, Zheng; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2013-06-01

    In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

  5. Measurement of lentiviral vector titre and copy number by cross-species duplex quantitative PCR.

    PubMed

    Christodoulou, I; Patsali, P; Stephanou, C; Antoniou, M; Kleanthous, M; Lederer, C W

    2016-01-01

    Lentiviruses are the vectors of choice for many preclinical studies and clinical applications of gene therapy. Accurate measurement of biological vector titre before treatment is a prerequisite for vector dosing, and the calculation of vector integration sites per cell after treatment is as critical to the characterisation of modified cell products as it is to long-term follow-up and the assessment of risk and therapeutic efficiency in patients. These analyses are typically based on quantitative real-time PCR (qPCR), but as yet compromise accuracy and comparability between laboratories and experimental systems, the former by using separate simplex reactions for the detection of endogene and lentiviral sequences and the latter by designing different PCR assays for analyses in human cells and animal disease models. In this study, we validate in human and murine cells a qPCR system for the single-tube assessment of lentiviral vector copy numbers that is suitable for analyses in at least 33 different mammalian species, including human and other primates, mouse, pig, cat and domestic ruminants. The established assay combines the accuracy of single-tube quantitation by duplex qPCR with the convenience of one-off assay optimisation for cross-species analyses and with the direct comparability of lentiviral transduction efficiencies in different species.

  6. Molecular mechanisms of retroviral integration site selection

    PubMed Central

    Kvaratskhelia, Mamuka; Sharma, Amit; Larue, Ross C.; Serrao, Erik; Engelman, Alan

    2014-01-01

    Retroviral replication proceeds through an obligate integrated DNA provirus, making retroviral vectors attractive vehicles for human gene-therapy. Though most of the host cell genome is available for integration, the process of integration site selection is not random. Retroviruses differ in their choice of chromatin-associated features and also prefer particular nucleotide sequences at the point of insertion. Lentiviruses including HIV-1 preferentially integrate within the bodies of active genes, whereas the prototypical gammaretrovirus Moloney murine leukemia virus (MoMLV) favors strong enhancers and active gene promoter regions. Integration is catalyzed by the viral integrase protein, and recent research has demonstrated that HIV-1 and MoMLV targeting preferences are in large part guided by integrase-interacting host factors (LEDGF/p75 for HIV-1 and BET proteins for MoMLV) that tether viral intasomes to chromatin. In each case, the selectivity of epigenetic marks on histones recognized by the protein tether helps to determine the integration distribution. In contrast, nucleotide preferences at integration sites seem to be governed by the ability for the integrase protein to locally bend the DNA duplex for pairwise insertion of the viral DNA ends. We discuss approaches to alter integration site selection that could potentially improve the safety of retroviral vectors in the clinic. PMID:25147212

  7. 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

  8. Comprehensive, integrated, remote sensing at DOE sites

    SciTech Connect

    Lackey, J.G.; Burson, Z.G.

    1984-01-01

    The Department of Energy has established a program called Comprehensive, Integrated Remote Sensing (CIRS). The overall objective is to provide a state-of-the-art data base of remotely sensed data for all users of such information at large DOE sites. The primary types of remote sensing provided consist of the following: (1) large format aerial photography; (2) video from aerial platforms; (3) multispectral scanning; and (4) airborne nuclear radiometric surveys. Implementation of the CIRS Program began with field operations at the Savannah River Plant in 1982 and is continuing at that DOE site at a level of effort of about $1.5 m per year. Integrated remote sensing studies were subsequently extended to the West Valley Demonstration Project in the summer and fall of 1984. It is expected that the Program will eventually be extended to cover all large DOE sites on a continuing basis. 2 figures.

  9. Retroviral integration: Site matters: Mechanisms and consequences of retroviral integration site selection.

    PubMed

    Demeulemeester, Jonas; De Rijck, Jan; Gijsbers, Rik; Debyser, Zeger

    2015-11-01

    Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co-opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine-grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success. © 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.

  10. Launch site integration of Liquid Rocket Boosters

    NASA Technical Reports Server (NTRS)

    Scott, Leland P.; Dickinson, William J.

    1989-01-01

    The impacts of introducing Liquid Rocket Boosters (LRB) into the STS/KSC launch environment are identified and evaluated. Proposed ground systems configurations are presented along with a launch site requirements summary. Pre-launch processing scenarios are described and the required facility modifications and new facility requirements are analyzed. Flight vehicle design recommendations to enhance launch processing are discussed. Processing approaches to integrate LRB with existing STS launch operations are evaluated. The key features and significance of launch site transition to a new STS configuration in parallel with ongoing launch activities are enumerated.

  11. Targeted, homology-driven gene insertion in stem cells by ZFN-loaded ‘all-in-one’ lentiviral vectors

    PubMed Central

    Cai, Yujia; Laustsen, Anders; Zhou, Yan; Sun, Chenglong; Anderson, Mads Valdemar; Li, Shengting; Uldbjerg, Niels; Luo, Yonglun; Jakobsen, Martin R; Mikkelsen, Jacob Giehm

    2016-01-01

    Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et al., 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZFN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded ‘all-in-one’ IDLVs for site-directed gene insertion in stem cell-based gene therapies. DOI: http://dx.doi.org/10.7554/eLife.12213.001 PMID:27278774

  12. Driving DNA transposition by lentiviral protein transduction

    PubMed Central

    Cai, Yujia; Mikkelsen, Jacob Giehm

    2014-01-01

    Gene vectors derived from DNA transposable elements have become powerful molecular tools in biomedical research and are slowly moving into the clinic as carriers of therapeutic genes. Conventional uses of DNA transposon-based gene vehicles rely on the intracellular production of the transposase protein from transfected nucleic acids. The transposase mediates mobilization of the DNA transposon, which is typically provided in the context of plasmid DNA. In recent work, we established lentiviral protein transduction from Gag precursors as a new strategy for direct delivery of the transposase protein. Inspired by the natural properties of infecting viruses to carry their own enzymes, we loaded lentivirus-derived particles not only with vector genomes carrying the DNA transposon vector but also with hundreds of transposase subunits. Such particles were found to drive efficient transposition of the piggyBac transposable element in a range of different cell types, including primary cells, and offer a new transposase delivery approach that guarantees short-term activity and limits potential cytotoxicity. DNA transposon vectors, originally developed and launched as a non-viral alternative to viral integrating vectors, have truly become viral. Here, we briefly review our findings and speculate on the perspectives and potential advantages of transposase delivery by lentiviral protein transduction. PMID:25057443

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

    PubMed Central

    McAllery, Samantha A; Ahlenstiel, Chantelle L; Suzuki, Kazuo; Symonds, Geoff P; Kelleher, Anthony D; Turville, Stuart G

    2016-01-01

    While current antiretroviral therapy has significantly improved, challenges still remain in life-long targeting of HIV-1 reservoirs. Lentiviral gene therapy has the potential to deliver protective genes into the HIV-1 reservoir. However, inefficient reverse transcription (RT) occurs in HIV-1 reservoirs during lentiviral gene delivery. The viral protein Vpx is capable of increasing lentiviral RT by antagonizing the restriction factor SAMHD1. Incorporating Vpx into lentiviral vectors could substantially increase gene delivery into the HIV-1 reservoir. The feasibility of this Vpx approach was tested in resting cell models utilizing macrophages and dendritic cells. Our results showed Vpx exposure led to increased permissiveness of cells over a period that exceeded 2 weeks. Consequently, significant lower potency of HIV-1 antiretrovirals inhibiting RT and integration was observed. When Vpx was incorporated with anti-HIV-1 genes inhibiting either pre-RT or post-RT stages of the viral life-cycle, transduction levels significantly increased. However, a stronger antiviral effect was only observed with constructs that inhibit pre-RT stages of the viral life cycle. In conclusion this study demonstrates a way to overcome the major delivery obstacle of gene delivery into HIV-1 reservoir cell types. Importantly, incorporating Vpx with pre-RT anti-HIV-1 genes, demonstrated the greatest protection against HIV-1 infection. PMID:27790625

  14. Clinical-scale lentiviral vector transduction of PBL for TCR gene therapy and potential for expression in less differentiated cells

    PubMed Central

    Yang, Shicheng; Rosenberg, Steven A.; Morgan, Richard A.

    2012-01-01

    Summary In human gene therapy applications, lentiviral vectors may have advantages over gamma-retroviral vectors because of their ability to transduce non-dividing cells, their resistance to gene silencing, and a lack of integration site preference. In this study, we utilized VSV-G pseudotype third generation lentiviral vectors harboring specific anti-tumor T-cell receptor (TCR) to establish clinical-scale lentiviral transduction of PBL. Spinoculation (1000 × g, 32°C for 2 h) in the presence of protamine sulfate represents the most efficient and economical approach to transduce a large number of PBLs compared to RetroNectin-based methods. Up to 20 million cells per well of a 6-well plate were efficiently transduced and underwent an average 50-fold expansion in two weeks. TCR transduced PBL mediated specific anti-tumor activities including IFN-γ release and cell lysis. Compared to gamma-retroviral vectors, the TCR transgene could be preferentially expressed on a less-differentiated cell population. PMID:18833004

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

    PubMed Central

    Romero, Zulema; Campo-Fernandez, Beatriz; Wherley, Jennifer; Kaufman, Michael L; Urbinati, Fabrizia; Cooper, Aaron R; Hoban, Megan D; Baldwin, Kismet M; Lumaquin, Dianne; Wang, Xiaoyan; Senadheera, Shantha; Hollis, Roger P; Kohn, Donald B

    2015-01-01

    Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies. PMID:26029723

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

    PubMed

    Romero, Zulema; Campo-Fernandez, Beatriz; Wherley, Jennifer; Kaufman, Michael L; Urbinati, Fabrizia; Cooper, Aaron R; Hoban, Megan D; Baldwin, Kismet M; Lumaquin, Dianne; Wang, Xiaoyan; Senadheera, Shantha; Hollis, Roger P; Kohn, Donald B

    2015-01-01

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

  17. New protocol for lentiviral vector mass production.

    PubMed

    Segura, María Mercedes; Garnier, Alain; Durocher, Yves; Ansorge, Sven; Kamen, Amine

    2010-01-01

    Multiplasmid transient transfection is the most widely used technique for the generation of lentiviral vectors. However, traditional transient transfection protocols using 293 T adherent cells and calcium phosphate/DNA co-precipitation followed by ultracentrifugation are tedious, time-consuming, and difficult to scale up. This chapter describes a streamlined protocol for the fast mass production of lentiviral vectors and their purification by affinity chromatography. Lentiviral particles are generated by transient transfection of suspension growing HEK 293 cells in serum-free medium using polyethylenimine (PEI) as transfection reagent. Lentiviral vector production is carried out in Erlenmeyer flasks agitated on orbital shakers requiring minimum supplementary laboratory equipment. Alternatively, the method can be easily scaled up to generate larger volumes of vector stocks in bioreactors. Heparin affinity chromatography allows for selective concentration and purification of lentiviral particles in a singlestep directly from vector supernatants. The method is suitable for the production and purification of different vector pseudotypes.

  18. New-generation multicistronic expression platform: pTRIDENT vectors containing size-optimized IRES elements enable homing endonuclease-based cistron swapping into lentiviral expression vectors.

    PubMed

    Fux, Cornelia; Langer, Dominik; Kelm, Jens M; Weber, Wilfried; Fussenegger, Martin

    2004-04-20

    Capitalizing on a proven multicistronic expression vector platform we have designed novel pTRIDENT vectors which (1). enable coordinated expression of three desired transgenes, (2). are size-optimized, (3). take advantage of small highly efficient internal ribosome entry sites of the GTX or Rbm3 type, (4). harbor various sites specific for homing endonucleases facilitating promoter/multicistronic expression unit/polyadenylation site swapping as well as (5). straightforward integration into human HIV-l-based lentiviral expression vectors tailored to contain compatible homing endonucleases. Multicistronic expression profiles of novel pTRIDENT vectors engineered for different tricistronic expression configurations encoding human low-molecular-weight urokinase-type plasminogen activator (u-PA(LMW)) or Bacillus stearothermophilus-derived alpha-amylase (SAMY), human vascular endothelial growth factor (hVEGF), and human placental secreted alkaline phosphatase (SEAP) have been quantified in Chinese hamster ovary cells (CHO-K1), mouse fibroblasts (NIH/3T3), and/or human fibrosarcoma (HT-1080) cells. In addition, a pTRIDENT-derived SAMY-VEGF-SEAP expression cassette transferred into a compatible lentiviral expression vector enabled simultaneous high-level transgene expression following transduction of transgenic lentiviral particles into primary human chondrocytes. Copyright 2004 Wiley Periodicals, Inc.

  19. Overview of the HIV-1 Lentiviral Vector System.

    PubMed

    Ramezani, Ali; Hawley, Robert G

    2002-11-01

    Replication-defective oncoretroviral vectors have been the most widely used vehicles for gene-transfer studies because of their capacity to efficiently introduce and stably express transgenes in mammalian cells. A limitation of oncoretroviral vectors is that cell division is required for proviral integration into the host genome. By comparison, lentiviruses such as human immunodeficiency virus type 1 (HIV-1) have evolved a nuclear-import machinery that allows them to infect nondividing as well as dividing cells. This unique property has led to the development of lentiviral vectors for gene delivery to a variety of nondividing or slowly dividing cells including neurons and glial cells of the central nervous system and others. This unit is intended to provide an overview of HIV-1 molecular biology and an introduction to successive generations of HIV-1-based lentiviral vectors.

  20. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.

    PubMed

    Biffi, Alessandra; Montini, Eugenio; Lorioli, Laura; Cesani, Martina; Fumagalli, Francesca; Plati, Tiziana; Baldoli, Cristina; Martino, Sabata; Calabria, Andrea; Canale, Sabrina; Benedicenti, Fabrizio; Vallanti, Giuliana; Biasco, Luca; Leo, Simone; Kabbara, Nabil; Zanetti, Gianluigi; Rizzo, William B; Mehta, Nalini A L; Cicalese, Maria Pia; Casiraghi, Miriam; Boelens, Jaap J; Del Carro, Ubaldo; Dow, David J; Schmidt, Manfred; Assanelli, Andrea; Neduva, Victor; Di Serio, Clelia; Stupka, Elia; Gardner, Jason; von Kalle, Christof; Bordignon, Claudio; Ciceri, Fabio; Rovelli, Attilio; Roncarolo, Maria Grazia; Aiuti, Alessandro; Sessa, Maria; Naldini, Luigi

    2013-08-23

    Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.

  1. Sustained high-level polyclonal hematopoietic marking and transgene expression 4 years after autologous transplantation of rhesus macaques with SIV lentiviral vector–transduced CD34+ cells

    PubMed Central

    Kim, Yoo-Jin; Kim, Yoon-Sang; Larochelle, Andre; Renaud, Gabriel; Wolfsberg, Tyra G.; Adler, Rima; Donahue, Robert E.; Hematti, Peiman; Hong, Bum-Kee; Roayaei, Jean; Akagi, Keiko; Riberdy, Janice M.; Nienhuis, Arthur W.; Persons, Derek A.

    2009-01-01

    We previously reported that lentiviral vectors derived from the simian immunodeficiency virus (SIV) were efficient at transducing rhesus hematopoietic repopulating cells. To evaluate the persistence of vector-containing and -expressing cells long term, and the safety implications of SIV lentiviral vector–mediated gene transfer, we followed 3 rhesus macaques for more than 4 years after transplantation with transduced CD34+ cells. All 3 animals demonstrated significant vector marking and expression of the GFP transgene in T cells, B cells, and granulocytes, with mean GFP+ levels of 6.7% (range, 3.3%-13.0%), 7.4% (4.2%-13.4%), and 5.6% (3.1%-10.5%), respectively. There was no vector silencing in hematopoietic cells over time. Vector insertion site analysis of granulocytes demonstrated sustained highly polyclonal reconstitution, with no evidence for progression to oligoclonality. A significant number of clones were found to contribute at both 1-year and 3- or 4-year time points. No vector integrations were detected in the MDS1/EVI1 region, in contrast to our previous findings with a γ-retroviral vector. These data show that lentiviral vectors can mediate stable and efficient long-term expression in the progeny of transduced hematopoietic stem cells, with an integration profile that may be safer than that of standard Moloney murine leukemia virus (MLV)–derived retroviral vectors. PMID:19339698

  2. Targeting retroviral and lentiviral vectors.

    PubMed

    Sandrin, V; Russell, S J; Cosset, F L

    2003-01-01

    Retroviral vectors capable of efficient in vivo gene delivery to specific target cell types or to specific locations of disease pathology would greatly facilitate many gene therapy applications. The surface glycoproteins of membrane-enveloped viruses stand among the choice candidates to control the target cell receptor recognition and host range of retroviral vectors onto which they are incorporated. This can be achieved in many ways, such as the exchange of glycoprotein by pseudotyping, their biochemical modifications, their conjugation with virus-cell bridging agents or their structural modifications. Understanding the fundamental properties of the viral glycoproteins and the molecular mechanism of virus entry into cells has been instrumental in the functional alteration of their tropism. Here we briefly review the current state of our understanding of the structure and function of viral envelope glycoproteins and we discuss the emerging targeting strategies based on retroviral and lentiviral vector systems.

  3. Lentiviral vectors in cancer immunotherapy.

    PubMed

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

    2015-01-01

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

  4. PBO Integrated Real-Time Observing Sites at Volcanic Sites

    NASA Astrophysics Data System (ADS)

    Mencin, D.; Jackson, M.; Borsa, A.; Feaux, K.; Smith, S.

    2009-05-01

    The Plate Boundary Observatory, an element of NSF's EarthScope program, has six integrated observatories in Yellowstone and four on Mt St Helens. These observatories consist of some combination of borehole strainmeters, borehole seismometers, GPS, tiltmeters, pore pressure, thermal measurements and meteorological data. Data from all these instruments have highly variable data rates and formats, all synchronized to GPS time which can cause significant congestion of precious communication resources. PBO has been experimenting with integrating these data streams to both maximize efficiency and minimize latency through the use of software that combines the streams, like Antelope, and VPN technologies.

  5. Lentiviral vector engineering for anti-HIV RNAi gene therapy.

    PubMed

    ter Brake, Olivier; Westerink, Jan-Tinus; Berkhout, Ben

    2010-01-01

    RNA interference or RNAi-based gene therapy for the treatment of HIV-1 infection has recently emerged as a highly effective antiviral approach. The lentiviral vector system is a good candidate for the expression of antiviral short hairpin RNAs (shRNA) in HIV-susceptible cells. However, this strategy can give rise to vector problems because the anti-HIV shRNAs can also target the HIV-based lentiviral vector system. In addition, there may be self-targeting of the shRNA-encoding sequences within the vector RNA genome in the producer cell. The insertion of microRNA (miRNA) cassettes in the vector may introduce Drosha cleavage sites that will also result in the destruction of the vector genome during the production and/or the transduction process. Here, we describe possible solutions to these lentiviral-RNAi problems. We also describe a strategy for multiple shRNA expression to establish a combinatorial RNAi therapy.

  6. Biosafety challenges for use of lentiviral vectors in gene therapy.

    PubMed

    Rothe, Michael; Modlich, Ute; Schambach, Axel

    2013-12-01

    Lentiviral vectors are promising tools for the genetic modification of cells in biomedical research and gene therapy. Their use in recent clinical trials for the treatment of adrenoleukodystrophy, β-thalassemia, Wiskott-Aldrich- Syndrome and metachromatic leukodystrophy underlined their efficacy for therapies especially in case of hereditary diseases. In comparison to gammaretroviral LTR-driven vectors, which were employed in the first clinical trials, lentiviral vectors present with some favorable features like the ability to transduce also non-dividing cells and a potentially safer insertion profile. However, genetic modification with viral vectors in general and stable integration of the therapeutic gene into the host cell genome bear concerns with respect to different levels of personal or environmental safety. Among them, insertional mutagenesis by enhancer mediated dysregulation of neighboring genes or aberrant splicing is still the biggest concern. However, also risks like immunogenicity of vector particles, the phenotoxicity of the transgene and potential vertical or horizontal transmission by replication competent retroviruses need to be taken into account. This review will give an overview on biosafety aspects that are relevant to the use of lentiviral vectors for genetic modification and gene therapy. Furthermore, assay systems aiming at evaluating biosafety in preclinical settings and recent promising clinical trials including efforts of monitoring of patients after gene therapy will be discussed.

  7. Integration of Biomass Harvesting and Site Preparation

    Treesearch

    Bryce J. Stokes; William F. Watson

    1986-01-01

    This study was conducted to assess the costs of various site preparation methods with various levels of harvesting Site impacts, soil compaction and disturbance were examined. Three hawesting methods rare evaluated in pine pulpwood plantation and pine sawtimber stands. The harvesting methods tested were (1) conventional - harvesting all roundwood. (2) two-pass - first...

  8. Lentiviral Vectors and Cystic Fibrosis Gene Therapy

    PubMed Central

    Castellani, Stefano; Conese, Massimo

    2010-01-01

    Cystic fibrosis (CF) is a chronic autosomic recessive syndrome, caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, a chloride channel expressed on the apical side of the airway epithelial cells. The lack of CFTR activity brings a dysregulated exchange of ions and water through the airway epithelium, one of the main aspects of CF lung disease pathophysiology. Lentiviral (LV) vectors, of the Retroviridae family, show interesting properties for CF gene therapy, since they integrate into the host genome and allow long-lasting gene expression. Proof-of-principle that LV vectors can transduce the airway epithelium and correct the basic electrophysiological defect in CF mice has been given. Initial data also demonstrate that LV vectors can be repeatedly administered to the lung and do not give rise to a gross inflammatory process, although they can elicit a T cell-mediated response to the transgene. Future studies will clarify the efficacy and safety profile of LV vectors in new complex animal models with CF, such as ferrets and pigs. PMID:21994643

  9. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.

    PubMed

    Aiuti, Alessandro; Biasco, Luca; Scaramuzza, Samantha; Ferrua, Francesca; Cicalese, Maria Pia; Baricordi, Cristina; Dionisio, Francesca; Calabria, Andrea; Giannelli, Stefania; Castiello, Maria Carmina; Bosticardo, Marita; Evangelio, Costanza; Assanelli, Andrea; Casiraghi, Miriam; Di Nunzio, Sara; Callegaro, Luciano; Benati, Claudia; Rizzardi, Paolo; Pellin, Danilo; Di Serio, Clelia; Schmidt, Manfred; Von Kalle, Christof; Gardner, Jason; Mehta, Nalini; Neduva, Victor; Dow, David J; Galy, Anne; Miniero, Roberto; Finocchi, Andrea; Metin, Ayse; Banerjee, Pinaki P; Orange, Jordan S; Galimberti, Stefania; Valsecchi, Maria Grazia; Biffi, Alessandra; Montini, Eugenio; Villa, Anna; Ciceri, Fabio; Roncarolo, Maria Grazia; Naldini, Luigi

    2013-08-23

    Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.

  10. Lentiviral Delivery of Proteins for Genome Engineering.

    PubMed

    Cai, Yujia; Mikkelsen, Jacob Giehm

    2016-01-01

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

  11. Integrated Propulsion Data System Public Web Site

    NASA Technical Reports Server (NTRS)

    Hamilton, Kimberly

    2001-01-01

    The Integrated Propulsion Data System's (IPDS) focus is to provide technologically-advanced philosophies of doing business at SSC that will enhance the existing operations, engineering and management strategies and provide insight and metrics to assess their daily impacts, especially as related to the Propulsion Test Directorate testing scenarios for the 21st Century.

  12. Targeting lentiviral vectors for cancer immunotherapy

    PubMed Central

    Arce, Frederick; Breckpot, Karine; Collins, Mary; Escors, David

    2012-01-01

    Delivery of tumour-associated antigens (TAA) in a way that induces effective, specific immunity is a challenge in anti-cancer vaccine design. Circumventing tumour-induced tolerogenic mechanisms in vivo is also critical for effective immunotherapy. Effective immune responses are induced by professional antigen presenting cells, in particular dendritic cells (DC). This requires presentation of the antigen to both CD4+ and CD8+ T cells in the context of strong co-stimulatory signals. Lentiviral vectors have been tested as vehicles, for both ex vivo and in vivo delivery of TAA and/or activation signals to DC, and have been demonstrated to induce potent T cell mediated immune responses that can control tumour growth. This review will focus on the use of lentiviral vectors for in vivo gene delivery to DC, introducing strategies to target DC, either targeting cell entry or gene expression to improve safety of the lentiviral vaccine or targeting dendritic cell activation pathways to enhance performance of the lentiviral vaccine. In conclusion, this review highlights the potential of lentiviral vectors as a generally applicable ‘off-the-shelf’ anti-cancer immunotherapeutic. PMID:22983382

  13. Surface modification via strain-promoted click reaction facilitates targeted lentiviral transduction.

    PubMed

    Chu, Yanjie; Oum, Yoon Hyeun; Carrico, Isaac S

    2016-01-01

    As a result of their ability to integrate into the genome of both dividing and non-dividing cells, lentiviruses have emerged as a promising vector for gene delivery. Targeted gene transduction of specific cells and tissues by lentiviral vectors has been a major goal, which has proven difficult to achieve. We report a novel targeting protocol that relies on the chemoselective attachment of cancer specific ligands to unnatural glycans on lentiviral surfaces. This strategy exhibits minimal perturbation on virus physiology and demonstrates remarkable flexibility. It allows for targeting but can be more broadly useful with applications such as vector purification and immunomodulation.

  14. Deciphering the Code for Retroviral Integration Target Site Selection

    PubMed Central

    Santoni, Federico Andrea; Hartley, Oliver; Luban, Jeremy

    2010-01-01

    Upon cell invasion, retroviruses generate a DNA copy of their RNA genome and integrate retroviral cDNA within host chromosomal DNA. Integration occurs throughout the host cell genome, but target site selection is not random. Each subgroup of retrovirus is distinguished from the others by attraction to particular features on chromosomes. Despite extensive efforts to identify host factors that interact with retrovirion components or chromosome features predictive of integration, little is known about how integration sites are selected. We attempted to identify markers predictive of retroviral integration by exploiting Precision-Recall methods for extracting information from highly skewed datasets to derive robust and discriminating measures of association. ChIPSeq datasets for more than 60 factors were compared with 14 retroviral integration datasets. When compared with MLV, PERV or XMRV integration sites, strong association was observed with STAT1, acetylation of H3 and H4 at several positions, and methylation of H2AZ, H3K4, and K9. By combining peaks from ChIPSeq datasets, a supermarker was identified that localized within 2 kB of 75% of MLV proviruses and detected differences in integration preferences among different cell types. The supermarker predicted the likelihood of integration within specific chromosomal regions in a cell-type specific manner, yielding probabilities for integration into proto-oncogene LMO2 identical to experimentally determined values. The supermarker thus identifies chromosomal features highly favored for retroviral integration, provides clues to the mechanism by which retrovirus integration sites are selected, and offers a tool for predicting cell-type specific proto-oncogene activation by retroviruses. PMID:21124862

  15. Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

    PubMed Central

    Wu, Yuntao

    2010-01-01

    Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells. PMID:20972394

  16. Stable and functional lymphoid reconstitution in artemis-deficient mice following lentiviral artemis gene transfer into hematopoietic stem cells.

    PubMed

    Benjelloun, Fatine; Garrigue, Alexandrine; Demerens-de Chappedelaine, Corinne; Soulas-Sprauel, Pauline; Malassis-Séris, Michele; Stockholm, Daniel; Hauer, Julia; Blondeau, Johanna; Rivière, Julie; Lim, Annick; Le Lorc'h, Marc; Romana, Serge; Brousse, Nicole; Pâques, Frederique; Galy, Anne; Charneau, Pierre; Fischer, Alain; de Villartay, Jean-Pierre; Cavazzana-Calvo, Marina

    2008-08-01

    Patients with mutations in the Artemis gene display a complete absence of T- and B lymphocytes, together with increased cellular radiosensitivity; this leads to a radiosensitive severe combined immunodeficiency (RS-SCID). Allogenic hematopoietic stem-cell (HSC) transplantation is only partially successful in the absence of an human leukocyte antigen-genoidentical donor, and this has prompted a search for alternative therapeutic approaches such as gene therapy. In this study, a self-inactivated lentiviral vector expressing Artemis was used to complement the Artemis knockout mouse (Art(-/-)). Transplantation of Artemis-transduced HSCs into irradiated Art(-/-) mice restored a stable (over a 15-month period of follow-up) and functional T- and cell repertoire that was comparable to that of control mice. The success of secondary transplantations demonstrated that the HSCs had been transduced. One of thirteen mice developed a thymoma 6 months after gene therapy. Although thymic cells were seen to be carrying two lentiviral integration sites, there was no evidence of lentivirus-driven oncogene activation. The Art(-/-) mice were found to be prone to develop T-cell lymphomas, either spontaneously or after irradiation. These data indicate that the observed lymphoproliferation was probably the consequence of the chromosomal instability associated with the Artemis-deficient background. As a whole, our work provides a basis for supporting the gene therapy approach in Artemis-deficient SCID.

  17. Integration Site Choice of a Feline Immunodeficiency Virus Vector

    PubMed Central

    Kang, Yubin; Moressi, Christopher J.; Scheetz, Todd E.; Xie, Litao; Tran, Diane Thi; Casavant, Thomas L.; Ak, Prashanth; Benham, Craig J.; Davidson, Beverly L.; McCray, Paul B.

    2006-01-01

    We mapped 226 unique integration sites in human hepatoma cells following gene transfer with a feline immunodeficiency virus (FIV)-based lentivirus vector. FIV integrated across the entire length of the transcriptional units. Microarray data indicated that FIV integration favored actively transcribed genes. Approximately 21% of FIV integrations within transcriptional units occurred in genes regulated by the LEDGF/p75 transcriptional coactivator. DNA in regions of FIV insertion sites exhibited a “bendable” structure and a pattern of duplex destabilization favoring strand separation. FIV integration preferences are more similar to those of primate lentiviruses and distinct from those of Moloney murine leukemia virus, avian sarcoma leukosis virus, and foamy virus. PMID:16912328

  18. Converting Maturing Nuclear Sites to Integrated Power Production Islands

    DOE PAGES

    Solbrig, Charles W.

    2011-01-01

    Nuclear islands, which are integrated power production sites, could effectively sequester and safeguard the US stockpile of plutonium. A nuclear island, an evolution of the integral fast reactor, utilizes all the Transuranics (Pu plus minor actinides) produced in power production, and it eliminates all spent fuel shipments to and from the site. This latter attribute requires that fuel reprocessing occur on each site and that fast reactors be built on-site to utilize the TRU. All commercial spent fuel shipments could be eliminated by converting all LWR nuclear power sites to nuclear islands. Existing LWR sites have the added advantage ofmore » already possessing a license to produce nuclear power. Each could contribute to an increase in the nuclear power production by adding one or more fast reactors. Both the TRU and the depleted uranium obtained in reprocessing would be used on-site for fast fuel manufacture. Only fission products would be shipped to a repository for storage. The nuclear island concept could be used to alleviate the strain of LWR plant sites currently approaching or exceeding their spent fuel pool storage capacity. Fast reactor breeding ratio could be designed to convert existing sites to all fast reactors, or keep the majority thermal.« less

  19. Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion

    PubMed Central

    Zheng, Yongxiang; Yu, Fei; Wu, Yiming; Si, Longlong; Xu, Huan; Zhang, Chuanling; Xia, Qing; Xiao, Sulong; Wang, Qi; He, Qiuchen; Chen, Peng; Wang, Jiangyun; Taira, Kazunari; Zhang, Lihe; Zhou, Demin

    2015-01-01

    With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure–function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus–host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications. PMID:25765642

  20. Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion.

    PubMed

    Zheng, Yongxiang; Yu, Fei; Wu, Yiming; Si, Longlong; Xu, Huan; Zhang, Chuanling; Xia, Qing; Xiao, Sulong; Wang, Qi; He, Qiuchen; Chen, Peng; Wang, Jiangyun; Taira, Kazunari; Zhang, Lihe; Zhou, Demin

    2015-06-23

    With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.

  1. TECHNOLOGY INTEGRATION FOR CONTAMINATED SITE REMEDIATION: CLEANUP GOALS & PERFORMANCE CRITERIA

    EPA Science Inventory

    There is a need to develop and field-test integrated remediation technologies that operate in a synergistic manner for cost-effective treatment of contaminated sites to achieve risk-based and rational endpoints. Aggressive technologies designed for rapid source-zone remediation m...

  2. TECHNOLOGY INTEGRATION FOR CONTAMINATED SITE REMEDIATION: CLEANUP GOALS & PERFORMANCE CRITERIA

    EPA Science Inventory

    There is a need to develop and field-test integrated remediation technologies that operate in a synergistic manner for cost-effective treatment of contaminated sites to achieve risk-based and rational endpoints. Aggressive technologies designed for rapid source-zone remediation m...

  3. Site-specific recombinases as tools for heterologous gene integration.

    PubMed

    Hirano, Nobutaka; Muroi, Tetsurou; Takahashi, Hideo; Haruki, Mitsuru

    2011-10-01

    Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering, and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic manipulation, especially manipulations involving site-specific gene integration into heterologous genomes.

  4. Marker-free site-specific gene integration in plants.

    PubMed

    Srivastava, Vibha; Ow, David W

    2004-12-01

    For nearly 15 years, the use of site-specific recombination systems in plants has focused on the deletion or integration of DNA. Each of these applications offers practical solutions to two problems in biotechnology: the presence of unneeded DNA in the transgene locus and variation in the locus structure among independent transgenic lines. Given that each of these separate applications is becoming more practical for commercial use, it is time to consider combining them into an integrated technology. Here we propose a strategy for a "combined step" method that makes use of two site-specific recombination systems: one for integrating the DNA and a second for removing sequences that are not needed after DNA transfer. This strategy is based on published data and exemplifies the use of the Cre-lox, FLP-FRT and R-RS inducible systems.

  5. Modification site localization scoring integrated into a search engine.

    PubMed

    Baker, Peter R; Trinidad, Jonathan C; Chalkley, Robert J

    2011-07-01

    Large proteomic data sets identifying hundreds or thousands of modified peptides are becoming increasingly common in the literature. Several methods for assessing the reliability of peptide identifications both at the individual peptide or data set level have become established. However, tools for measuring the confidence of modification site assignments are sparse and are not often employed. A few tools for estimating phosphorylation site assignment reliabilities have been developed, but these are not integral to a search engine, so require a particular search engine output for a second step of processing. They may also require use of a particular fragmentation method and are mostly only applicable for phosphorylation analysis, rather than post-translational modifications analysis in general. In this study, we present the performance of site assignment scoring that is directly integrated into the search engine Protein Prospector, which allows site assignment reliability to be automatically reported for all modifications present in an identified peptide. It clearly indicates when a site assignment is ambiguous (and if so, between which residues), and reports an assignment score that can be translated into a reliability measure for individual site assignments.

  6. Hanford Site waste treatment/storage/disposal integration

    SciTech Connect

    MCDONALD, K.M.

    1999-02-24

    In 1998 Waste Management Federal Services of Hanford, Inc. began the integration of all low-level waste, mixed waste, and TRU waste-generating activities across the Hanford site. With seven contractors, dozens of generating units, and hundreds of waste streams, integration was necessary to provide acute waste forecasting and planning for future treatment activities. This integration effort provides disposition maps that account for waste from generation, through processing, treatment and final waste disposal. The integration effort covers generating facilities from the present through the life-cycle, including transition and deactivation. The effort is patterned after the very successful DOE Complex EM Integration effort. Although still in the preliminary stages, the comprehensive onsite integration effort has already reaped benefits. These include identifying significant waste streams that had not been forecast, identifying opportunities for consolidating activities and services to accelerate schedule or save money; and identifying waste streams which currently have no path forward in the planning baseline. Consolidation/integration of planned activities may also provide opportunities for pollution prevention and/or avoidance of secondary waste generation. A workshop was held to review the waste disposition maps, and to identify opportunities with potential cost or schedule savings. Another workshop may be held to follow up on some of the long-term integration opportunities. A change to the Hanford waste forecast data call would help to align the Solid Waste Forecast with the new disposition maps.

  7. Integrated grassland observation sites and integrated cropland observation sites at El Reno, Oklahoma

    USDA-ARS?s Scientific Manuscript database

    With the financial support from the National Science Foundation and the USDA National Institute of Food and Agriculture, a team of researchers from the University of Oklahoma and the USDA ARS Grazinglands Research Laboratory have worked together and established two Integrated Grassland Observation s...

  8. Development and use of SIV-based Integrase defective lentiviral vector for immunization

    PubMed Central

    Michelini, Zuleika; Negri, Donatella RM; Baroncelli, Silvia; Spada, Massimo; Leone, Pasqualina; Bona, Roberta; Klotman, Mary E.; Cara, Andrea

    2009-01-01

    Integrase (IN) defective lentiviral vectors have a high safety profile and might prove useful as immunizing agents especially against HIV-1. However, IN defective SIV-based vectors must be developed in order to test their potential in the non human primate models (NHP) of AIDS. To this aim we tested a novel SIV-based IN defective lentiviral vector for its ability to induce sustained immune responses in mice. BALB/c mice were immunized once intramuscularly with a SIV-based IN defective lentiviral vector expressing the model antigen enhanced green fluorescence protein (eGFP). Immune responses were evaluated 90 days after the injection and compared with those elicited with the IN competent counterpart. The IN defective vector was able to efficiently elicit specific and long-lasting polyfunctional immune responses as evaluated by enzyme-linked immunospot (ELISPOT) assays for interferon-γ (IFN-γ) in spleens, bone marrow (BM) and draining lymph nodes, and by intracellular staining (ICS) for IFN-γ, Interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) in both splenocytes and BM cells without integration of the vector into the host genome. This is the first demonstration that an IN defective SIV-based lentiviral vector provides effective immunization, thus paving the way for the construction of IN defective vectors expressing SIV antigen(s) and test their efficacy against a SIV virus challenge in the NHP model of AIDS. PMID:19523909

  9. Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol.

    PubMed

    Horn, Peter A; Keyser, Kirsten A; Peterson, Laura J; Neff, Tobias; Thomasson, Bobbie M; Thompson, Jesse; Kiem, Hans-Peter

    2004-05-15

    The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34(+) hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)- and granulocyte-colony stimulating factor (G-CSF)-primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.

  10. Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity.

    PubMed

    Gay, Virginie; Moreau, Karen; Hong, Saw-See; Ronfort, Corinne

    2012-02-01

    A human immunodeficiency virus type (HIV-1)-based lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein and encoding the GFP reporter gene was used to evaluate different methods of lentiviral vector titration. GFP expression, viral DNA quantification and the efficiency of vector DNA integration were assayed after infection of conventional HIV-1-permissive cell lines and human primary adult fibroblasts with the vector. We found that vector titers based on GFP expression determined by flow cytometry may vary by more than 50-fold depending on the cell type and the promoter-cell combination used. Interestingly, we observed that the viral integration process in primary HDFa cells was significantly more efficient compared to that in SupT1 or 293T cells. We propose that determination of the amount of integrated viral DNA by quantitative PCR be used in combination with the reporter gene expression assay.

  11. Integration of Environmental Compliance at the Savannah River Site - 13024

    SciTech Connect

    Hoel, David; Griffith, Michael

    2013-07-01

    The Savannah River Site (SRS) is a large federal installation hosting diverse missions and multiple organizations with competing regulatory needs. Accordingly, there was a need to integrate environmental compliance strategies to ensure the consistent flow of information between Department of Energy-Savannah River (DOE-SR), the regulatory agencies and other interested parties. In order to meet this objective, DOE and major SRS contractors and tenants have committed to a strategy of collaboratively working together to ensure that a consistent, integrated, and fully coordinated approach to environmental compliance and regulator relationships is maintained. DOE-SR and Savannah River Nuclear Solutions, LLC, the SRS management and operations contractor, have established an environmental compliance integration process that provides for the consistent flow down of requirements to projects, facilities, SRS contractors, and subcontractors as well as the upward flow of information to assist in the early identification and resolution of environmental regulatory issues and enhancement of compliance opportunities. In addition, this process strongly fosters teamwork to collaboratively resolve complex regulatory challenges, promote pollution prevention and waste minimization opportunities to advance site missions in a manner that balances near-term actions with the long-term site vision, while being protective of human health and the environment. Communication tools are being utilized, some with enhancements, to ensure appropriate information is communicated to all levels with environmental responsibility at SRS. SRS internal regulatory integration is accomplished through a variety of informational exchange forums (e.g., Challenges, Opportunities and Resolution (COR) Team, DOE's Joint Site Regulatory Integration Team, and the Senior Environmental Managers Council (SEMC)). SRS communications and problem-solving with the regulatory agencies have been enhanced through formation of an

  12. Integrated Patient Education on U.S. Hospital Web Sites.

    PubMed

    Huang, Edgar; Wu, Kerong; Edwards, Kelsey

    2016-01-01

    Based on a census of the 2015 Most Wired Hospitals, this content analysis aimed to find out how patient education has been integrated on these best IT hospitals' Web sites to serve the purposes of marketing and meeting online visitors' needs. This study will help hospitals to understand where the weaknesses are in their interactive patient education implementation and come up with a smart integration strategy. The study found that 70% of these hospitals had adopted interactive patient education contents, 76.6% of such contents were from a third-party developer, and only 20% of the hospitals linked their patient education contents to one or more of the hospital's resources while 26% cross-references such contents. The authors concluded that more hospitals should take advantage of modern information communication technology to cross-reference their patient education contents and to integrate such contents into their overall online marketing strategy to benefit patients and themselves.

  13. Comparing DNA integration site clusters with scan statistics

    PubMed Central

    Berry, Charles C.; Ocwieja, Karen E.; Malani, Nirav; Bushman, Frederic D.

    2014-01-01

    Motivation: Gene therapy with retroviral vectors can induce adverse effects when those vectors integrate in sensitive genomic regions. Retroviral vectors are preferred that target sensitive regions less frequently, motivating the search for localized clusters of integration sites and comparison of the clusters formed by integration of different vectors. Scan statistics allow the discovery of spatial differences in clustering and calculation of false discovery rates providing statistical methods for comparing retroviral vectors. Results: A scan statistic for comparing two vectors using multiple window widths is proposed with software to detect clustering differentials and compute false discovery rates. Application to several sets of experimentally determined HIV integration sites demonstrates the software. Simulated datasets of various sizes and signal strengths are used to determine the power to discover clusters and evaluate a convenient lower bound. This provides a toolkit for planning evaluations of new gene therapy vectors. Availability and implementation: The geneRxCluster R package containing a simple tutorial and usage hints is available from http://www.bioconductor.org. Contact: ccberry@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24489369

  14. Lentiviral Effector Pathways of TRIM Proteins.

    PubMed

    Turrini, Filippo; Di Pietro, Andrea; Vicenzi, Elisa

    2014-04-01

    The human tripartite motif (TRIM) family, composed of more than 77 members, encompasses an emerging group of innate antiviral factors. Most TRIM proteins are characterized by being E3 ubiquitin ligases, but also engage in specific interactions with a variety of cellular and viral partners. They are involved in many cellular processes, including cell differentiation, transcriptional regulation, cytoskeleton remodeling, intracellular trafficking, membrane repair, and oncogenesis. In regard to antiviral immunity, they restrict both retroviruses and lentiviruses as well as other DNA and RNA viruses. This review will focus on the TRIM members endowed with anti-retroviral and anti-lentiviral activities and, in particular, human immunodeficiency virus.

  15. The Inside Out of Lentiviral Vectors

    PubMed Central

    Durand, Stéphanie; Cimarelli, Andrea

    2011-01-01

    Lentiviruses induce a wide variety of pathologies in different animal species. A common feature of the replicative cycle of these viruses is their ability to target non-dividing cells, a property that constitutes an extremely attractive asset in gene therapy. In this review, we shall describe the main basic aspects of the virology of lentiviruses that were exploited to obtain efficient gene transfer vectors. In addition, we shall discuss some of the hurdles that oppose the efficient genetic modification mediated by lentiviral vectors and the strategies that are being developed to circumvent them. PMID:22049307

  16. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems

    PubMed Central

    Béguin, Pierre; Charpin, Nicole; Koonin, Eugene V.; Forterre, Patrick; Krupovic, Mart

    2016-01-01

    Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei. Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3′ end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1–Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems. PMID:27655632

  17. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems.

    PubMed

    Béguin, Pierre; Charpin, Nicole; Koonin, Eugene V; Forterre, Patrick; Krupovic, Mart

    2016-12-01

    Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3' end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1-Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems.

  18. Opportunities for Launch Site Integrated System Health Engineering and Management

    NASA Technical Reports Server (NTRS)

    Waterman, Robert D.; Langwost, Patricia E.; Waterman, Susan J.

    2005-01-01

    The launch site processing flow involves operations such as functional verification, preflight servicing and launch. These operations often include hazards that must be controlled to protect human life and critical space hardware assets. Existing command and control capabilities are limited to simple limit checking durig automated monitoring. Contingency actions are highly dependent on human recognition, decision making, and execution. Many opportunities for Integrated System Health Engineering and Management (ISHEM) exist throughout the processing flow. This paper will present the current human-centered approach to health management as performed today for the shuttle and space station programs. In addition, it will address some of the more critical ISHEM needs, and provide recommendations for future implementation of ISHEM at the launch site.

  19. Hanford Site waste management and environmental restoration integration plan

    SciTech Connect

    Merrick, D.L.

    1990-04-30

    The Hanford Site Waste Management and Environmental Restoration Integration Plan'' describes major actions leading to waste disposal and site remediation. The primary purpose of this document is to provide a management tool for use by executives who need to quickly comprehend the waste management and environmental restoration programs. The Waste Management and Environmental Restoration Programs have been divided into missions. Waste Management consists of five missions: double-shell tank (DST) wastes; single-shell tank (SST) wastes (surveillance and interim storage, stabilization, and isolation); encapsulated cesium and strontium; solid wastes; and liquid effluents. Environmental Restoration consists of two missions: past practice units (PPU) (including characterization and assessment of SST wastes) and surplus facilities. For convenience, both aspects of SST wastes are discussed in one place. A general category of supporting activities is also included. 20 refs., 14 figs., 7 tabs.

  20. Host factors in retroviral integration and the selection of integration target sites

    PubMed Central

    Craigie, Robert; Bushman, Frederic D.

    2015-01-01

    In order to replicate, a retrovirus must integrate a DNA copy of the viral RNA genome into a chromosome of the host cell. The study of retroviral integration has advanced considerably in the last few years. Here we focus on host factor interactions and the linked area of integration targeting. Genome-wide screens for cellular factors affecting HIV replication have identified a series of host cell proteins that may mediate subcellular trafficking of integration complexes, nuclear import, and integration target site selection. The cell transcriptional co-activator protein LEDGF/p75 has been identified as a tethering factor important for HIV integration, and recently, BET proteins (Brd2, 4, and 4) have been identified as tethering factors for the gammaretroviruses. A new class of HIV inhibitors has been developed targeting the HIV-1 IN-LEDGF binding site, though surprisingly these inhibitors appear to block assembly late during replication and do not act at the integration step. Going forward, genome-wide studies of HIV-host interactions offer many new starting points to investigate HIV replication and identify potential new inhibitor targets. PMID:26104434

  1. Characteristics of the volatile organic compounds -- Arid Integrated Demonstration Site

    SciTech Connect

    Last, G.V.; Lenhard, R.J.; Bjornstad, B.N.; Evans, J.C.; Roberson, K.R.; Spane, F.A.; Amonette, J.E.; Rockhold, M.L.

    1991-10-01

    The Volatile Organic Compounds -- Arid Integrated Demonstration Program (VOC-Arid ID) is targeted at demonstration and testing of technologies for the evaluation and cleanup of volatile organic compounds and associated contaminants at arid DOE sites. The initial demonstration site is an area of carbon tetrachloride (CCl{sub 4}) contamination located near the center of the Hanford Site. The movement of CCl{sub 4} and other volatile organic contaminants in the subsurface is very complex. The problem at the Hanford Site is further complicated by the concurrent discharge of other waste constituents including acids, lard oil, organic phosphates, and transuranic radionuclides. In addition, the subsurface environment is very complex, with large spatial variabilities in hydraulic properties. A thorough understanding of the problem is essential to the selection of appropriate containment, retrieval, and/or in situ remedial technologies. The effectiveness of remedial technologies depends on knowing where the contaminants are, how they are held up in a given physical and chemical subsurface environment; and knowing the physical, chemical, and microbiological changes that are induced by the various remedial technologies.

  2. Histone Deacetylase Inhibition Rescues Gene Knockout Levels Achieved with Integrase-Defective Lentiviral Vectors Encoding Zinc-Finger Nucleases

    PubMed Central

    Pelascini, Laetitia P.L.; Maggio, Ignazio; Liu, Jin; Holkers, Maarten; Cathomen, Toni

    2013-01-01

    Abstract Zinc-finger nucleases (ZFNs) work as dimers to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. In doing so, they constitute powerful triggers to edit and to interrogate the function of genomic sequences in higher eukaryotes. A preferred route to introduce ZFNs into somatic cells relies on their cotransduction with two integrase-defective lentiviral vectors (IDLVs) each encoding a monomer of a functional heterodimeric pair. The episomal nature of IDLVs diminishes the risk of genotoxicity and ensures the strict transient expression profile necessary to minimize deleterious effects associated with long-term ZFN activity. However, by deploying IDLVs and conventional lentiviral vectors encoding HPRT1- or eGFP-specific ZFNs, we report that DSB formation at target alleles is limited after IDLV-mediated ZFN transfer. This IDLV-specific underperformance stems, to a great extent, from the activity of chromatin-remodeling histone deacetylases (HDACs). Importantly, the prototypic and U.S. Food and Drug Administration–approved inhibitors of metal-dependent HDACs, trichostatin A and vorinostat, respectively, did not hinder illegitimate recombination-mediated repair of targeted chromosomal DSBs. This allowed rescuing IDLV-mediated site-directed mutagenesis to levels approaching those achieved by using their isogenic chromosomally integrating counterparts. Hence, HDAC inhibition constitutes an efficacious expedient to incorporate in genome-editing strategies based on transient IDLV-mediated ZFN expression. Finally, we compared two of the most commonly used readout systems to measure targeted gene knockout activities based on restriction and mismatch-sensitive endonucleases. These experiments indicate that these enzymatic assays display a similar performance. PMID:24059449

  3. Hanford and Savannah River Site Programmatic and Technical Integration

    SciTech Connect

    Ramsey, William Gene

    2013-08-15

    Abstract only. The Hanford Site and the Savannah River Site (SRS) were the primary plutonium production facilities within the U.S. nuclear weapons complex. Radioactive wastes were generated as part of these missions and are stored in similar fashion. The majority of radioactivity maintained by the two sites is located in underground carbon steel tanks in the physical form of supernatant, saltcake, or sludge. Disposition of SRS tank waste is ongoing by converting it into glass (pathway for sludge and radionuclides separated from supernatant or dissolved saltcake) or cement (pathway for the decontaminated supernatant and dissolved saltcake). Tank closure activity has also begun at SRS and will continue for the duration of mission. The Hanford tank waste inventory is roughly 2/3rds larger than SRS's by volume- but nominally half the radioactivity. The baseline disposition path includes high-level and low-activity waste vitrification with separate disposition of contact-handled transuranic tank waste. Retrieval of tank waste from aging single­ shell tanks (SSTs) into double-shell tanks (DSTs) is currently ongoing. As vitrification commences later this decade, Hanford will be in a similar operations mode as SRS. Site integration is increasing as the missions align. The ongoing integration is centered on key issues that impact both sites- regardless of mission timeframe. Three recent workshop exchanges have been held to improve communication with the primary intent of improving operations and technical work organization. The topics of these workshops are as follows: DST space utilization, optimization, and closure; Waste Feed Qualification; and, Cementitious Waste Forms. Key goals for these and future exchanges include aligning research and technology, preparing for joint initiatives (to maximize budgetary value for the customer), and reviewing lessons learned. Each site has played a leading role in the development of technology and operational practices that can be

  4. Research at Hanford's 300 Area Integrated Field Challenge Site

    NASA Astrophysics Data System (ADS)

    Zachara, J.; Rockhold, M.; Fredrickson, J.; Vermeul, V.; Ward, A.; Liu, C.; McKinley, J.; Bjornstad, B.; Freshley, M.; Haggerty, R.; Kent, D.; Lichtner, P.; Rubin, Y.; Versteeg, R.; Zheng, C.

    2008-12-01

    The U.S. Department of Energy - Environmental Remediation Sciences Division is supporting an Integrated Field Challenge (IFC) Site at Hanford's 300 Area. This site, immediately adjacent to the Columbia R., is the location of a groundwater uranium plume that resulted from past discharges of liquid effluent to unlined disposal ponds and trenches. Plume concentrations have persisted above the drinking water standard in spite of the cessation of all liquid discharges more than 15 years ago and significant efforts to excavate and remove contaminated sediments. The persistence of the uranium plume is postulated to be a result of a complex interplay between hydrological, geochemical, and microbiological processes, and rate-limited mass transfer in the highly heterogeneous sediments. An IFC research site has been established in the area of one of the former disposal ponds to provide the infrastructure for developing improved understanding of the mechanisms responsible for the uranium plume persistence, with an ultimate goal of providing a robust, scientific basis for future remediation decisions. Thirty-five wells were installed at the site in FY08 for subsurface characterization and monitoring of field experiments. Detailed characterization studies have been performed or are currently underway using a variety of hydrological, geophysical, geochemical, and microbiological methods. In addition to field experiments, a series of column and bench-scale transport experiments are being performed to measure process interactions at smaller scales under well-controlled laboratory conditions, and to parameterize mechanistically-based model representations of these processes. This presentation gives an overview of guiding hypotheses for the 300 Area IFC Site, the well layout and instrumentation, initial characterization results, and ongoing or planned experiments and modeling activities.

  5. Integrated fate and toxicity assessment for site contaminants

    SciTech Connect

    MacDonell, Margaret; Peterson, John; Finster, Molly; Douglas, R.

    2007-07-01

    Understanding the fate and toxicity of environmental contaminants is essential to framing practical management decisions. Forms and bioavailable concentrations often change over time due to natural physical, chemical, and biological processes. For some sites, hundreds of contaminants may be of initial interest, and even small projects can involve a substantial number of contaminants. With multiple assessments common, attention to effectiveness and efficiency is important, and integrating fate and toxicity information provides a valuable way to focus the analyses. Fate assessments help identify what forms may be present where and when, while toxicity information indicates what health effects could result if people were exposed. The integration process is illustrated by an application for the Hanford site, to support long-term management decisions for the cesium and strontium capsules. Fate data, health-based benchmarks, and related toxicity information were effectively combined to indicate performance targets for chemicals and radionuclides identified for capsule leachate that could migrate to groundwater. More than 50 relevant benchmarks and toxicity context were identified for 15 of the 17 study contaminants; values for chronic drinking water exposure provided the common basis for selected indicators. For two chemicals, toxicity information was identified from the scientific literature to guide the performance targets. (authors)

  6. Arid sites stakeholder participation in evaluating innovative technologies: VOC-Arid Site Integrated Demonstration

    SciTech Connect

    Peterson, T.S.; McCabe, G.H.; Brockbank, B.R.

    1995-05-01

    Developing and deploying innovative environmental cleanup technologies is an important goal for the U.S. Department of Energy (DOE), which faces challenging remediation problems at contaminated sites throughout the United States. Achieving meaningful, constructive stakeholder involvement in cleanup programs, with the aim of ultimate acceptance of remediation decisions, is critical to meeting those challenges. DOE`s Office of Technology Development sponsors research and demonstration of new technologies, including, in the past, the Volatile Organic Compounds Arid Site Integrated Demonstration (VOC-Arid ID), hosted at the Hanford Site in Washington State. The purpose of the VOC-Arid ID has been to develop and demonstrate new technologies for remediating carbon tetrachloride and other VOC contamination in soils and ground water. In October 1994 the VOC-Arid ID became a part of the Contaminant Plume Containment and Remediation Focus Area (Plume Focus Area). The VOC Arid ID`s purpose of involving stakeholders in evaluating innovative technologies will now be carried on in the Plume Focus Area in cooperation with Site Technology Coordination Groups and Site Specific Advisory Boards. DOE`s goal is to demonstrate promising technologies once and deploy those that are successful across the DOE complex. Achieving that goal requires that the technologies be acceptable to the groups and individuals with a stake in DOE facility cleanup. Such stakeholders include groups and individuals with an interest in cleanup, including regulatory agencies, Native American tribes, environmental and civic interest groups, public officials, environmental technology users, and private citizens. This report documents the results of the stakeholder involvement program, which is an integral part of the VOC-Arid ID.

  7. Integrated Geophysical Analysis at a Legacy Test Site

    NASA Astrophysics Data System (ADS)

    Yang, X.; Mellors, R. J.; Sweeney, J. J.; Sussman, A. J.

    2015-12-01

    We integrate magnetic, electromagnetic (EM), gravity, and seismic data to develop a unified and consistent model of the subsurface at the U20ak site on Pahute Mesa at the Nevada National Nuclear Security Site (NNSS). The 1985 test, conducted in tuff at a depth of approximately 600 m did not collapse to the surface or produce a crater. The purpose of the geophysical measurements is to characterize the subsurface above and around the presumed explosion cavity. The magnetic data are used to locate steel borehole casings and pipes and are correlated with surface observations. The EM data show variation in lithology at depth and clear signatures from borehole casings and surface cables. The gravity survey detects a clear gravity low in the area of the explosion. The seismic data indicates shallow low velocity zone and indications of a deeper low velocity zones. In this study, we conduct 2D inversion of EM data for better characterization of site geology and use a common 3D density model to jointly interpret both the seismic and gravity data along with constraints on lithology boundaries from the EM. The integration of disparate geophysical datasets allows improved understanding of the non-prompt physical signatures of an underground nuclear explosion (UNE). LLNL Release Number: LLNL-ABS-675677. The authors express their gratitude to the National Nuclear Security Administration, Defense Nuclear Nonproliferation Research and Development, and the Comprehensive Inspection Technologies and UNESE working group, a multi-institutional and interdisciplinary group of scientists and engineers. This work was performed by Lawrence Livermore National Laboratory and Los Alamos National Laboratory under award number DE-AC52-06NA25946.

  8. Commercial integration and partnering at Savannah River Site

    SciTech Connect

    Steele, J.R.; Babione, R.A.; Shikashio, L.A.; Wacaster, A.J.; Paterson, A.D.

    1994-06-01

    Savannah River Site (SRS), particularly the Savannah River Technology Center (SRTC) with the experience from the first successful Integrated Technology Demonstration, can provide an excellent foundation for meeting DOE-EM`s objectives with the new DOE-EM five focus area approach. With this in mind, SRTC established an activity to pursue full commercialization of environmental technologies. This report is an assessment of the status of commercialization at SRS and provides recommendations for enhancement as well as some tools critical to implementation. A review was made of the current situation at SRS with regards to taking technology development to commercial fruition. This was done from the perspective of comparing it to known commercialization models and processes. It was found that SRTC already works through many of the steps in these processes. With integration and action-oriented efforts of the inclusion of business and market factors, SRTC could become an aggressive, successful developer of commercialized technologies. Commercial success criteria tools were developed with regards to integrating them with SRTC selection criteria to ensure that all critical factors are covered in technology commercialization project evaluations. Private investors are very clear that their interest lies in funding commercial enterprises, not merely technologies. Mobilizing private capital is critical to real job growth and long-term economic development. Also, potential industry partners were identified that are willing to be involved with SRS` technology applications and regional development efforts. As another important component to success, regional support organizations were reviewed and evaluated.

  9. Demonstration of Eastman Christensen horizontal drilling system -- Integrated Demonstration Site, Savannah River Site

    SciTech Connect

    Not Available

    1992-12-01

    An innovative horizontal drilling system was used to install two horizontal wells as part of an integrated demonstration project at the Savannah River Site (SRS), Aiken, South Carolina. The SRS is located in south-central South Carolina in the upper Coastal Plain physiographic province. The demonstration site is located near the A/M Area, and is currently known as the Integated Demonstration Site. The Department of Energy`s Office of Technology Development initiated an integrated demonstration of innovative technologies for cleanup of volatile organic compounds (VOCS) in soils and groundwater at the SRS in 1989. The overall goal of the program is to demonstrate, at a single location, multiple technologies in the fields of drilling, characterization, monitoring, and remediation. Innovative technologies are compared to one another and to baseline technologies in terms of technical performance and cost effectiveness. Transfer of successfully demonstrated technologies and systems to DOE environmental restoration organizations, to other government agencies, and to industry is a critical part of the program.

  10. Demonstration of Eastman Christensen horizontal drilling system -- Integrated Demonstration Site, Savannah River Site

    SciTech Connect

    Not Available

    1992-12-01

    An innovative horizontal drilling system was used to install two horizontal wells as part of an integrated demonstration project at the Savannah River Site (SRS), Aiken, South Carolina. The SRS is located in south-central South Carolina in the upper Coastal Plain physiographic province. The demonstration site is located near the A/M Area, and is currently known as the Integated Demonstration Site. The Department of Energy's Office of Technology Development initiated an integrated demonstration of innovative technologies for cleanup of volatile organic compounds (VOCS) in soils and groundwater at the SRS in 1989. The overall goal of the program is to demonstrate, at a single location, multiple technologies in the fields of drilling, characterization, monitoring, and remediation. Innovative technologies are compared to one another and to baseline technologies in terms of technical performance and cost effectiveness. Transfer of successfully demonstrated technologies and systems to DOE environmental restoration organizations, to other government agencies, and to industry is a critical part of the program.

  11. Overview of the Ridge 2000 Integrated Studies Sites

    NASA Astrophysics Data System (ADS)

    Fisher, C.

    2005-12-01

    The Ridge 2000 program is in its fourth year and fieldwork at each of the Integrated Studies Sites (ISS) is in full swing. Multidisciplinary monitoring continues at the EPR ISS with seismic, temperature, and current data being continuously recorded. Long-term fluid sampling programs aimed at furthering our understanding of temporal variations in the chemistry of high-temperature hydrothermal vents are continuing. In situ fluid chemistry monitors have been deployed for weeks, and longer deployments are planned as the technology matures. Nested within these monitoring studies are experiments addressing larval dispersal and changes in microbial and macrobiological communities. In early 2006, geodetic monitoring will begin, with an array of pressure gauges as well as a detailed compliance study. By early 2007, a 3-D multichannel seismic survey will have provided unprecedented details of the crustal structure at 9°50'N. Together these studies provide a strong framework for an interdisciplinary understanding of the links between the forces that produce a mid-ocean ridge spreading center and their manifestation on the seafloor. Fieldwork on the Endeavour segment of the Juan de Fuca ridge in 2005 also included a balance of monitoring, experimental, and sampling programs across a wide range of disciplines. Four interdisciplinary field programs were conducted to maintain and expand ongoing Ridge 2000 and proto-NEPTUNE experiments. These research programs continued development and testing in situ chemical and microbial sensors, conducted co-registered sampling of fluids, fauna, and chimney material, and recovered moorings that measured heat and chemical fluxes at the segment scale. High-resolution mapping was also completed at this site, which has been chosen for one of the two initial NEPTUNE Canada nodes to prepare the way for the collaborative, cabled observatory projects. The mapping cruise included 5 secondary school teachers as part of the REVEL outreach and education

  12. Envelope Determinants of Equine Lentiviral Vaccine Protection

    PubMed Central

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

    2013-01-01

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

  13. Nevada National Security Site Integrated Groundwater Sampling Plan, Revision 0

    SciTech Connect

    Marutzky, Sam; Farnham, Irene

    2014-10-01

    The purpose of the Nevada National Security Site (NNSS) Integrated Sampling Plan (referred to herein as the Plan) is to provide a comprehensive, integrated approach for collecting and analyzing groundwater samples to meet the needs and objectives of the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Field Office (NNSA/NFO) Underground Test Area (UGTA) Activity. Implementation of this Plan will provide high-quality data required by the UGTA Activity for ensuring public protection in an efficient and cost-effective manner. The Plan is designed to ensure compliance with the UGTA Quality Assurance Plan (QAP). The Plan’s scope comprises sample collection and analysis requirements relevant to assessing the extent of groundwater contamination from underground nuclear testing. This Plan identifies locations to be sampled by corrective action unit (CAU) and location type, sampling frequencies, sample collection methodologies, and the constituents to be analyzed. In addition, the Plan defines data collection criteria such as well-purging requirements, detection levels, and accuracy requirements; identifies reporting and data management requirements; and provides a process to ensure coordination between NNSS groundwater sampling programs for sampling of interest to UGTA. This Plan does not address compliance with requirements for wells that supply the NNSS public water system or wells involved in a permitted activity.

  14. Transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector.

    PubMed

    Hiripi, L; Negre, D; Cosset, F-L; Kvell, K; Czömpöly, T; Baranyi, M; Gócza, E; Hoffmann, O; Bender, B; Bosze, Zs

    2010-10-01

    Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit.

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

    PubMed Central

    Farley, Daniel C; McCloskey, Laura; Thorne, Barbara A; Tareen, Semih U; Nicolai, Christopher J; Campbell, David J; Bannister, Richard; Stewart, Hannah J; Pearson, Laura JE; Moyer, Bentley J; Robbins, Scott H; Zielinski, Leah; Kim, Tae; Radcliffe, Pippa A; Mitrophanous, Kyriacos A; Gombotz, Wayne R; Miskin, James E; Kelley-Clarke, Brenna

    2015-01-01

    It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector. PMID:26029728

  16. Selective transduction of astrocytic and neuronal CNS subpopulations by lentiviral vectors pseudotyped with Chikungunya virus envelope.

    PubMed

    Eleftheriadou, Ioanna; Dieringer, Michael; Poh, Xuan Ying; Sanchez-Garrido, Julia; Gao, Yunan; Sgourou, Argyro; Simmons, Laura E; Mazarakis, Nicholas D

    2017-04-01

    Lentiviral vectors are gene delivery vehicles that integrate into the host genome of dividing and non-dividing mammalian cells facilitating long-term transgene expression. Lentiviral vector versatility is greatly increased by incorporating heterologous viral envelope proteins onto the vector particles instead of the native envelope, conferring on these pseudotyped vectors a modified tropism and host range specificity. We investigated the pseudotyping efficiency of HIV-1 based lentiviral vectors with alphaviral envelope proteins from the Chikungunya Virus (CHIKV-G) and Sindbis Virus (SINV-G). Following vector production optimisation, titres for the CHIKV-G pseudotype were comparable to the VSV-G pseudotype but those for the SINV-G pseudotype were significantly lower. High titre CHIKV-G pseudotyped vector efficiently transduced various human and mouse neural cell lines and normal human astrocytes (NHA) in vitro. Although transduction was broad, tropism for NHAs was observed. In vivo stereotaxic delivery in striatum, thalamus and hippocampus respectively in the adult rat brain revealed localised transduction restricted to striatal astrocytes and hippocampal dentate granule neurons. Transduction of different subtypes of granule neurons from precursor to post-mitotic stages of differentiation was evident in the sub-granular zone and dentate granule cell layer. No significant inflammatory response was observed, but comparable to that of VSV-G pseudotyped lentiviral vectors. Robust long-term expression followed for three months post-transduction along with absence of neuroinflammation, coupled to the selective and unique neuron/glial tropism indicates that these vectors could be useful for modelling and gene therapy studies in the CNS.

  17. IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes.

    PubMed

    Verhoeyen, Els; Dardalhon, Valerie; Ducrey-Rundquist, Odile; Trono, Didier; Taylor, Naomi; Cosset, François-Loïc

    2003-03-15

    Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription, nuclear import, and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell-based gene therapy trials performed to date, cells have been activated via their cognate antigen receptor. To couple activation with gene transfer, we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However, transduction of highly purified resting T cells with these anti-CD3-displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here, we describe interleukin-7 (IL-7)-displaying HIV-1-derived vectors. Like recombinant IL-7, these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore, a single exposure to the IL-7-displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells, preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether, these results suggest that IL-7-displaying vectors could constitute interesting tools for T-cell-targeted gene therapy.

  18. Desmin-regulated Lentiviral Vectors for Skeletal Muscle Gene Transfer

    PubMed Central

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

    2009-01-01

    Lentiviral vectors (LVs) are highly attractive as a gene therapy agent as they are able to stably integrate their genomes in both dividing and nondividing cells and, in principle, provide long-term therapeutic benefit. However, their performance in skeletal muscle in adult animals has, to date, been disappointing. In order to gain clearer insight into their utility in this tissue type, we have conducted an extensive quantitative comparison of constitutive and muscle-specific promoter activities in skeletal muscle and nonmuscle systems following LV delivery in cell lines and neonatal mice. Our data show that LV delivery to hind leg skeletal muscle of neonatal mouse results in long-term transgene expression in adulthood. We find that the human desmin (DES) promoter/enhancer is the first muscle-specific control region to match the activity of the highly active constitutive human cytomegalovirus (hCMV) promoter/enhancer in skeletal muscle within a LV context both in vitro and in vivo. Furthermore, the DES promoter/enhancer provides six- to eightfold greater expression per viral copy than the muscle-specific human muscle creatine kinase (CKM) promoter/enhancer. DES also confers a more reproducible and tissue-specific transgene expression profile compared to CKM and is therefore a highly attractive regulatory element for use in muscle gene therapy vectors. PMID:19935780

  19. Lentiviral vectors for the treatment of primary immunodeficiencies.

    PubMed

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

    2014-07-01

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

  20. BIOTIC INTEGRITY OF STREAMS IN THE SAVANNAH RIVER SITE INTEGRATOR OPERABLE UNITS, 1996 TO 2003

    SciTech Connect

    Paller, M; Susan Dyer, S

    2004-11-08

    The Savannah River Site (SRS) has been divided into six Integrator Operable Units (IOUs) that correspond to the watersheds of the five major streams on the SRS (Upper Three Runs, Fourmile Branch, Pen Branch, Steel Creek, and Lower Three Runs) and the portions of the Savannah River and Savannah River Swamp associated with the SRS. The streams are the primary integrators within each IOU because they potentially receive, through surface or subsurface drainage, soluble contaminants from all waste sites within their watersheds. If these contaminants reach biologically significant levels, they would be expected to effect the numbers, types, and health of stream organisms. In this study, biological sampling was conducted within each IOU as a measure of the cumulative ecological effects of the waste sites within the IOUs. The use of information from biological sampling to assess environmental quality is often termed bioassessment. The IOU bioassessment program included 38 sites in SRS streams and nine sites in the Savannah River. Sampling was conducted in 1996 to 1998, 2000, and 2003. Four bioassessment methods were used to evaluate ecological conditions in the IOU streams: the Index of Biotic Integrity, the Fish Health Assessment Index, measurement of fish tissue contaminant levels, and two benthic macroinvertebrate indices. The Index of Biotic Integrity (IBI) is an EPA supported method based on comparison of ecologically important and sensitive fish assemblage variables between potentially disturbed and reference (i.e., undisturbed) sites. It is designed to assess the ability of a stream to support a self-sustaining biological community and ecological processes typical of undisturbed, natural conditions. Since many types of contaminants can bioaccumulate, fish tissue contaminant data were used to determine the types of chemicals fish were exposed to and their relative magnitudes among IOUs. The Fish Health Assessment Index (HAI) is an EPA supported method for assessing

  1. Repeat Hydrography at the Endeavour Integrated Study Site, 2004 - 2006

    NASA Astrophysics Data System (ADS)

    Kellogg, J. P.; McDuff, R. E.; Thomson, R. E.; Stahr, F. R.

    2006-12-01

    Significant differences exist between hydrographic transects made in the summers from 2004 to 2006 at the Endeavour Segment Integrated Study Site on the Juan de Fuca Ridge. Along and across axis sections describe the hydrographic conditions above the segment in three dimensions. The resulting sections allow for rapid evaluation of the characteristics of the neutrally buoyant plume over each of the vent fields and its location relative to the ridge axis. Results indicate heat content over the northern vent fields, Salty Dawg and Sasquatch, significantly increased between the summers of 2004 and 2005. In 2004, the plumes over these vent fields were barely discernable while in 2005 prominent plumes existed with potential temperature anomalies over 0.1°C. At the time of a rapid response cruise in March 2005, no significant change in the heat content of the water column was detected. By July 2005, dramatic changes had occurred in the overlying water column structure. The potential temperature anomaly section from 2005 is indicative of a thicker (about 75 m) neutrally buoyant plume with and substantially more heat at the north end of the valley. In 2004, the shallowest plume depth was 1900 m contrasted with 1830 m in 2005. Vent data being obtained by other RIDGE 2000 and UW Keck investigators will help constrain the underlying causes of these changes. New hydrography will be collected in August September 2006.

  2. Repeat Hydrography at the Endeavour Integrated Study Site, 2004 - 2005

    NASA Astrophysics Data System (ADS)

    Kellogg, J. P.; McDuff, R. E.; Thomson, R. E.; Stahr, F. R.

    2005-12-01

    Significant differences exist between hydrographic transects made in 2004 and 2005 at the Endeavour Segment Integrated Study Site on the Juan de Fuca Ridge. Sections that describe the conditions above the segment utilize twenty-one nearly uniformly spaced hydrographic stations from south of Mothra to north of the Sasquatch hydrothermal vent fields. Criteria used in choosing station locations included depth, ~500 m spacing from other stations, and being centrally located in the valley. The resulting sections allow for rapid evaluation of the characteristics of the neutrally buoyant plume over each of the vent fields. Preliminary results indicate heat content over the northern vent fields, Salty Dawg and Sasquatch, significantly increased between the summers of 2004 and 2005. In 2004, the plumes over these vent fields were barely discernable while in 2005 prominent plumes existed with potential temperature anomalies over 0.1°C. Vent data being obtained by other RIDGE 2000 and UW Keck investigators will help constrain the underlying causes of these changes. Isopycnals in the 2005 sections are also elevated along the entire length of the transect by approximately 50 m or more. The potential temperature anomaly section from 2005 is indicative of a thicker (about 75 m) neutrally buoyant plume and substantially more heat at the north end of the valley. In 2004, the shallowest plume depth was 1900 m contrasted with 1830 m in 2005.

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

    PubMed

    Hare, Stephen; Shun, Ming-Chieh; Gupta, Saumya Shree; Valkov, Eugene; Engelman, Alan; Cherepanov, Peter

    2009-01-01

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

  4. Oviduct-specific expression of human neutrophil defensin 4 in lentivirally generated transgenic chickens.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Current Research at the Endeavour Ridge 2000 Integrated Studies Site

    NASA Astrophysics Data System (ADS)

    Butterfield, D. A.; Kelley, D. S.; Ridge 2000 Community, R.

    2004-12-01

    Integrated geophysical, geological, chemical, and biological studies are being conducted on the Endeavour segment with primary support from NSF, the W.M. Keck Foundation, and NSERC (Canada). The research includes a seismic network, physical and chemical sensors, high-precision mapping and time-series sampling. Several research expeditions have taken place at the Endeavour ISS in the past year. In June 2003, an NSF-sponsored cruise with R.V. al T.G.Thompson/ROV al Jason2 installed microbial incubators in drill-holes in the sides of active sulfide chimneys and sampled rocks, fluids, and microbes in the Mothra and Main Endeavour Field (MEF). In July 2003, with al Thompson/Jason2, an NSF-LEXEN project at Baby Bare on Endeavour east flank conducted sampling through seafloor-penetrating probes, plus time-series sampling of fluids, microbes, and rocks at the MEF. In September 2003, with al Thompson/ROV al ROPOS, the Keck Proto-Neptune project installed a seismic network consisting of 1 broadband and 7 short-period seismometers, installation of chemical/physical sensors and time-series samplers for chemistry and microbiology in the MEF and Clam Bed sites, collection of rocks, fluids, animals, and microbes. In May/June 2004, an NSF-sponsored al Atlantis/Alvin cruise recovered sulfide incubators installed in 2003, redeployed a sulfide incubator, mapped MEF and Mothra vent fields with high-resolution Imagenix sonar, sampled fluids from MEF, Mothra, and Clam Bed, recovered year-long time-series fluid and microbial samplers from MEF and Clam Bed, recovered and installed hot vent temperature-resistivity monitors, cleaned up the MEF and deployed new markers at major sulfide structures. In August 2004, there were two MBARI/Keck-sponsored cruises with R.V. al Western Flyer/ROV al Tiburon. The first cruise completed the seismic network with addition of two more broadband seismometers and serviced all 7 short-period seismometers. al Tiburon then performed microbial and chemical

  7. Bovine leukemia virus integration site selection in cattle that develop leukemia.

    PubMed

    Murakami, Hironobu; Yamada, Takahito; Suzuki, Miho; Nakahara, Yusuke; Suzuki, Kazuhiko; Sentsui, Hiroshi

    2011-03-01

    It is essential for efficient replication of retroviruses that the viral genome is integrated into the host genome after reverse transcription. Some retroviruses are preferentially integrated into certain genomic regions that may differ depending on the disease. In this study, we analyzed the integration site of bovine leukemia virus (BLV) in leukemic cells and 55 integration sites were determined. Although the integration sites were not located in a particular chromosome, the BLV provirus was integrated into transcription units at a frequency of 43.6% (24/55) and the transcriptional direction of the provirus was in accordance with that of the integrated host genes in 62.5% (15/24). The integration sites were located in introns of the host gene, excluding only one site, which was located in downstream from a stop codon. BLV provirus was never found in a protein coding sequence (CDS) in this study. Moreover, the BLV provirus did not favor integration near transcription start sites and CpG islands, or repetitive sequences such as transposons. Therefore, the possibility that the integration of the BLV provirus disrupts the host gene is very low. Although a hot spot was not found in the BLV provirus integration sites, the provirus favored the integration into regions disadvantageous for viral gene expression since no integration site was preferentially located into/near CDS, transcription start site or CpG island. It is suggested that the integration site of the BLV provirus in leukemic cells is related to the suppression of viral gene expression. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Genetic engineering of human embryonic stem cells with lentiviral vectors.

    PubMed

    Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E

    2005-08-01

    Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

  9. Optimized production and concentration of lentiviral vectors containing large inserts.

    PubMed

    al Yacoub, Nadya; Romanowska, Malgorzata; Haritonova, Natalie; Foerster, John

    2007-07-01

    Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Here we evaluate published protocols for lentivirus production on a range of transfer vectors differing in size (7.5-13.2 kb). We present a modified virus production protocol robustly yielding useful titers (up to 10(7)/ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. Moreover, we find that virus recovery after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. We describe a fast (4 h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors. The protocols outlined in the current report should be useful for many labs interested in producing and concentrating high titer lentiviral stocks.

  10. Maintaining Site Integrity for Breeding Least Bell's Vireos

    Treesearch

    James M. Greaves

    1989-01-01

    Least Bell's vireos (Vireo bellii pusillus) exhibit a high degree of between-year fidelity to breeding area. Use of many sites in an isolated population in Santa Barbara, California, spans at least 10 years. Males and females show different types and degrees of tenacity to territories, as well as to some nest-sites. More than 60 percent male and...

  11. Integrated bird conservation web site in the United States

    Treesearch

    Roxanne Bogart; Chris Eberly; Elizabeth Martin

    2005-01-01

    In working towards a vision of integrated bird conservation, scientists, conservationists, land managers, and administrators are faced with a variety of scientific, managerial, administrative, and logistical challenges and complexities. The broad scope of integrated bird conservation requires organizations to work together to conserve birds across taxonomic groups,...

  12. Air Vehicle Technology Integration Program (AVTIP). Delivery Order 0054: Opportune Landing Site (OLS) Critical Experiment

    DTIC Science & Technology

    2008-04-01

    AFRL-RB-WP-TR-2009-3118 AIR VEHICLE TECHNOLOGY INTEGRATION PROGRAM (AVTIP) Delivery Order 0054: Opportune Landing Site (OLS) Critical...VEHICLE TECHNOLOGY INTEGRATION PROGRAM (AVTIP) Delivery Order 0054: Opportune Landing Site (OLS) Critical Experiment 5a. CONTRACT NUMBER F33615-00

  13. Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system.

    PubMed

    Nandy, Soumen; Srivastava, Vibha

    2011-08-01

    Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.

  14. Lentiviral hematopoietic stem cell gene therapy in inherited metabolic disorders.

    PubMed

    Wagemaker, Gerard

    2014-10-01

    After more than 20 years of development, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and storage disorders. This brief review summarizes the development and applications, focusing on the lysosomal enzyme deficiencies, especially Pompe disease.

  15. Lentiviral Hematopoietic Stem Cell Gene Therapy in Inherited Metabolic Disorders

    PubMed Central

    2014-01-01

    Abstract After more than 20 years of development, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and storage disorders. This brief review summarizes the development and applications, focusing on the lysosomal enzyme deficiencies, especially Pompe disease. PMID:25184354

  16. HIV integration sites in latently infected cell lines: evidence of ongoing replication.

    PubMed

    Symons, Jori; Chopra, Abha; Malatinkova, Eva; De Spiegelaere, Ward; Leary, Shay; Cooper, Don; Abana, Chike O; Rhodes, Ajantha; Rezaei, Simin D; Vandekerckhove, Linos; Mallal, Simon; Lewin, Sharon R; Cameron, Paul U

    2017-01-13

    Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

  17. Viral Determinants of Integration Site Preferences of Simian Immunodeficiency Virus-Based Vectors

    PubMed Central

    Monse, Hella; Laufs, Stephanie; Kuate, Seraphin; Zeller, W. Jens; Fruehauf, Stefan; Überla, Klaus

    2006-01-01

    Preferential integration into transcriptionally active regions of genomes has been observed for retroviral vectors based on gamma-retroviruses and lentiviruses. However, differences in the integration site preferences were detected, which might be explained by differences in viral components of the preintegration complexes. Viral determinants of integration site preferences have not been defined. Therefore, integration sites of simian immunodeficiency virus (SIV)-based vectors produced in the absence of accessory genes or lacking promoter and enhancer elements were compared. Similar integration patterns for the different SIV vectors indicate that vif, vpr, vpx, nef, env, and promoter or enhancer elements are not required for preferential integration of SIV into transcriptionally active regions of genomes. PMID:16873270

  18. Integrated Remote Sensing Modalities for Classification at a Legacy Test Site

    NASA Astrophysics Data System (ADS)

    Lee, D. J.; Anderson, D.; Craven, J.

    2016-12-01

    Detecting, locating, and characterizing suspected underground nuclear test sites is of interest to the worldwide nonproliferation monitoring community. Remote sensing provides both cultural and surface geological information over a large search area in a non-intrusive manner. We have characterized a legacy nuclear test site at the Nevada National Security Site (NNSS) using an aerial system based on RGB imagery, light detection and ranging, and hyperspectral imaging. We integrate these different remote sensing modalities to perform pattern recognition and classification tasks on the test site. These tasks include detecting cultural artifacts and exotic materials. We evaluate if the integration of different remote sensing modalities improves classification performance.

  19. Collaborating sites for community-oriented integrated care and health promotion

    PubMed Central

    Thomas, Paul

    2017-01-01

    London Journal of Primary Care wishes to develop a network of collaborating sites to better understand how to achieve community-oriented integrated care and health promotion in different contexts. A collaborating site can do more than submit papers. It can develop its own domain on the LJPC website, contribute to the development of LJPC policy, and stimulate discussions with other collaborating sites. At any time a collaborating site can opt out. In addition to securing papers for publication, a site might nurture a network of supporters, teach people to use multiple research and quality improvement methods, develop a system of governance for locally led inquiries, develop case studies of community-oriented integrated care and health promotion and facilitate within-site and between-site learning and change. PMID:28356918

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

    PubMed

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

    2008-04-01

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

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

    PubMed

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

    2014-04-24

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

  2. Comparative analysis of molecular strategies attenuating positional effects in lentiviral vectors carrying multiple genes.

    PubMed

    Osti, Daniela; Marras, Emanuela; Ceriani, Isabella; Grassini, Greta; Rubino, Tiziana; Viganò, Daniela; Parolaro, Daniela; Perletti, Gianpaolo

    2006-09-01

    Efficient, high-level expression of multiple genes is often difficult to achieve in retroviral vectors, due to positional effects affecting transcription of adjacent sequences. Here we describe the comparative analysis of different strategies for co-expressing two model cDNA sequences in the context of a second generation lentiviral vector system. A first option was based on the generation of a polycistronic construct by subcloning an internal ribosome entry site (IRES) sequence between tandem cDNAs. IRES-dependent translation of the cDNA placed downstream (3') of the first transgene was poor, and the protein was barely detectable in transduced cells. A similar result was obtained when both transgenes were placed under the transcriptional control of two independent internal promoters. When these independent transcription units were separated by the 5'HS4 chromatin insulator of the chicken beta-globin locus, a marked increase of the expression of the downstream protein was observed. Similarly, insertion of a polyadenylation sequence between the tandem transcription units fully restored - in transfection experiments - the expression of the downstream sequence, whose protein pattern was identical to the single-gene control, suggesting that in this specific construct transcriptional interference was the likely cause of the observed positional effects. These results indicate that chromatin insulator sequences can be useful molecular tools to overcome positional effects in the context of lentiviral vectors.

  3. Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression.

    PubMed

    Stahlhut, Maike; Schwarzer, Adrian; Eder, Matthias; Yang, Min; Li, Zhixiong; Morgan, Michael; Schambach, Axel; Kustikova, Olga S

    2015-09-01

    Constitutive co-expression of cooperating transgenes using retroviral integrating vectors is frequently used for genetic modification of different cell types to establish therapeutic or cancer models. However, such approaches are unable to dissect the influence of dose, order and reversibility of transgene expression on the fate of newly developed therapeutic/malignant phenotypes. We present a modular lentiviral vector system, which provides expression of constitutive and inducible components. To demonstrate its functionality, we constitutively expressed the well-described transcription factor Meis1 followed by inducible co-expression of collaborating partner Hoxa9 under the control of tetracycline responsive promoters in murine fibroblasts and primary hematopoietic progenitor cells (HPCs). Fluorescent markers to track transgene co-expression revealed tightly controlled, efficiently inducible and reversible but cell type dependent gene transfer over time. We demonstrated dose-dependent blockade of myeloid differentiation when both Meis1/Hoxa9 were concomitantly overexpressed in primary HPCs in vitro, but the absence of the transformed phenotype in non-induced samples or when Hoxa9 expression was down-regulated. This system combines the advantages of lentiviral gene transfer and the opportunity for drug-controlled co-expression of multiple transgenes to dissect, among others, gene networks governing complex cell behavior, such as proto-oncogene dose-dependent leukemogenic pathways or collaborating mechanisms of genes enhancing competitive fitness of hematopoietic cells.

  4. Nonintegrating Lentiviral Vectors Can Effectively Deliver Ovalbumin Antigen for Induction of Antitumor Immunity

    PubMed Central

    Hu, Biliang; Yang, Haiguang; Dai, Bingbing; Tai, April

    2009-01-01

    Abstract It has been demonstrated that nonintegrating lentiviral vectors (NILVs) are efficient in maintaining transgene expression in vitro and in vivo. Gene delivery by NILVs can significantly reduce nonspecific vector integration, which has been shown to cause malignant transformation in patients receiving gene therapy for X-linked severe combined immunodeficiency. Strong and sustained immune responses were observed after a single immunization with NILVs carrying viral antigens. However, there is no report to date that evaluates the efficacy of NILVs in inducing antigen-specific antitumor immunity. Using a well-characterized tumor model, we tested in vivo immunization with a self-inactivating lentiviral vector harboring a defective integrase. A high frequency of ovalbumin peptide (OVAp1)-specific CD8+ T cells and a substantial antibody response were detected in naive mice immunized with an NILV encoding an OVA transgene. Furthermore, this immunization method completely protected the mice against the growth of E.G7 tumor cells expressing the OVA antigen. Thus, this study provides evidence that immunization using NILVs can be a safe and promising approach for exploring cancer immunotherapy. PMID:19663564

  5. Lentiviral vectors as tools to understand central nervous system biology in mammalian model organisms.

    PubMed

    Parr-Brownlie, Louise C; Bosch-Bouju, Clémentine; Schoderboeck, Lucia; Sizemore, Rachel J; Abraham, Wickliffe C; Hughes, Stephanie M

    2015-01-01

    Lentiviruses have been extensively used as gene delivery vectors since the mid-1990s. Usually derived from the human immunodeficiency virus genome, they mediate efficient gene transfer to non-dividing cells, including neurons and glia in the adult mammalian brain. In addition, integration of the recombinant lentiviral construct into the host genome provides permanent expression, including the progeny of dividing neural precursors. In this review, we describe targeted vectors with modified envelope glycoproteins and expression of transgenes under the regulation of cell-selective and inducible promoters. This technology has broad utility to address fundamental questions in neuroscience and we outline how this has been used in rodents and primates. Combining viral tract tracing with immunohistochemistry and confocal or electron microscopy, lentiviral vectors provide a tool to selectively label and trace specific neuronal populations at gross or ultrastructural levels. Additionally, new generation optogenetic technologies can be readily utilized to analyze neuronal circuit and gene functions in the mature mammalian brain. Examples of these applications, limitations of current systems and prospects for future developments to enhance neuroscience knowledge will be reviewed. Finally, we will discuss how these vectors may be translated from gene therapy trials into the clinical setting.

  6. Initial Characterization of Integrase-Defective Lentiviral Vectors for Pancreatic Cancer Gene Therapy.

    PubMed

    Hanoun, Naima; Gayral, Marion; Pointreau, Adeline; Buscail, Louis; Cordelier, Pierre

    2016-02-01

    The vast majority (85%) of pancreatic ductal adenocarcinomas (PDACs) are discovered at too of a late stage to allow curative surgery. In addition, PDAC is highly resistant to conventional methods of chemotherapy and radiotherapy, which only offer a marginal clinical benefit. Consequently, the prognosis of this cancer is devastating, with a 5-year survival rate of less than 5%. In this dismal context, we recently demonstrated that PDAC gene therapy using nonviral vectors is safe and feasible, with early signs of efficacy in selected patients. Our next step is to transfer to the clinic HIV-1-based lentiviral vectors (LVs) that outshine other therapeutic vectors to treat experimental models of PDAC. However, a primary safety issue presented by LVs that may delay their use in patients is the risk of oncogenesis after vector integration in the host's cell DNA. Thus, we developed a novel anticancerous approach based on integrase-defective lentiviral vectors (IDLVs) and demonstrated that IDLVs can be successfully engineered to transiently deliver therapeutic genes to inhibit pancreatic cancer cells proliferation. This work stems for the use of therapeutic IDLVs for the management of PDAC, in forthcoming early phase gene therapy clinical trial for this disease with no cure.

  7. Sites of retroviral DNA integration: From basic research to clinical applications.

    PubMed

    Serrao, Erik; Engelman, Alan N

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency.

  8. Sites of Retroviral DNA Integration: From Basic Research to Clinical Applications

    PubMed Central

    Serrao, Erik; Engelman, Alan N.

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of the viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with HIV-1 can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or AIDS patients on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency. PMID:26508664

  9. Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter.

    PubMed

    Cooper, Aaron R; Lill, Georgia R; Gschweng, Eric H; Kohn, Donald B

    2015-01-01

    Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors.

  10. An integrated remote sensing approach for identifying ecological range sites. [parker mountain

    NASA Technical Reports Server (NTRS)

    Jaynes, R. A.

    1983-01-01

    A model approach for identifying ecological range sites was applied to high elevation sagebrush-dominated rangelands on Parker Mountain, in south-central Utah. The approach utilizes map information derived from both high altitude color infrared photography and LANDSAT digital data, integrated with soils, geological, and precipitation maps. Identification of the ecological range site for a given area requires an evaluation of all relevant environmental factors which combine to give that site the potential to produce characteristic types and amounts of vegetation. A table is presented which allows the user to determine ecological range site based upon an integrated use of the maps which were prepared. The advantages of identifying ecological range sites through an integrated photo interpretation/LANDSAT analysis are discussed.

  11. Characterization of a third generation lentiviral vector pseudotyped with Nipah virus envelope proteins for endothelial cell transduction.

    PubMed

    Witting, S R; Vallanda, P; Gamble, A L

    2013-10-01

    Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, third generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared with vesicular stomatitis virus pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in third generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells.

  12. Dystrophin Delivery to Muscles of mdx Mice Using Lentiviral Vectors Leads to Myogenic Progenitor Targeting and Stable Gene Expression

    PubMed Central

    Kimura, En; Li, Sheng; Gregorevic, Paul; Fall, Brent M; Chamberlain, Jeffrey S

    2009-01-01

    To explore whether stable transduction of myogenic stem cells using lentiviral vectors could be of benefit for treating dystrophic muscles, we generated vectors expressing a functional microdystrophin/enhanced green fluorescence protein fusion (µDys/eGFP) gene. Lentiviral vector injection into neonatal mdx4cv muscles resulted in widespread and stable expression of dystrophin for at least 2 years. This expression resulted in a significant amelioration of muscle pathophysiology as assessed by a variety of histological and functional assays. To assess whether this long-term expression was accompanied by stable transduction of satellite cells, we harvested muscle mononuclear cells 1 year after vector injection. Up to 20% of the cultured myoblast colonies expressed the µDys/eGFP transgene following myotube formation. Furthermore, transplantation of the muscle mononuclear cells into secondary mdx4cv recipients showed their ability to regenerate dystrophin-expressing myofibers in vivo. The ability to isolate myogenic cells able to form dystrophin-positive myotubes or myofibers in vitro and in vivo >1 year postinjection indicates that the vectors stably transduced muscle satellite cells, or a progenitor of such cells, in neonatal mdx4cv muscles. These studies suggest that integrating lentiviral vectors have potential utility for gene therapy of muscular dystrophy. PMID:19888194

  13. Retrovirus Integration Database (RID): a public database for retroviral insertion sites into host genomes.

    PubMed

    Shao, Wei; Shan, Jigui; Kearney, Mary F; Wu, Xiaolin; Maldarelli, Frank; Mellors, John W; Luke, Brian; Coffin, John M; Hughes, Stephen H

    2016-07-04

    The NCI Retrovirus Integration Database is a MySql-based relational database created for storing and retrieving comprehensive information about retroviral integration sites, primarily, but not exclusively, HIV-1. The database is accessible to the public for submission or extraction of data originating from experiments aimed at collecting information related to retroviral integration sites including: the site of integration into the host genome, the virus family and subtype, the origin of the sample, gene exons/introns associated with integration, and proviral orientation. Information about the references from which the data were collected is also stored in the database. Tools are built into the website that can be used to map the integration sites to UCSC genome browser, to plot the integration site patterns on a chromosome, and to display provirus LTRs in their inserted genome sequence. The website is robust, user friendly, and allows users to query the database and analyze the data dynamically. https://rid.ncifcrf.gov ; or http://home.ncifcrf.gov/hivdrp/resources.htm .

  14. Production of germline transgenic pigs co-expressing double fluorescent proteins by lentiviral vector.

    PubMed

    Chen, Xiao-Yu; Zhu, Zhi-Wei; Yu, Fu-Xian; Huang, Jing; Hu, Xiao-Rui; Pan, Jian-Zhi

    2016-11-01

    Genomic integration of transgene by lentiviral vector has been proved an efficient method to produce single-transgenic animals. But it failed to create multi-gene transgenic offspring. Here, we have exploited lentivirus to generate the double-transgenic piglets through the female germline. The recombinant lentivirus containing fluorescent proteins genes (DsRed1 and Venus) were injected into the perivitelline space of 2-cell stage in vitro porcine embryos. Compared to control group, there was no significantly decreased in the proportion of blastocysts, and the two fluorescent protein genes were co-expressed in almost all the injected embryos. Total of 32 injected in vitro embryos were transferred to 2 recipients. One recipient gave birth of three live offspring, and one female piglet was identified as genomic transgene integration by PCR analysis. Subsequently, the female transgenic founder was mated naturally with a wild-type boar and gave birth of two litters of total 23 F(1) generation piglets, among which Venus and DsRed1 genes were detected in 11 piglets and 10 kinds of organs by PCR and RT-PCR respectively. The co-expression of two fluorescent proteins was visible in four different frozen tissue sections from the RT-PCR positive piglets, and 3 to 5 copies of the transgenes were detected to be integrated into the second generation genome by southern blotting analysis. The transgenes were heritable and stably integrated in the F(1) generation. The results indicated for the first time that lentiviral vector combined with natural mating has the potential to become a simple and practical technology to create germline double-transgenic livestock or biomedical animals.

  15. Genomic characterization of viral integration sites in HPV-related cancers.

    PubMed

    Bodelon, Clara; Untereiner, Michael E; Machiela, Mitchell J; Vinokurova, Svetlana; Wentzensen, Nicolas

    2016-11-01

    Persistent infection with carcinogenic human papillomaviruses (HPV) causes the majority of anogenital cancers and a subset of head and neck cancers. The HPV genome is frequently found integrated into the host genome of invasive cancers. The mechanisms of how it may promote disease progression are not well understood. Thoroughly characterizing integration events can provide insights into HPV carcinogenesis. Individual studies have reported limited number of integration sites in cell lines and human samples. We performed a systematic review of published integration sites in HPV-related cancers and conducted a pooled analysis to formally test for integration hotspots and genomic features enriched in integration events using data from the Encyclopedia of DNA Elements (ENCODE). Over 1,500 integration sites were reported in the literature, of which 90.8% (N = 1,407) were in human tissues. We found 10 cytobands enriched for integration events, three previously reported ones (3q28, 8q24.21 and 13q22.1) and seven additional ones (2q22.3, 3p14.2, 8q24.22, 14q24.1, 17p11.1, 17q23.1 and 17q23.2). Cervical infections with HPV18 were more likely to have breakpoints in 8q24.21 (p = 7.68 × 10(-4) ) than those with HPV16. Overall, integration sites were more likely to be in gene regions than expected by chance (p = 6.93 × 10(-9) ). They were also significantly closer to CpG regions, fragile sites, transcriptionally active regions and enhancers. Few integration events occurred within 50 Kb of known cervical cancer driver genes. This suggests that HPV integrates in accessible regions of the genome, preferentially genes and enhancers, which may affect the expression of target genes.

  16. Comparative fitness assessment of Anopheles stephensi transgenic lines receptive to site-specific integration.

    PubMed

    Amenya, D A; Bonizzoni, M; Isaacs, A T; Jasinskiene, N; Chen, H; Marinotti, O; Yan, G; James, A A

    2010-04-01

    Genetically modified mosquitoes that are unable to transmit pathogens offer opportunities for controlling vector-borne diseases such as malaria and dengue. Site-specific gene recombination technologies are advantageous in the development of these insects because antipathogen effector genes can be inserted at integration sites in the genome that cause the least alteration in mosquito fitness. Here we describe Anopheles stephensi transgenic lines containing phi C31 attP'docking' sites linked to a fluorescent marker gene. Chromosomal insertion sites were determined and life-table parameters were assessed for transgenic mosquitoes of each line. No significant differences in fitness between the transgenic and nontransgenic mosquitoes were detected in this study. These transgenic lines are suitable for future site-specific integrations of antiparasite transgenes into the attP sites.

  17. TG1 integrase-based system for site-specific gene integration into bacterial genomes.

    PubMed

    Muroi, Tetsurou; Kokuzawa, Takaaki; Kihara, Yoshihiko; Kobayashi, Ryuichi; Hirano, Nobutaka; Takahashi, Hideo; Haruki, Mitsuru

    2013-05-01

    Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ∼2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ∼10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ∼10-kbp DNA into the genome with an efficiency of ∼10(4) transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli "centromere" analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC-migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.

  18. Endogenous lentiviral elements in the weasel family (Mustelidae).

    PubMed

    Han, Guan-Zhu; Worobey, Michael

    2012-10-01

    Endogenous retroviruses provide molecular fossils for studying the ancient evolutionary history of retroviruses. Here, we report our independent discovery and analysis of endogenous lentiviral insertions (Mustelidae endogenous lentivirus [MELV]) within the genomes of weasel family (Mustelidae). Genome-scale screening identified MELV elements in the domestic ferret (Mustela putorius furo) genome (MELVmpf). MELVmpf exhibits a typical lentiviral genomic organization. Phylogenetic analyses position MELVmpf basal to either primate lentiviruses or feline immunodeficiency virus. Moreover, we verified the presence of MELV insertions in the genomes of several species of the Lutrinae and Mustelinae subfamilies but not the Martinae subfamily, suggesting that the invasion of MELV into the Mustelidae genomes likely took place between 8.8 and 11.8 Ma. The discovery of MELV in weasel genomes extends the host range of lentiviruses to the Caniformia (order Carnivora) and provides important insights into the prehistoric diversity of lentiviruses.

  19. Lentiviral vectors: turning a deadly foe into a therapeutic agent.

    PubMed

    Trono, D

    2000-01-01

    The past 3 years have witnessed the spectacular irruption of lentiviral vectors into the limelight of the gene therapy scene. Owing to their ability to deliver transgenes in tissues that had long appeared irremediably refractory to stable genetic manipulation, lentivectors have opened fresh perspectives for the genetic treatment of a wide array of hereditary as well as acquired disorders, and a concrete proposal for their clinical use seems imminent. This article traces the path that has led to this rapid development and describes the current state of the art in the design and production of lentiviral vectors. The important question of biosafety is discussed. This system seems to have the edge over other gene delivery tools for particular targets, however, there remain several issues to be resolved before lentivectors make it to the bedside. Gene Therapy (2000) 7, 20-23.

  20. Integration of geophysics within the Argonne expedited site characterization Program at a site in the southern High Plains

    SciTech Connect

    Hastings, B.; Hildebrandt, G.; Meyer, T.; Saunders, W.; Burton, J.C.

    1995-05-01

    An Argonne National Laboratory Expedited Site Characterization (ESC) program was carried out at a site in the central United States. The Argonne ESC process emphasizes an interdisciplinary approach in which all available information is integrated to produce as complete a picture as possible of the geologic and hydrologic controls on contaminant distribution and transport. As part of this process, all pertinent data that have been collected from previous investigations are thoroughly analyzed before a decision is made to collect additional information. A seismic reflection program recently concluded at the site had produced inconclusive results. Before we decided whether another acquisition program was warranted, we examined the existing data set to evaluate the quality of the raw data, the appropriateness of the processing sequence, and the integrity of the interpretation. We decided that the field data were of sufficient quality to warrant reprocessing and reinterpretation. The main thrust of the reprocessing effort was to enhance the continuity of a shallow, low-frequency reflection identified as a perching horizon within the Ogallala formation. The reinterpreted seismic data were used to locate the boundaries of the perched aquifer, which helped to guide the Argonne ESC drilling and sampling program. In addition, digitized geophysical well log data from previous drilling programs were reinterpreted and integrated into the geologic and hydrogeologic model.

  1. Measles virus glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both SLAM and CD46 entry receptors.

    PubMed

    Frecha, Cecilia; Lévy, Camille; Costa, Caroline; Nègre, Didier; Amirache, Fouzia; Buckland, Robin; Russell, Steven J; Cosset, François-Loïc; Verhoeyen, Els

    2011-06-01

    Gene transfer into quiescent T and B cells is of importance for gene therapy and immunotherapy approaches to correct hematopoietic disorders. Previously, we generated lentiviral vectors (LVs) pseudotyped with the Edmonston measles virus (MV) hemagglutinin and fusion glycoproteins (Hgps and Fgps) (H/F-LVs), which, for the first time, allowed efficient transduction of quiescent human B and T cells. These target cells express both MV entry receptors used by the vaccinal Edmonston strain, CD46 and signaling lymphocyte activation molecule (SLAM). Interestingly, LVs pseudotyped with an MV Hgp, blind for the CD46 binding site, were completely inefficient for resting-lymphocyte transduction. Similarly, SLAM-blind H mutants that recognize only CD46 as the entry receptor did not allow stable LV transduction of resting T cells. The CD46-tropic LVs accomplished vector-cell binding, fusion, entry, and reverse transcription at levels similar to those achieved by the H/F-LVs, but efficient proviral integration did not occur. Our results indicate that both CD46 and SLAM binding sites need to be present in cis in the Hgp to allow successful stable transduction of quiescent lymphocytes. Moreover, the entry mechanism utilized appears to be crucial: efficient transduction was observed only when CD46 and SLAM were correctly engaged and an entry mechanism that strongly resembles macropinocytosis was triggered. Taken together, our results suggest that although vector entry can occur through the CD46 receptor, SLAM binding and subsequent signaling are also required for efficient LV transduction of quiescent lymphocytes to occur.

  2. Analysis of Partial Recombinants in Lentiviral Vector Preparations

    PubMed Central

    Kuate, Seraphin; Marino, Michael P.

    2014-01-01

    Abstract The presence of replication-competent lentivirus (RCL) in lentiviral vector preparations is a major safety concern for clinical applications of such vectors. RCL are believed to emerge from rare recombinant vector genomes that are referred to as partial recombinants or Psi-Gag recombinants. To quantitatively determine the fraction of partial recombinants in lentiviral vector preparations and to analyze them at the DNA sequence level, we established a drug selection assay involving a lentiviral packaging construct containing a drug-resistance gene encoding blasticidin (BSD) resistance. Upon transduction of target cells, the BSD resistance gene confers BSD resistance to the transduced cells. The results obtained indicate that there were up to 156 BSD-resistant colonies in a total of 106 transducing vector particles. The predicted recombination events were verified by polymerase chain reaction using genomic DNA obtained from BSD-resistant cell clones and by DNA sequence analysis. In an attempt to reduce the emergence of partial recombinants, sequence overlaps between the packaging and the vector constructs were reduced by substituting the Rev response element (RRE) present in the vector construct using a heterologous RRE element derived from simian immunodeficiency virus (SIVmac239). The results obtained showed that a reduction of sequence overlaps resulted in an up to sevenfold reduction of the frequency of BSD-resistant colonies, indicating that the capacity to form partial recombinants was diminished. PMID:24367910

  3. Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

    PubMed

    Meneghini, Vasco; Lattanzi, Annalisa; Tiradani, Luigi; Bravo, Gabriele; Morena, Francesco; Sanvito, Francesca; Calabria, Andrea; Bringas, John; Fisher-Perkins, Jeanne M; Dufour, Jason P; Baker, Kate C; Doglioni, Claudio; Montini, Eugenio; Bunnell, Bruce A; Bankiewicz, Krystof; Martino, Sabata; Naldini, Luigi; Gritti, Angela

    2016-05-01

    Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non-human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe-affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD. © 2016

  4. Retrovirus DNA termini bound by integrase communicate in trans for full-site integration in vitro.

    PubMed

    McCord, M; Chiu, R; Vora, A C; Grandgenett, D P

    1999-07-05

    Integration of linear retrovirus DNA involves the concerted insertion of the viral termini (full-site integration) into the host chromosome. We investigated the interactions that occur between long terminal repeat (LTR) termini bound by avian retrovirus integrase (IN) for full-site integration in vitro. Wild-type (wt) or mutant LTR donors that possess gain-of-function ("G") or loss-of-function ("L") for full-site integration activity were used. G LTR termini are characterized as having significantly higher strand transfer activity than the wt and the L LTR termini. L LTR mutations are classified as partially or extremely defective for strand transfer activity. The L mutations were further classified by their ability to either permit or block the assembly of G or wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. We demonstrated that avian myeloblastosis virus IN bound to G LTR termini increased the incorporation of partially defective L LTR termini into nucleoprotein complexes that were capable of full-site integration. The observed full-site integration activity of these assembled nucleoprotein complexes appeared to be influenced by each individual IN-LTR complex in trans. In contrast, extremely defective L LTR termini exhibited the ability to effectively block the assembly of wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. Data from nonspecific DNA competition experiments suggested that IN had an apparent higher affinity for G LTR donor termini than for partially defective L LTR donor termini as measured by full-site integration activity. However, assembled nucleoprotein complexes containing either two G or two L LTR donors were stable, having a similar half-life of approximately 2 h on ice. The results suggest that LTR termini bound by IN exhibit an allosteric effect to modulate full-site integration in vitro. Similar regulatory controls also appear to exist in vivo between the wt U3 and wt U5 LTR

  5. Integration Site Selection by the Bacteroides Conjugative Transposon CTnBST▿

    PubMed Central

    Song, Bo; Shoemaker, Nadja B.; Gardner, Jeffrey F.; Salyers, Abigail A.

    2007-01-01

    A newly discovered Bacteroides conjugative transposon (CTn), CTnBST, integrates more site specifically than two other well-studied CTns, the Bacteroides CTn CTnDOT and the enterococcal CTn Tn916. Moreover, the integrase of CTnBST, IntBST, had the C-terminal 6-amino-acid signature that is associated with the catalytic regions of members of the tyrosine recombinase family, most of which integrate site specifically. Also, in most of these integrases, all of the conserved amino acids are required for integration. In the case of IntBST, however, we found that changing three of the six conserved amino acids in the signature, one of which was the presumed catalytic tyrosine, resulted in a 1,000-fold decrease in integration frequency. Changes in the other amino acids had little or no effect. Thus, although the CTnBST integrase still seems to be a member of the tyrosine recombinase family, it clearly differs to some extent from other members of the family in its catalytic site. We also determined the sequence requirements for CTnBST integration in the 18-bp region where the crossover occurs preferentially during integration. We found that CTnBST integrates in this preferred site about one-half of the time but can also use other sites. A consensus sequence was tentatively derived by comparison of a few secondary sites: AATCTGNNAAAT. We report here that within the consensus region, no single base change affected the frequency of integration. However, 3 bp at one end of the consensus sequence (CTG) proved to be essential for integration into the preferred site. This sequence appeared to be at one end of a 7-bp crossover region, CTGNNAA. The other bases could vary without affecting either integration frequency or specificity. Thus, in contrast to well-studied site-specific recombinases which require homology throughout the crossover region, integration of CTnBST requires homology at one end of the crossover region but not at the other end. PMID:17616597

  6. HIV promoter integration site primarily modulates transcriptional burst size rather than frequency.

    PubMed

    Skupsky, Ron; Burnett, John C; Foley, Jonathan E; Schaffer, David V; Arkin, Adam P

    2010-09-30

    Mammalian gene expression patterns, and their variability across populations of cells, are regulated by factors specific to each gene in concert with its surrounding cellular and genomic environment. Lentiviruses such as HIV integrate their genomes into semi-random genomic locations in the cells they infect, and the resulting viral gene expression provides a natural system to dissect the contributions of genomic environment to transcriptional regulation. Previously, we showed that expression heterogeneity and its modulation by specific host factors at HIV integration sites are key determinants of infected-cell fate and a possible source of latent infections. Here, we assess the integration context dependence of expression heterogeneity from diverse single integrations of a HIV-promoter/GFP-reporter cassette in Jurkat T-cells. Systematically fitting a stochastic model of gene expression to our data reveals an underlying transcriptional dynamic, by which multiple transcripts are produced during short, infrequent bursts, that quantitatively accounts for the wide, highly skewed protein expression distributions observed in each of our clonal cell populations. Interestingly, we find that the size of transcriptional bursts is the primary systematic covariate over integration sites, varying from a few to tens of transcripts across integration sites, and correlating well with mean expression. In contrast, burst frequencies are scattered about a typical value of several per cell-division time and demonstrate little correlation with the clonal means. This pattern of modulation generates consistently noisy distributions over the sampled integration positions, with large expression variability relative to the mean maintained even for the most productive integrations, and could contribute to specifying heterogeneous, integration-site-dependent viral production patterns in HIV-infected cells. Genomic environment thus emerges as a significant control parameter for gene expression

  7. DNFSB Recommendation 94-1 Hanford Site Integrated Stabilization Management Plan. Volume 1

    SciTech Connect

    Gerber, E.W.

    1995-10-01

    The US Department of Energy (DOE) has developed an Integrated Program Plan (IPP) to address concerns identified in Defense Nuclear Facilities Safety Board Recommendation 94-1. The IPP describes the actions that DOE plans to implement at its various sites to convert excess fissile materials to forms or conditions suitable for safe interim storage. The baseline IPP was issued as DOE`s Defense Nuclear Facilities Safety Board (DNFSB) Recommendation 94-1 Implementation Plan (IP), which was transmitted to the DNFSB on February 28, 1995. The IPP is being further developed to include complex-wide requirements for research and development and a long-range facility requirements section. The planned additions to the baseline IPP are being developed based on a systems engineering approach that integrates facilities and capabilities at the various DOE sites and focuses on attaining safe interim storage with minimum safety risks and environmental impacts. Each affected DOE site has developed a Site Integrated Stabilization Management Plan (SISMP) to identify individual site plans to implement the DNFSB Recommendation 94-1 and to provide a basis for formulating planned additions to the IPP. The SISMPs were developed based on the objectives, requirements, and commitments identified in the baseline DNFSB Recommendation 94-1 IPP. The SISMPs will be periodically updated to reflect improved integration between DOE sites as identified during the IPP systems engineering evaluations.

  8. The use of HIV-1 integration site analysis information in clinical studies aiming at HIV cure.

    PubMed

    Kiselinova, Maja; De Spiegelaere, Ward; Vandekerckhove, Linos

    2016-07-01

    The mechanisms for the establishment and the persistence of the latent HIV-1 reservoir remain to be completely defined. HIV-1 infection is characterised by the integration of the reverse transcribed proviral DNA into the host's genome. This integrated proviral DNA can remain replication silent, but a small part of it is fully competent to restart viral replication when treatment is interrupted. Hence, this replication-competent provirus is the cause of viral rebound and is called the viral reservoir. The exact site of proviral integration within the host's cellular chromosome may affect the transcriptional activity of HIV. Thanks to recent technological advances, HIV-1 integration site analysis has been used to assess HIV-1 reservoirs in HIV-infected individuals. Analysis of HIV-1 integration sites in infected individuals undergoing suppressive ART led to identification of expanded clonal cell populations, indicating that clonal proliferation of the proviral reservoir may contribute to the long-term persistence of viral reservoirs. Here we describe the findings of several clinical studies, where a comprehensive HIV-1 integration site analysis was performed.

  9. Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells.

    PubMed

    Lévy, Camille; Verhoeyen, Els; Cosset, François-Loïc

    2015-10-01

    Since they allow gene integration into their host genome, lentiviral vectors (LVs) have strong therapeutic potentials, as emphasized by recent clinical trials. The surface-display of the pantropic vesicular stomatitis virus G glycoprotein (VSV-G) on LVs resulted in powerful tools for fundamental and clinical research. However, improved LVs are required either to genetically modify cell types not permissive to classical VSV-G-LVs or to restrict entry to specific cell types. Incorporation of heterologous viral glycoproteins (gps) on LVs often require modification of their cytoplasmic tails and ligands can be inserted into their ectodomain to target LVs to specific receptors. Recently, measles virus (MV) gps have been identified as strong candidates for LV-retargeting to multiple cell types, with the potential to evolve toward clinical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Therapeutic benefit of lentiviral-mediated neonatal intracerebral gene therapy in a mouse model of globoid cell leukodystrophy

    PubMed Central

    Lattanzi, Annalisa; Salvagno, Camilla; Maderna, Claudio; Benedicenti, Fabrizio; Morena, Francesco; Kulik, Willem; Naldini, Luigi; Montini, Eugenio; Martino, Sabata; Gritti, Angela

    2014-01-01

    Globoid cell leukodystrophy (GLD) is an inherited lysosomal storage disease caused by β-galactocerebrosidase (GALC) deficiency. Gene therapy (GT) should provide rapid, extensive and lifetime GALC supply in central nervous system (CNS) tissues to prevent or halt irreversible neurologic progression. Here we used a lentiviral vector (LV) to transfer a functional GALC gene in the brain of Twitcher mice, a severe GLD model. A single injection of LV.GALC in the external capsule of Twitcher neonates resulted in robust transduction of neural cells with minimal and transient activation of inflammatory and immune response. Importantly, we documented a proficient transduction of proliferating and post-mitotic oligodendroglia, a relevant target cell type in GLD. GALC activity (30–50% of physiological levels) was restored in the whole CNS of treated mice as early as 8 days post-injection. The early and stable enzymatic supply ensured partial clearance of storage and reduction of psychosine levels, translating in amelioration of histopathology and enhanced lifespan. At 6 months post-injection in non-affected mice, LV genome persisted exclusively in the injected region, where transduced cells overexpressed GALC. Integration site analysis in transduced brain tissues showed no aberrant clonal expansion and preferential targeting of neural-specific genes. This study establishes neonatal LV-mediated intracerebral GT as a rapid, effective and safe therapeutic intervention to correct CNS pathology in GLD and provides a strong rationale for its application in this and similar leukodystrophies, alone or in combination with therapies targeting the somatic pathology, with the final aim of providing an effective and timely treatment of these global disorders. PMID:24463623

  11. Preclinical demonstration of lentiviral vector-mediated correction of immunological and metabolic abnormalities in models of adenosine deaminase deficiency.

    PubMed

    Carbonaro, Denise A; Zhang, Lin; Jin, Xiangyang; Montiel-Equihua, Claudia; Geiger, Sabine; Carmo, Marlene; Cooper, Aaron; Fairbanks, Lynette; Kaufman, Michael L; Sebire, Neil J; Hollis, Roger P; Blundell, Michael P; Senadheera, Shantha; Fu, Pei-Yu; Sahaghian, Arineh; Chan, Rebecca Y; Wang, Xiaoyan; Cornetta, Kenneth; Thrasher, Adrian J; Kohn, Donald B; Gaspar, H Bobby

    2014-03-01

    Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA(-/-) mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA(-/-) mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34(+) cells transduced with 1-5 × 10(7) TU/ml had 1-3 vector copies/cell and expressed 1-2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis.

  12. Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas

    PubMed Central

    Justice, James F.; Morgan, Robin W.

    2015-01-01

    ABSTRACT Avian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes—MYC, MYB, Mir-155, and TERT—have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also analyze the genome-wide ALV integration landscape in vivo and find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integration in vivo in the chicken genome. PMID:26670384

  13. A Nonintegrative Lentiviral Vector-Based Vaccine Provides Long-Term Sterile Protection against Malaria

    PubMed Central

    Coutant, Frédéric; Sanchez David, Raul Yusef; Félix, Tristan; Boulay, Aude; Caleechurn, Laxmee; Souque, Philippe; Thouvenot, Catherine; Bourgouin, Catherine

    2012-01-01

    Trials testing the RTS,S candidate malaria vaccine and radiation-attenuated sporozoites (RAS) have shown that protective immunity against malaria can be induced and that an effective vaccine is not out of reach. However, longer-term protection and higher protection rates are required to eradicate malaria from the endemic regions. It implies that there is still a need to explore new vaccine strategies. Lentiviral vectors are very potent at inducing strong immunological memory. However their integrative status challenges their safety profile. Eliminating the integration step obviates the risk of insertional oncogenesis. Providing they confer sterile immunity, nonintegrative lentiviral vectors (NILV) hold promise as mass pediatric vaccine by meeting high safety standards. In this study, we have assessed the protective efficacy of NILV against malaria in a robust pre-clinical model. Mice were immunized with NILV encoding Plasmodium yoelii Circumsporozoite Protein (Py CSP) and challenged with sporozoites one month later. In two independent protective efficacy studies, 50% (37.5–62.5) of the animals were fully protected (p = 0.0072 and p = 0.0008 respectively when compared to naive mice). The remaining mice with detectable parasitized red blood cells exhibited a prolonged patency and reduced parasitemia. Moreover, protection was long-lasting with 42.8% sterile protection six months after the last immunization (p = 0.0042). Post-challenge CD8+ T cells to CSP, in contrast to anti-CSP antibodies, were associated with protection (r = −0.6615 and p = 0.0004 between the frequency of IFN-g secreting specific T cells in spleen and parasitemia). However, while NILV and RAS immunizations elicited comparable immunity to CSP, only RAS conferred 100% of sterile protection. Given that a better protection can be anticipated from a multi-antigen vaccine and an optimized vector design, NILV appear as a promising malaria vaccine. PMID:23133649

  14. Archaeological site protection: An integral component of the Exxon Valdez shoreline cleanup

    SciTech Connect

    Wooley, C.B.; Haggarty, J.C.

    1995-12-31

    A major cultural site identification and protection program in Prince William Sound and the Gulf of Alaska was conducted as part of the Exxon Valdez spill response. In cooperation with state and federal agencies and Native corporations with historic preservation mandates, the four-year program was designed to identify archaeological sites in the area of the spill, determine the effect of planned cleanup on them, and mitigate impacts to sites during cleanup. Archaeological site protection constraints, augmented by an extensive cultural resource training program, were an integral part of each shoreline-specific cleanup plan. As a result, impacts attributable to the cleanup were limited to minor disturbances and two vandalism incidents. Impacts from oiling were minimal largely because most intertidal cultural sites had lost their fragile constituents and contextual integrity as a result of prespill erosion. State and federal studies confirmed the efficacy of the site identification and protection program, finding negligible impacts attributable to either direct oiling or the cleanup at intact sites. The Cultural Resource Program also developed innovative management strategies with implications for future emergency responses involving complex land management and site protection issues. The program greatly enhanced the knowledge of the area`s history by collecting and synthesizing considerable new archaeological information. 27 refs., 5 figs., 1 tab.

  15. Demonstration of innovative monitoring technologies at the Savannah River Integrated Demonstration Site

    SciTech Connect

    Rossabi, J.; Jenkins, R.A.; Wise, M.B.

    1993-12-31

    The Department of Energy`s Office of Technology Development initiated an Integrated Demonstration Program at the Savannah River Site in 1989. The objective of this program is to develop, demonstrate, and evaluate innovative technologies that can improve present-day environmental restoration methods. The Integrated Demonstration Program at SRS is entitled ``Cleanup of Organics in Soils and Groundwater at Non-Arid Sites.`` New technologies in the areas of drilling, characterization, monitoring, and remediation are being demonstrated and evaluated for their technical performance and cost effectiveness in comparison with baseline technologies. Present site characterization and monitoring methods are costly, time-consuming, overly invasive, and often imprecise. Better technologies are required to accurately describe the subsurface geophysical and geochemical features of a site and the nature and extent of contamination. More efficient, nonintrusive characterization and monitoring techniques are necessary for understanding and predicting subsurface transport. More reliable procedures are also needed for interpreting monitoring and characterization data. Site characterization and monitoring are key elements in preventing, identifying, and restoring contaminated sites. The remediation of a site cannot be determined without characterization data, and monitoring may be required for 30 years after site closure.

  16. AGU Launches Web Site for New Scientific Integrity and Professional Ethics Policy

    NASA Astrophysics Data System (ADS)

    Townsend, Randy

    2013-03-01

    AGU's Scientific Integrity and Professional Ethics policy, approved by the AGU Board of Directors and Council in December 2012, is now available online on a new Web site, http://ethics.agu.org. As the Web site states, the policy embodies a "set of guidelines for scientific integrity and professional ethics for the actions of the members and the governance of the Union in its internal activities; in its public persona; and most importantly, in the research and peer review processes of its scientific publications, its communications and outreach, and its scientific meetings."

  17. New ΦBT1 site-specific integrative vectors with neutral phenotype in Streptomyces.

    PubMed

    Gonzalez-Quiñonez, Nathaly; López-García, María Teresa; Yagüe, Paula; Rioseras, Beatriz; Pisciotta, Annalisa; Alduina, Rosa; Manteca, Ángel

    2016-03-01

    Integrative plasmids are one of the best options to introduce genes in low copy and in a stable form into bacteria. The ΦC31-derived plasmids constitute the most common integrative vectors used in Streptomyces. They integrate at different positions (attB and pseudo-attB sites) generating different mutations. The less common ΦBT1-derived vectors integrate at the unique attB site localized in the SCO4848 gene (S. coelicolor genome) or their orthologues in other streptomycetes. This work demonstrates that disruption of SCO4848 generates a delay in spore germination. SCO4848 is co-transcribed with SCO4849, and the spore germination phenotype is complemented by SCO4849. Plasmids pNG1-4 were created by modifying the ΦBT1 integrative vector pMS82 by introducing a copy of SCO4849 under the control of the promoter region of SCO4848. pNG2 and pNG4 also included a copy of the P ermE * in order to facilitate gene overexpression. pNG3 and pNG4 harboured a copy of the bla gene (ampicillin resistance) to facilitate selection in E. coli. pNG1-4 are the only integrative vectors designed to produce a neutral phenotype when they are integrated into the Streptomyces genome. The experimental approach developed in this work can be applied to create phenotypically neutral integrative plasmids in other bacteria.

  18. Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus.

    PubMed

    Bryda, Elizabeth C; Pearson, Michael; Agca, Yuksel; Bauer, Beth A

    2006-12-01

    Transgene delivery systems, particularly those involving retroviruses, often result in the integration of multiple copies of the transgene throughout the host genome. Since site-specific silencing of trangenes can occur; it becomes important to identify the number and chromosomal location of the multiple copies of the transgenes in order to correlate inheritance of the transgene at a particular chromosomal site with a specific and robust phenotype. Using a technique that combines restriction endonuclease digest and several rounds of PCR amplification followed by nucleotide sequencing, it is possible to identify multiple chromosomal integration sites in transgenic founder animals. By designing genotyping assays to detect each individual integration site in the offspring of these founders, the inheritance of transgenes integrated at specific chromosomal locations can be followed efficiently as the transgenes randomly segregate in subsequent generations. Phenotypic characteristics can then be correlated with inheritance of a transgene integrated at a particular chromosomal location to allow rational selection of breeding animals in order to establish the transgenic line.

  19. A phage integrase directs efficient site-specific integration in human cells

    PubMed Central

    Groth, Amy C.; Olivares, Eric C.; Thyagarajan, Bhaskar; Calos, Michele P.

    2000-01-01

    The integrase from the Streptomyces phage φC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the φC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein–Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications. PMID:10801973

  20. Using the Choquet integral for screening geological CO2 storage sites

    SciTech Connect

    Zhang, Y.

    2011-03-01

    For geological CO{sub 2} storage site selection, it is desirable to reduce the number of candidate sites through a screening process before detailed site characterization is performed. Screening generally involves defining a number of criteria which then need to be evaluated for each site. The importance of each criterion to the final evaluation will generally be different. Weights reflecting the relative importance of these criteria can be provided by experts. To evaluate a site, each criterion must be evaluated and scored, and then aggregated, taking into account the importance of the criteria. We propose the use of the Choquet integral for aggregating the scores. The Choquet integral considers the interactions among criteria, i.e. whether they are independent, complementary to each other, or partially repetitive. We also evaluate the Shapley index, which demonstrates how the importance of a given piece of information may change if it is considered by itself or together with other available information. An illustrative example demonstrates how the Choquet integral properly accounts for the presence of redundancy in two site-evaluation criteria, making the screening process more defensible than the standard weighted-average approach.

  1. Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.

    PubMed Central

    Grandgenett, D P; Inman, R B; Vora, A C; Fitzgerald, M L

    1993-01-01

    Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction. Images PMID:8474165

  2. HTLV-1 proviral integration sites differ between asymptomatic carriers and patients with HAM/TSP.

    PubMed

    Niederer, Heather A; Laydon, Daniel J; Melamed, Anat; Elemans, Marjet; Asquith, Becca; Matsuoka, Masao; Bangham, Charles R M

    2014-09-30

    HTLV-1 causes proliferation of clonal populations of infected T cells in vivo, each clone defined by a unique proviral integration site in the host genome. The proviral load is strongly correlated with odds of the inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is evidence that asymptomatic HTLV-1 carriers (ACs) have a more effective CD8 + T cell response, including a higher frequency of HLA class I alleles able to present peptides from a regulatory protein of HTLV-1, HBZ. We have previously shown that specific features of the host genome flanking the proviral integration site favour clone survival and spontaneous expression of the viral transactivator protein Tax in naturally infected PBMCs ex vivo. However, the previous studies were not designed or powered to detect differences in integration site characteristics between ACs and HAM/TSP patients. Here, we tested the hypothesis that the genomic environment of the provirus differs systematically between ACs and HAM/TSP patients, and between individuals with strong or weak HBZ presentation. We used our recently described high-throughput protocol to map and quantify integration sites in 95 HAM/TSP patients and 68 ACs from Kagoshima, Japan, and 75 ACs from Kumamoto, Japan. Individuals with 2 or more HLA class I alleles predicted to bind HBZ peptides were classified 'strong' HBZ binders; the remainder were classified 'weak binders'. The abundance of HTLV-1-infected T cell clones in vivo was correlated with proviral integration in genes and in areas with epigenetic marks associated with active regulatory elements. In clones of equivalent abundance, integration sites in genes and active regions were significantly more frequent in ACs than patients with HAM/TSP, irrespective of HBZ binding and proviral load. Integration sites in genes were also more frequent in strong HBZ binders than weak HBZ binders. Clonal abundance is correlated with integration in a

  3. Integrating Risk Analyses and Tools at the U.S. Department of Energy Hanford Site

    SciTech Connect

    Lober, R. W.; Yasek, R. M.; Morse, J. G.; Buck, J. W.; Henderson, C. C.; Sams, T. L.; Bunn, A. L.; Vaughn, P.

    2002-02-26

    Risk assessment and environmental impact analysis at the U.S. Department of Energy (DOE) Hanford Site in Washington State has made significant progress in refining the strategy for using risk analysis to support closing of several hundred waste sites plus 149 single-shell tanks at the Hanford Site. A Single-Shell Tank System Closure Work Plan outlines the current basis for closing the single-shell tank systems. An analogous site approach has been developed to address closure of aggregated groups of similar waste sites. Because of the complexity, decision time frames, proximity of non-tank farm waste sites to tank farms, scale, and regulatory considerations, various projects are providing integrated assessments to support risk analyses and decision-making. This paper will describe the approach for using risk assessment to support waste site and tank closure decisions, the tools being developed, and how integration of these risk assessments and analyses are being performed to ad dress near-term and longterm decisions.

  4. Preclinical evaluation of efficacy and safety of an improved lentiviral vector for the treatment of β-thalassemia and sickle cell disease.

    PubMed

    Negre, Olivier; Bartholomae, Cynthia; Beuzard, Yves; Cavazzana, Marina; Christiansen, Lauryn; Courne, Céline; Deichmann, Annette; Denaro, Maria; de Dreuzy, Edouard; Finer, Mitchell; Fronza, Raffaele; Gillet-Legrand, Béatrix; Joubert, Christophe; Kutner, Robert; Leboulch, Philippe; Maouche, Leïla; Paulard, Anaïs; Pierciey, Francis J; Rothe, Michael; Ryu, Byoung; Schmidt, Manfred; von Kalle, Christof; Payen, Emmanuel; Veres, Gabor

    2015-01-01

    A previously published clinical trial demonstrated the benefit of autologous CD34(+) cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered β-globin gene (β(A-T87Q)-globin) in a subject with β thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.

  5. Biogeochemical cycling of carbon, nitrogen, and sulfur at the Howland Integrated Forest Study site, Howland, Maine

    Treesearch

    James W. McLaughlin; Ivan J. Fernandez; Stewart M. Goltz; Lindsey E. Rustad; Larry Zibilske

    1996-01-01

    The biogeochemistry of C, N, and S was studied for six years at the Howland Integrated Forest Study (HIFS) site by measuring those constituents in major above- and below-ground pools and fluxes. Leaching losses of C from the solum were much less than CO2 efflux, with a mean annual leaching rate of 31.2 kg ha-1 yr

  6. TECHNOLOGY INTEGRATION FOR CONTAMINATED SITE REMEDIATION: CLEANUP GOALS AND PERFORMANCE CRITERIA

    EPA Science Inventory

    There is a need to develop and field-test integrated remediation technologies that operate in a synergistic manner for cost-effective treatment of contaminated sites to achieve risk-based and rational endpoints. Aggressive technologies designed for rapid source-zone remediation m...

  7. Technology development for phosphoric acid fuel cell powerplant (phase 2). [on site integrated energy systems

    NASA Technical Reports Server (NTRS)

    Christner, L.

    1980-01-01

    Progress is reported in the development of material, cell components, and reformers for on site integrated energy systems. Internal resistance and contact resistance were improved. Dissolved gases (O2, N2, and CO2) were found to have no effect on the electrochemical corrosion of phenolic composites. Stack performance was increased by 100 mV over the average 1979 level.

  8. TECHNOLOGY INTEGRATION FOR CONTAMINATED SITE REMEDIATION: CLEANUP GOALS AND PERFORMANCE CRITERIA

    EPA Science Inventory

    There is a need to develop and field-test integrated remediation technologies that operate in a synergistic manner for cost-effective treatment of contaminated sites to achieve risk-based and rational endpoints. Aggressive technologies designed for rapid source-zone remediation m...

  9. Integrated decision support, sensor networks and adaptive control for wireless site-specific sprinkler irrigation

    USDA-ARS?s Scientific Manuscript database

    The development of site-specific sprinkler irrigation water management systems will be a major factor in future efforts to improve the various efficiencies of water-use and to support a sustainable irrigated environment. The challenge is to develop fully integrated management systems with supporting...

  10. Integrated Decision Support, Sensor Networks and Adaptive Control for Wireless Site-specific Sprinkler Irrigation

    USDA-ARS?s Scientific Manuscript database

    The development of site-specific sprinkler irrigation water management systems will be a major factor in future efforts to improve the various efficiencies of water-use and to support a sustainable irrigated environment. The challenge is to develop fully integrated management systems with supporting...

  11. How Do I Do That? Integrating Web Sites into the Gifted Education Classroom

    ERIC Educational Resources Information Center

    Besnoy, Kevin

    2006-01-01

    The purpose of this article is twofold: (1) to provide teachers of the gifted with a reference for adopting IT pedagogical standards, strategies, and tools, specifically Web sites, into the gifted education curriculum; and (2) to show gifted educators the need for research-based standards regarding the integration of websites into the gifted…

  12. Integration of aerial imaging and variable-rate technology for site-specific aerial herbicide application

    USDA-ARS?s Scientific Manuscript database

    As remote sensing and variable rate technology are becoming more available for aerial applicators, practical methodologies on effective integration of these technologies are needed for site-specific aerial applications of crop production and protection materials. The objectives of this study were to...

  13. Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

    PubMed

    Juhas, Mario; Evans, Lewis D B; Frost, Joe; Davenport, Peter W; Yarkoni, Orr; Fraser, Gillian M; Ajioka, James W

    2014-01-01

    E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

  14. Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits

    PubMed Central

    Juhas, Mario; Evans, Lewis D. B.; Frost, Joe; Davenport, Peter W.; Yarkoni, Orr; Fraser, Gillian M.; Ajioka, James W.

    2014-01-01

    E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site. PMID:25350000

  15. Development of the integrated programme for the cleanup of the Sellafield (site)

    SciTech Connect

    Wylie, P.

    2007-07-01

    The Sellafield site in the United Kingdom is highly complex and integrated site. It has two operating reprocessing plants, a fuel fabrication facility and significant processing facilities for the conditioning of high level, intermediate level and low level wastes. It plays a major role in supporting the UK nuclear generation and dealing with the legacy from the early nuclear programmes. Sellafield also supports a number of international nuclear programmes. The UK nuclear industry is undergoing an intense programme of change with many nuclear facilities coming to the end of their lives, new reactors being discussed and a different organisational structure for running the UK nuclear programme. Common to all of this is increased determination to decommission and cleanup historic nuclear wastes. The challenge for the site has been how to put together a cleanup programme for the site whilst recognising the ongoing international strategic value of many of the facilities on the site. The work to produce an integrated plan for Sellafield is now complete and extends from the detailed consideration of facilities and processes through to socio economic impact on the local community. Extensive use has been made of operational research tools and strategic models that cover costs, resources and technical aspects for over 300 active facilities, and over 1000 waste streams. Key strategic decisions facing the site have been analysed and decision calendars produced. These illustrate the options available for each strategic decision and the latest date by which decisions can be taken. Many of the most significant decisions affecting the site need to be progressed within the next few years. The paper describes the process by which the integrated plan for Sellafield has been produced, some of the tools and techniques used in analysing them and the key decisions facing the site. (authors)

  16. Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology.

    PubMed

    Liu, Ying; Lu, Zheming; Xu, Ruiping; Ke, Yang

    2016-02-02

    Integration of human papillomavirus (HPV) DNA into the host genome can be a driver mutation in cervical carcinoma. Identification of HPV integration at base resolution has been a longstanding technical challenge, largely due to sensitivity masking by HPV in episomes or concatenated forms. The aim was to enhance the understanding of the precise localization of HPV integration sites using an innovative strategy. Using HPV capture technology combined with next generation sequencing, HPV prevalence and the exact integration sites of the HPV DNA in 47 primary cervical cancer samples and 2 cell lines were investigated. A total of 117 unique HPV integration sites were identified, including HPV16 (n = 101), HPV18 (n = 7), and HPV58 (n = 9). We observed that the HPV16 integration sites were broadly located across the whole viral genome. In addition, either single or multiple integration events could occur frequently for HPV16, ranging from 1 to 19 per sample. The viral integration sites were distributed across almost all the chromosomes, except chromosome 22. All the cervical cancer cases harboring more than four HPV16 integration sites showed clinical diagnosis of stage III carcinoma. A significant enrichment of overlapping nucleotides shared between the human genome and HPV genome at integration breakpoints was observed, indicating that it may play an important role in the HPV integration process. The results expand on knowledge from previous findings on HPV16 and HPV18 integration sites and allow a better understanding of the molecular basis of the pathogenesis of cervical carcinoma.

  17. Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology

    PubMed Central

    Xu, Ruiping; Ke, Yang

    2016-01-01

    Integration of human papillomavirus (HPV) DNA into the host genome can be a driver mutation in cervical carcinoma. Identification of HPV integration at base resolution has been a longstanding technical challenge, largely due to sensitivity masking by HPV in episomes or concatenated forms. The aim was to enhance the understanding of the precise localization of HPV integration sites using an innovative strategy. Using HPV capture technology combined with next generation sequencing, HPV prevalence and the exact integration sites of the HPV DNA in 47 primary cervical cancer samples and 2 cell lines were investigated. A total of 117 unique HPV integration sites were identified, including HPV16 (n = 101), HPV18 (n = 7), and HPV58 (n = 9). We observed that the HPV16 integration sites were broadly located across the whole viral genome. In addition, either single or multiple integration events could occur frequently for HPV16, ranging from 1 to 19 per sample. The viral integration sites were distributed across almost all the chromosomes, except chromosome 22. All the cervical cancer cases harboring more than four HPV16 integration sites showed clinical diagnosis of stage III carcinoma. A significant enrichment of overlapping nucleotides shared between the human genome and HPV genome at integration breakpoints was observed, indicating that it may play an important role in the HPV integration process. The results expand on knowledge from previous findings on HPV16 and HPV18 integration sites and allow a better understanding of the molecular basis of the pathogenesis of cervical carcinoma. PMID:26735580

  18. Lentiviral Vector-Mediated RNA Silencing in the Central Nervous System

    PubMed Central

    Foster, Edmund; Moon, Lawrence D.F.

    2014-01-01

    Abstract RNA silencing is an established method for investigating gene function and has attracted particular interest because of the potential for generating RNA-based therapeutics. Using lentiviral vectors as an efficient delivery system that offers stable, long-term expression in postmitotic cells further enhances the applicability of an RNA-based gene therapy for the CNS. In this review we provide an overview of both lentiviral vectors and RNA silencing along with design considerations for generating lentiviral vectors capable of RNA silencing. We go on to describe the current preclinical data regarding lentiviral vector-mediated RNA silencing for CNS disorders and discuss the concerns of side effects associated with lentiviral vectors and small interfering RNAs and how these might be mitigated. PMID:24090197

  19. DNFSB Recommendation 94-1 Hanford Site Integrated Stabilization Management Plan. Volume 2

    SciTech Connect

    McCormack, R.L.

    1995-08-01

    The Hanford Site Integrated Stabilization Management Plan (SISMP) is being developed in support of the US Department of Energy`s (DOE) Defense Nuclear Facilities Safety Board (DNFSB) Recommendation 94-1 Integrated Program Plan (IPP). Volume 1 of the SISMP identifies the technical scope and costs associated with Hanford Site plans to resolve concerns identified in DNFSB Recommendation 94-1. Volume 2 of the SISMP provides the Resource Loaded Integrated Schedules for Spent Nuclear Fuel Project and Plutonium Finishing Plant activities identified in Volume 1 of the SISMP. Appendix A provides the schedules and progress curves related to spent nuclear fuel management. Appendix B provides the schedules and progress curves related to plutoniumbearing material management. Appendix C provides programmatic logic diagrams that were referenced in Volume 1 of the SISMP.

  20. DNFSB Recommendation 94-1 Hanford Site Integrated Stabilization Management Plan. Volume 2

    SciTech Connect

    Gerber, E.W.

    1995-10-01

    The Hanford Site Integrated Stabilization Management Plan (SISMP) was developed in support of the US Department of Energy`s (DOE) Defense Nuclear Facilities Safety Board (DNFSB) Recommendation 94-1 Integrated Program Plan (IPP). Volume 1 of the SISMP identifies the technical scope and costs associated with Hanford Site plans to resolve concerns identified in DNFSB Recommendation 94-1. Volume 2 of the SISMP provides the Resource Loaded Integrated Schedules for Spent Nuclear Fuel Project and Plutonium Finishing Plant activities identified in Volume 1 of the SISMP. Appendix A provides the schedules and progress curves related to spent nuclear fuel management. Appendix B provides the schedules and progress curves related to plutonium-bearing material management. Appendix C provides programmatic logic diagrams that were referenced in Volume 1 of the SISMP.

  1. Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines

    SciTech Connect

    Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S.

    1994-09-01

    Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viral DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.

  2. Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces.

    PubMed

    Boccard, F; Smokvina, T; Pernodet, J L; Friedmann, A; Guérineau, M

    1989-01-01

    pSAM2 is an 11-kb plasmid integrated in the Streptomyces ambofaciens ATCC23877 and ATCC15154 genomes and found additionally as a free replicon in an uv derivative. After transfer into S. ambofaciens DSM40697 (devoid of pSAM2) or into Streptomyces lividans, specific integration of pSAM2 occurred very efficiently. A 58-bp sequence (att) present in both pSAM2 (attP) and S. ambofaciens strain DSM40697 (attB) attachment regions is found at the boundaries (attL and attR) of integrated pSAM2 in S. ambofaciens strain ATCC23877. The S. lividans chromosomal integration zone contained an imperfectly conserved att sequence (attB), and the integration event of pSAM2 was located within a 49-bp sequence of attB. Only one primary functional attB sequence was present in the S. lividans or S. ambofaciens DSM40697 total DNA. The integration zone of S. lividans hybridized with the integration zone of S. ambofaciens DSM40697. The two integration zones were homologous only to the right side of the att sequence. The conserved region contained an open reading frame (ORF A) with a stop codon located 99 bp from the attB sequence in both strains. S. ambofaciens DSM40697 contained DNA sequences related to pSAM2 on the left side of the att site. The att sequence was included in a region conserved in Streptomyces antibioticus, Streptomyces actuosus, Streptomyces bikiniensis, Streptomyces coelicolor, Streptomyces glaucescens, and Streptomyces parvulus. Site-specific integration of a pSAM2 derivative was characterized in another unrelated strain, Streptomyces griseofuscus. This strain contained an imperfectly conserved 58-bp attB sequence, and the integration event took place within a 45-bp sequence of attB. Site-specific integration of pSAM2 in three nonrelated Streptomyces strains suggests the wide host range of pSAM2 integration in Streptomyces.

  3. An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites.

    PubMed

    Ma, Sanyuan; Liu, Yue; Liu, Yuanyuan; Chang, Jiasong; Zhang, Tong; Wang, Xiaogang; Shi, Run; Lu, Wei; Xia, Xiaojuan; Zhao, Ping; Xia, Qingyou

    2017-02-09

    Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects.

  4. Heat recovery subsystem and overall system integration of fuel cell on-site integrated energy systems

    NASA Technical Reports Server (NTRS)

    Mougin, L. J.

    1983-01-01

    The best HVAC (heating, ventilating and air conditioning) subsystem to interface with the Engelhard fuel cell system for application in commercial buildings was determined. To accomplish this objective, the effects of several system and site specific parameters on the economic feasibility of fuel cell/HVAC systems were investigated. An energy flow diagram of a fuel cell/HVAC system is shown. The fuel cell system provides electricity for an electric water chiller and for domestic electric needs. Supplemental electricity is purchased from the utility if needed. An excess of electricity generated by the fuel cell system can be sold to the utility. The fuel cell system also provides thermal energy which can be used for absorption cooling, space heating and domestic hot water. Thermal storage can be incorporated into the system. Thermal energy is also provided by an auxiliary boiler if needed to supplement the fuel cell system output. Fuel cell/HVAC systems were analyzed with the TRACE computer program.

  5. Heat recovery subsystem and overall system integration of fuel cell on-site integrated energy systems

    NASA Astrophysics Data System (ADS)

    Mougin, L. J.

    1983-07-01

    The best HVAC (heating, ventilating and air conditioning) subsystem to interface with the Engelhard fuel cell system for application in commercial buildings was determined. To accomplish this objective, the effects of several system and site specific parameters on the economic feasibility of fuel cell/HVAC systems were investigated. An energy flow diagram of a fuel cell/HVAC system is shown. The fuel cell system provides electricity for an electric water chiller and for domestic electric needs. Supplemental electricity is purchased from the utility if needed. An excess of electricity generated by the fuel cell system can be sold to the utility. The fuel cell system also provides thermal energy which can be used for absorption cooling, space heating and domestic hot water. Thermal storage can be incorporated into the system. Thermal energy is also provided by an auxiliary boiler if needed to supplement the fuel cell system output. Fuel cell/HVAC systems were analyzed with the TRACE computer program.

  6. Lentiviral-mediated phenotypic correction of cystic fibrosis pigs

    PubMed Central

    Cooney, Ashley L.; Abou Alaiwa, Mahmoud H.; Shah, Viral S.; Bouzek, Drake C.; Stroik, Mallory R.; Powers, Linda S.; Gansemer, Nick D.; Meyerholz, David K.; Welsh, Michael J.; Stoltz, David A.; Sinn, Patrick L.; McCray, Paul B.

    2016-01-01

    Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in CF transmembrane conductance regulator (CFTR), resulting in defective anion transport. Regardless of the disease-causing mutation, gene therapy is a strategy to restore anion transport to airway epithelia. Indeed, viral vector–delivered CFTR can complement the anion channel defect. In this proof-of-principle study, functional in vivo CFTR channel activity was restored in the airways of CF pigs using a feline immunodeficiency virus–based (FIV-based) lentiviral vector pseudotyped with the GP64 envelope. Three newborn CF pigs received aerosolized FIV-CFTR to the nose and lung. Two weeks after viral vector delivery, epithelial tissues were analyzed for functional correction. In freshly excised tracheal and bronchus tissues and cultured ethmoid sinus cells, we observed a significant increase in transepithelial cAMP-stimulated current, evidence of functional CFTR. In addition, we observed increases in tracheal airway surface liquid pH and bacterial killing in CFTR vector–treated animals. Together, these data provide the first evidence to our knowledge that lentiviral delivery of CFTR can partially correct the anion channel defect in a large-animal CF model and validate a translational strategy to treat or prevent CF lung disease. PMID:27656681

  7. Lentiviral-mediated gene correction of mucopolysaccharidosis type IIIA

    PubMed Central

    Anson, Donald S; McIntyre, Chantelle; Thomas, Belinda; Koldej, Rachel; Ranieri, Enzo; Roberts, Ainslie; Clements, Peter R; Dunning, Kylie; Byers, Sharon

    2007-01-01

    Background Mucopolysaccharidosis type IIIA (MPS IIIA) is the most common of the mucopolysaccharidoses. The disease is caused by a deficiency of the lysosomal enzyme sulphamidase and results in the storage of the glycosaminoglycan (GAG), heparan sulphate. MPS IIIA is characterised by widespread storage and urinary excretion of heparan sulphate, and a progressive and eventually profound neurological course. Gene therapy is one of the few avenues of treatment that hold promise of a sustainable treatment for this disorder. Methods The murine sulphamidase gene cDNA was cloned into a lentiviral vector and high-titre virus produced. Human MPS IIIA fibroblast cultures were transduced with the sulphamidase vector and analysed using molecular, enzymatic and metabolic assays. High-titre virus was intravenously injected into six 5-week old MPS IIIA mice. Three of these mice were pre-treated with hyperosmotic mannitol. The weight of animals was monitored and GAG content in urine samples was analysed by polyacrylamide gel electrophoresis. Results Transduction of cultured MPS IIIA fibroblasts with the sulphamidase gene corrected both the enzymatic and metabolic defects. Sulphamidase secreted by gene-corrected cells was able to cross correct untransduced MPS IIIA cells. Urinary GAG was found to be greatly reduced in samples from mice receiving the vector compared to untreated MPS IIIA controls. In addition, the weight of treated mice became progressively normalised over the 6-months post-treatment. Conclusion Lentiviral vectors appear promising vehicles for the development of gene therapy for MPS IIIA. PMID:17227588

  8. A dynamic multimedia fuzzy-stochastic integrated environmental risk assessment approach for contaminated sites management.

    PubMed

    Hu, Yan; Wen, Jing-Ya; Li, Xiao-Li; Wang, Da-Zhou; Li, Yu

    2013-10-15

    A dynamic multimedia fuzzy-stochastic integrated environmental risk assessment approach was developed for contaminated sites management. The contaminant concentrations were simulated by a validated interval dynamic multimedia fugacity model, and different guideline values for the same contaminant were represented as a fuzzy environmental guideline. Then, the probability of violating environmental guideline (Pv) can be determined by comparison between the modeled concentrations and the fuzzy environmental guideline, and the constructed relationship between the Pvs and environmental risk levels was used to assess the environmental risk level. The developed approach was applied to assess the integrated environmental risk at a case study site in China, simulated from 1985 to 2020. Four scenarios were analyzed, including "residential land" and "industrial land" environmental guidelines under "strict" and "loose" strictness. It was found that PAH concentrations will increase steadily over time, with soil found to be the dominant sink. Source emission in soil was the leading input and atmospheric sedimentation was the dominant transfer process. The integrated environmental risks primarily resulted from petroleum spills and coke ovens, while the soil environmental risks came from coal combustion. The developed approach offers an effective tool for quantifying variability and uncertainty in the dynamic multimedia integrated environmental risk assessment and the contaminated site management.

  9. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome.

    PubMed

    Cui, Pin; Löber, Ulrike; Alquezar-Planas, David E; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N; Lenz, Dorina; Helgen, Kristofer M; Roca, Alfred L; Hartman, Stefanie; Greenwood, Alex D

    2016-01-01

    Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small.

  10. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome

    PubMed Central

    Alquezar-Planas, David E.; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N.; Lenz, Dorina; Helgen, Kristofer M.; Roca, Alfred L.; Hartman, Stefanie

    2016-01-01

    Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small. PMID:27069793

  11. Erace--an integrated system for treating organic-contaminated sites

    SciTech Connect

    Caley, S.M.; Heath, W.O.; Bergsman, T.M.; Gauglitz, P.A.; Pillay, C.; Moss, R.W.; Shah, R.R.; Goheen, S.C.; Camiaoni, D.M.

    1994-11-01

    The U.S. Department of Energy`s (DOE) Pacific Northwest Laboratory (PNL) is developing a suite of electrical technologies for treating sites contaminated with hazardous organic compounds. These include: (1) Six-Phase Soil Heating (SPSH) to remove volatile and semi-volatile organic compounds from soils; (2) In Situ Corona (ISC) to decompose nonvolatile and bound organic contaminants in soils; (3) High-Energy Corona (HEC) to treat contaminated off-gases; and (4) Liquid Corona (LC) to treat contaminated liquids. These four technologies comprise ERACE (Electrical Remediation at Contaminated Environments), an integrated system for accomplishing site remediation with little or no secondary wastes produced that would require off-site treatment or disposal. Each ERACE technology can be employed individually as a stand-alone treatment process, or combined as a system for total site remediation. For example, an ERACE system for treating sites contaminated with volatile organics would integrate SPSH to remove the contaminants from the soil, LC to continuously treat an aqueous stream condensed out of the soil off-gas, and HEC to treat non-condensibles remaining in the off-gas, before atmospheric release.

  12. Sal-Site: integrating new and existing ambystomatid salamander research and informational resources.

    PubMed

    Smith, Jeramiah J; Putta, Srikrishna; Walker, John A; Kump, D Kevin; Samuels, Amy K; Monaghan, James R; Weisrock, David W; Staben, Chuck; Voss, S Randal

    2005-12-16

    Salamanders of the genus Ambystoma are a unique model organism system because they enable natural history and biomedical research in the laboratory or field. We developed Sal-Site to integrate new and existing ambystomatid salamander research resources in support of this model system. Sal-Site hosts six important resources: 1) Salamander Genome Project: an information-based web-site describing progress in genome resource development, 2) Ambystoma EST Database: a database of manually edited and analyzed contigs assembled from ESTs that were collected from A. tigrinum tigrinum and A. mexicanum, 3) Ambystoma Gene Collection: a database containing full-length protein-coding sequences, 4) Ambystoma Map and Marker Collection: an image and database resource that shows the location of mapped markers on linkage groups, provides information about markers, and provides integrating links to Ambystoma EST Database and Ambystoma Gene Collection databases, 5) Ambystoma Genetic Stock Center: a website and collection of databases that describe an NSF funded salamander rearing facility that generates and distributes biological materials to researchers and educators throughout the world, and 6) Ambystoma Research Coordination Network: a web-site detailing current research projects and activities involving an international group of researchers. Sal-Site is accessible at http://www.ambystoma.org.

  13. Sal-Site: Integrating new and existing ambystomatid salamander research and informational resources

    PubMed Central

    Smith, Jeramiah J; Putta, Srikrishna; Walker, John A; Kump, D Kevin; Samuels, Amy K; Monaghan, James R; Weisrock, David W; Staben, Chuck; Voss, S Randal

    2005-01-01

    Salamanders of the genus Ambystoma are a unique model organism system because they enable natural history and biomedical research in the laboratory or field. We developed Sal-Site to integrate new and existing ambystomatid salamander research resources in support of this model system. Sal-Site hosts six important resources: 1) Salamander Genome Project: an information-based web-site describing progress in genome resource development, 2) Ambystoma EST Database: a database of manually edited and analyzed contigs assembled from ESTs that were collected from A. tigrinum tigrinum and A. mexicanum, 3) Ambystoma Gene Collection: a database containing full-length protein-coding sequences, 4) Ambystoma Map and Marker Collection: an image and database resource that shows the location of mapped markers on linkage groups, provides information about markers, and provides integrating links to Ambystoma EST Database and Ambystoma Gene Collection databases, 5) Ambystoma Genetic Stock Center: a website and collection of databases that describe an NSF funded salamander rearing facility that generates and distributes biological materials to researchers and educators throughout the world, and 6) Ambystoma Research Coordination Network: a web-site detailing current research projects and activities involving an international group of researchers. Sal-Site is accessible at . PMID:16359543

  14. Oak Ridge National Laboratory Management & Integration Perspective Subcontractors as Partners in Site Restoration

    SciTech Connect

    Brill, A.; Eidam, G.

    2002-02-26

    In 1997, the U. S. Department of Energy (DOE) Oak Ridge Operations (ORO) Office awarded the Management and Integration (M&I) contract for all five of their Oak Ridge Operations facilities to Bechtel Jacobs Company, LLC (BJC). This paper will focus on the success and challenges of several of the M&I projects at the Oak Ridge National Laboratory (ORNL). The initial goals for BJC were to transition up to 93% of their staff to the subcontract community as they moved away from operations to ''integration.'' The perspectives of BJC and one of their Remedial Action/Decontamination & Decommissioning (RADD) subcontractors will be combined in this paper to share with others how ''partnering'' together was essential for success. Projects completed by Safety and Ecology Corporation (SEC) under their RADD subcontract will be used to illustrate the process and the challenges/successes to completion. These projects will include pond remediation, tank remediation, and building cleanup for reuse. All these projects were ''fixed price'' with defined milestones keyed into award fee for BJC and regulatory milestones for DOE. By working together to form integrated teams focused on site remediation without sacrificing safety, all milestones were met. This paper will discuss the following items associated with the M&I environmental restoration projects at ORNL: overview of the M&I Contract; challenges in transitioning from ''operations'' to ''integration''; subcontracting strategies; subcontractor pre-qualification process; overview of ORNL Projects; and integrated team effort required to achieve site restoration goals.

  15. Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene.

    PubMed

    Luo, Yumei; Zhu, Detu; Zhang, Zhizhuo; Chen, Yaoyong; Sun, Xiaofang

    2015-01-01

    Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB) and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction.

  16. Integration of HIV in the Human Genome: Which Sites Are Preferential? A Genetic and Statistical Assessment

    PubMed Central

    Gonçalves, Juliana; Moreira, Elsa; Sequeira, Inês J.; Rodrigues, António S.; Rueff, José; Brás, Aldina

    2016-01-01

    Chromosomal fragile sites (FSs) are loci where gaps and breaks may occur and are preferential integration targets for some viruses, for example, Hepatitis B, Epstein-Barr virus, HPV16, HPV18, and MLV vectors. However, the integration of the human immunodeficiency virus (HIV) in Giemsa bands and in FSs is not yet completely clear. This study aimed to assess the integration preferences of HIV in FSs and in Giemsa bands using an in silico study. HIV integration positions from Jurkat cells were used and two nonparametric tests were applied to compare HIV integration in dark versus light bands and in FS versus non-FS (NFSs). The results show that light bands are preferential targets for integration of HIV-1 in Jurkat cells and also that it integrates with equal intensity in FSs and in NFSs. The data indicates that HIV displays different preferences for FSs compared to other viruses. The aim was to develop and apply an approach to predict the conditions and constraints of HIV insertion in the human genome which seems to adequately complement empirical data. PMID:27294106

  17. Distribution and characteristics of bovine leukemia virus integration sites in the host genome at three different clinical stages of infection.

    PubMed

    Miyasaka, T; Oguma, K; Sentsui, H

    2015-01-01

    Bovine leukemia virus (BLV) is an oncogenic retrovirus closely related to human T-cell lymphotropic virus. BLV-infected cattle are categorized as asymptomatic carriers or as having persistent lymphocytosis or enzootic bovine leukemia, depending on the clinical stage. We investigated the BLV integration site distribution at three BLV clinical stages and examined genome sequence features around the integration sites. In all, 264 BLV integration sites, at various locations on each chromosome, were identified in 28 cattle by inverse PCR and BLAST searches. Approximately one-third of BLV proviruses were independently integrated within transcriptional units, and approximately 10 % were integrated near transcription start sites. Moreover, less than 7 % of BLV integration sites were located near CpG islands. BLV did not preferentially integrate into transcriptionally active regions during any of the clinical stages. At the nucleotide level, regions around BLV integration points were significantly A/T rich with weak sequence consensus. BLV preferentially integrated within long interspersed nuclear repeat elements. Although BLV integration sites may not be associated with disease progression, integration is selective at the nucleotide level.

  18. Study of component technologies for fuel cell on-site integrated energy system. Volume 2: Appendices

    NASA Technical Reports Server (NTRS)

    Lee, W. D.; Mathias, S.

    1980-01-01

    This data base catalogue was compiled in order to facilitate the analysis of various on site integrated energy system with fuel cell power plants. The catalogue is divided into two sections. The first characterizes individual components in terms of their performance profiles as a function of design parameters. The second characterizes total heating and cooling systems in terms of energy output as a function of input and control variables. The integrated fuel cell systems diagrams and the computer analysis of systems are included as well as the cash flows series for baseline systems.

  19. Background Information for the Nevada National Security Site Integrated Sampling Plan, Revision 0

    SciTech Connect

    Farnham, Irene; Marutzky, Sam

    2014-12-01

    This document describes the process followed to develop the Nevada National Security Site (NNSS) Integrated Sampling Plan (referred to herein as the Plan). It provides the Plan’s purpose and objectives, and briefly describes the Underground Test Area (UGTA) Activity, including the conceptual model and regulatory requirements as they pertain to groundwater sampling. Background information on other NNSS groundwater monitoring programs—the Routine Radiological Environmental Monitoring Plan (RREMP) and Community Environmental Monitoring Program (CEMP)—and their integration with the Plan are presented. Descriptions of the evaluations, comments, and responses of two Sampling Plan topical committees are also included.

  20. HPV integration detection in CaSki and SiHa using detection of integrated papillomavirus sequences and restriction-site PCR.

    PubMed

    Raybould, Rachel; Fiander, Alison; Wilkinson, Gavin W G; Hibbitts, Sam

    2014-09-01

    Human Papillomavirus (HPV) infection is the primary cause of cervical neoplasia. HPV DNA is integrated into the human genome in the majority of cervical cancers. The nature of integration may differ with integration incorporating a single copy of HPV or occurring in concatenated form. Our understanding of HPV tumorigenesis is largely based on studies using characterised cell lines with defined integration sites; these cell lines provide an invaluable standard for validation of diagnostic assays. Cell lines also further understanding of integration mechanisms in clinical samples. The objective of this study was to explore integration assays and to investigate integration events in cell lines where HPV is integrated in concatenated form. Restriction site PCR and detection of integrated papillomavirus sequences were performed on DNA from SiHa and CaSki. A novel integration site on Xq27.3 and HPV genome rearrangements were detected in CaSki DNA. However, where integration was previously detected by FISH in CaSki, and reported to be integrated in concatenated form, integration was not detected by DIPS or RS-PCR. The data presented illustrate that HPV copy number can hinder integration detection; this needs consideration when interpreting results from tests applied to clinical samples.

  1. Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations.

    PubMed

    Denning, Warren; Das, Suvendu; Guo, Siqi; Xu, Jun; Kappes, John C; Hel, Zdenek

    2013-03-01

    Lentiviral vectors are widely used as effective gene-delivery vehicles. Optimization of the conditions for efficient lentiviral transduction is of a high importance for a variety of research applications. Presence of positively charged polycations reduces the electrostatic repulsion forces between a negatively charged cell and an approaching enveloped lentiviral particle resulting in an increase in the transduction efficiency. Although a variety of polycations are commonly used to enhance the transduction with retroviruses, the relative effect of various types of polycations on the efficiency of transduction and on the potential bias in the determination of titer of lentiviral vectors is not fully understood. Here, we present data suggesting that DEAE-dextran provides superior results in enhancing lentiviral transduction of most tested cell lines and primary cell cultures. Specific type and source of serum affects the efficiency of transduction of target cell populations. Non-specific binding of enhanced green fluorescent protein (EGFP)-containing membrane aggregates in the presence of DEAE-dextran does not significantly affect the determination of the titer of EGFP-expressing lentiviral vectors. In conclusion, various polycations and types of sera should be tested when optimizing lentiviral transduction of target cell populations.

  2. Integrated system for gathering, processing, and reporting data relating to site contamination

    DOEpatents

    Long, Delmar D.; Goldberg, Mitchell S.; Baker, Lorie A.

    1997-01-01

    An integrated screening system comprises an intrusive sampling subsystem, a field mobile laboratory subsystem, a computer assisted design/geographical information subsystem, and a telecommunication linkup subsystem, all integrated to provide synergistically improved data relating to the extent of site soil/groundwater contamination. According to the present invention, data samples related to the soil, groundwater or other contamination of the subsurface material are gathered and analyzed to measure contaminants. Based on the location of origin of the samples in three-dimensional space, the analyzed data are transmitted to a location display. The data from analyzing samples and the data from the locating the origin are managed to project the next probable sample location. The next probable sample location is then forwarded for use as a guide in the placement of ensuing sample location, whereby the number of samples needed to accurately characterize the site is minimized.

  3. Integrated system for gathering, processing, and reporting data relating to site contamination

    DOEpatents

    Long, D.D.; Goldberg, M.S.; Baker, L.A.

    1997-11-11

    An integrated screening system comprises an intrusive sampling subsystem, a field mobile laboratory subsystem, a computer assisted design/geographical information subsystem, and a telecommunication linkup subsystem, all integrated to provide synergistically improved data relating to the extent of site soil/groundwater contamination. According to the present invention, data samples related to the soil, groundwater or other contamination of the subsurface material are gathered and analyzed to measure contaminants. Based on the location of origin of the samples in three-dimensional space, the analyzed data are transmitted to a location display. The data from analyzing samples and the data from the locating the origin are managed to project the next probable sample location. The next probable sample location is then forwarded for use as a guide in the placement of ensuing sample location, whereby the number of samples needed to accurately characterize the site is minimized. 10 figs.

  4. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish.

    PubMed

    Kawahara, Atsuo; Hisano, Yu; Ota, Satoshi; Taimatsu, Kiyohito

    2016-05-13

    The zebrafish (Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish.

  5. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish

    PubMed Central

    Kawahara, Atsuo; Hisano, Yu; Ota, Satoshi; Taimatsu, Kiyohito

    2016-01-01

    The zebrafish (Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish. PMID:27187373

  6. Site-specific integration by the adeno-associated virus rep protein.

    PubMed

    Recchia, Alessandra; Mavilio, Fulvio

    2011-10-01

    Inserting genetic information at precise locations into the human genome has been the goal of gene transfer technology for almost two decades. The spectacular progress of mammalian genetics has led to the development of technology for genome editing and homologous recombination in human somatic cells that is finally approaching efficiency compatible with clinical application. Site-specific integration, or the insertion of genes at known locations by enzymes with target recognition capacity, has progressed slowly but steadily in recent years, and could very well be the basis of the next generation of gene transfer technology. This review focuses on the use of Rep, the replicase/integrase of the adeno-associated virus (AAV), to insert genes at the natural AAV integration site on human chromosome 19. This region (AAVS1) has characteristics that make it an ideal target for somatic transgenesis.

  7. DNFSB recommendation 94-1 Hanford site integrated stabilization management plan

    SciTech Connect

    McCormack, R.L.

    1997-05-07

    In May 1994, the Defense Nuclear Facilities Safety Board (DNFSB) issued DNFSB Recommendation 94-1 (Conway 1994), which identified concerns related to US Department of Energy (DOE) management of legacy fissile materials remaining from past defense production activities. The DNFSB expressed concern about the existing storage conditions for these materials and the slow pace at which the conditions were being remediated. The DNFSB also expressed its belief that additional delays in stabilizing these fissile materials would be accompanied by further deterioration of safety and unnecessary increased risks to workers and the public. In February 1995, DOE issued the DNFSB Recommendation 94-1 Implementation Plan (O`Leary 1995) to address the concerns identified in DNFSB Recommendation 94-1. The Implementation Plan (IP) identifies several DOE commitments to achieve safe interim storage for the legacy fissile materials, and constitutes DOE`s baseline DNFSB Recommendation 94-1 Integrated Program Plan (IPP). The IPP describes the actions DOE plans to implement within the DOE complex to convert its excess fissile materials to forms or conditions suitable for safe interim storage. The IPP was subsequently supplemented with an Integrated Facilities Plan and a Research and Development Plan, which further develop complex-wide research and development and long-range facility requirements and plans. The additions to the baseline IPP were developed based on a systems engineering approach that integrated facilities and capabilities at the various DOE sites and focused on attaining safe interim storage with minimum safety risks and environmental impacts. Each affected DOE site has developed a Site Integrated Stabilization Management Plan (SISMP) to identify individual site plans to implement the DNFSB Recommendation 94-1 IPP. The SISMPs were developed based on the objectives, requirements, and commitments identified in the DNFSB Recommendation 94-1 IP. The SISMPs also supported

  8. Gene transfer in ovarian cancer cells: a comparison between retroviral and lentiviral vectors.

    PubMed

    Indraccolo, Stefano; Habeler, Walter; Tisato, Veronica; Stievano, Laura; Piovan, Erich; Tosello, Valeria; Esposito, Giovanni; Wagner, Ralf; Uberla, Klaus; Chieco-Bianchi, Luigi; Amadori, Alberto

    2002-11-01

    Local gene therapy could be a therapeutic option for ovarian carcinoma, a life-threatening malignancy, because of disease containment within the peritoneal cavity in most patients. Lentiviral vectors, which are potentially capable of stable transgene expression, may be useful to vehicle therapeutic molecules requiring long-term production in these tumors. To investigate this concept, we used lentiviral vectors to deliver the enhanced green fluorescent protein (EGFP) gene to ovarian cancer cells. Their efficiency of gene transfer was compared with that of a retroviral vector carrying the same envelope. In vitro, both vectors infected ovarian cancer cells with comparable efficiency under standard culture conditions; however, the lentiviral vector was much more efficient in transducing growth-arrested cells when compared with the retroviral vector. Gene transfer was fully neutralized by an anti-VSV-G antibody, and in vitro stability was similar. In vivo, the lentiviral vector delivered the transgene 10-fold more efficiently to ovarian cancer cells growing i.p. in SCID mice, as evaluated by real-time PCR analysis of the tumors. Confocal microscopy analysis of tumor sections showed a dramatic difference at the level of transgene expression, because abundant EGFP(+) cells were detected only in mice receiving the lentiviral vector. Quantitative analysis by flow cytometry confirmed this and indicated 0.05 and 5.6% EGFP(+) tumor cells after administration of the retroviral and lentiviral vector, respectively. Injection of ex vivo transduced tumor cells, sorted for EGFP expression, indicated that the lentiviral vector was considerably more resistant to in vivo silencing in comparison with the retroviral vector. Finally, multiple administrations of a murine IFN-alpha(1)-lentiviral vector to ovarian carcinoma-bearing mice significantly prolonged the animals' survival, indicating the therapeutic efficacy of this approach. These findings indicate that lentiviral vectors deserve

  9. Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses.

    PubMed

    Kuate, Seraphin; Stefanou, Daniela; Hoffmann, Dennis; Wildner, Oliver; Uberla, Klaus

    2004-11-01

    The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.

  10. Sustaining expression of B domain-deleted human factor VIII mediated by using lentiviral vectors in NOD/SCID mouse.

    PubMed

    Li, Yan-Jie; Chen, Chong; Zeng, Ling-Yu; Cao, Jiang; Xu, Kai-Lin

    2012-06-01

    Recently, gene therapy has been become a promising approach to cure hemophilia A, a most common recessive bleeding disease. The aim of this study was to determine the perspective of lentiviral vector in hemophilia A gene therapy in vitro and in NOD/SCID mice. Lentivirus transfer vector pXZ9/BDDFVIII containing human B-domain-deleted Factor VIII-IRES-eGFP coding sequence and mock control pXZ9 were constructed. Lentivirus was prepared by co-transfecting 3 plasmids into 293FT cells. 293FT, HLF, human bone marrow mesenchymal stem cells and Chang-liver cells were transfected with the prepared virus. Coagulant activity of human FVIII, human FVIII antigen, human FVIII mRNA transcription and genomic integration were assayed by ELISA, one-step method, RT-PCR and PCR after infection. Lentiviral particles were concentrated by ultracentrifugation and NOD/SCID mice were transfected via portal vein injection. Human FVIII antigen in mouse blood plasma was analyzed by ELISA. eGFP expression was observed by fluorescent microscopy and human FVIII transcription in mouse liver was analyzed by RT-PCR at one month after transduction. The results showed that the high titer of recombinant virus was prepared and used to efficiently transduce the target cells in vitro. At 72 h after transfection, high levels of FVIII activity and FVIII antigen were detected. Human FVIII gene transcription could be detected in the liver of NOD/SCID mice received lentiviral particles carrying FVIII gene. Mouse hepatocytes were transfected with recombinant lentivirus efficiently in vivo. Human FVIII level in mouse blood plasma reached to (49 ± 6) mU, (54 ± 8) mU and (23 ± 4) mU at 72 h, one week and one month after transfection respectively. It is concluded that the lentiviral particles carrying BDDhFVIII gene can high efficiently transfect the target cells both in vitro and in vivo, and the transfected target cells can secrete hFVIII efficiently. The sustained expression of human FVIII in NOD/SCID mice is

  11. Develop and test fuel cell powered on-site integrated total energy system

    NASA Technical Reports Server (NTRS)

    Kaufman, A.; Feigenbaum, H.; Wang, C. L.; Werth, J.; Whelan, J. A.

    1983-01-01

    Test results are presented for a 24 cell, two sq ft (4kW) stack. This stack is a precursor to a 25kW stack that is a key milestone. Results are discussed in terms of cell performance, electrolyte management, thermal management, and reactant gas manifolding. The results obtained in preliminary testing of a 50kW methanol processing subsystem are discussed. Subcontracting activities involving application analysis for fuel cell on site integrated energy systems are updated.

  12. Develop and test fuel cell powered on-site integrated total energy system

    NASA Technical Reports Server (NTRS)

    1983-01-01

    Test results are given for a 5 kW stack and initial results for an integrated, grid connected system operating from methanol fuel. Site selection criteria are presented for future demonstration of a 50 or 100 kW OS/IES. Preliminary results are also given with approximate internal rates of return to the building owner. Progress in development and construction of a 50 kW modular methanol/steam reformer is reported.

  13. Safe genetic modification of cardiac stem cells using a site-specific integration technique.

    PubMed

    Lan, Feng; Liu, Junwei; Narsinh, Kazim H; Hu, Shijun; Han, Leng; Lee, Andrew S; Karow, Marisa; Nguyen, Patricia K; Nag, Divya; Calos, Michele P; Robbins, Robert C; Wu, Joseph C

    2012-09-11

    Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

  14. A plasmid from the methylotrophic actinomycete Amycolatopsis methanolica capable of site-specific integration.

    PubMed Central

    Vrijbloed, J W; Madoń, J; Dijkhuizen, L

    1994-01-01

    Amycolatopsis methanolica contains a 13.3-kb plasmid (pMEA300) which is present both in the free state and integrated at a unique genomic location. A 2.1-kb pMEA300 DNA fragment was sequenced, revealing the putative attP site and two open reading frames, xis and int, showing similarity to genes encoding excisionases and integrases, respectively. Images PMID:7961475

  15. Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique

    PubMed Central

    Lan, Feng; Liu, Junwei; Narsinh, Kazim H.; Hu, Shijun; Han, Leng; Lee, Andrew S.; Karow, Marisa; Nguyen, Patricia K.; Nag, Divya; Calos, Michele P.; Robbins, Robert C.; Wu, Joseph C.

    2012-01-01

    Background Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. Methods and Results We employed the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells (hECs). Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared to unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging (BLI), and positron emission tomography (PET). Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function two weeks after cell delivery, as assessed by echocardiography (P = 0.002) and magnetic resonance imaging (P = 0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated hECs, which enhanced hindlimb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. Conclusions The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types. PMID:22965984

  16. Evi-2, a common integration site involved in murine myeloid leukemogenesis.

    PubMed Central

    Buchberg, A M; Bedigian, H G; Jenkins, N A; Copeland, N G

    1990-01-01

    BXH-2 mice have the highest incidence of spontaneous retrovirally induced myeloid leukemia of any known inbred strain and, as such, represent a valuable model system for identifying cellular proto-oncogenes involved in myeloid disease. Chronic murine leukemia viruses often induce disease by insertional activation or mutation of cellular proto-oncogenes. These loci are identified as common viral integration sites in tumor DNAs. Here we report on the characterization of a novel common viral integration site in BXH-2 myeloid leukemias, designated Evi-2. Within the cluster of viral integration sites that define Evi-2, we identified a gene that has the potential for encoding a novel protein of 223 amino acids. This putative proto-oncogene possesses all of the structural features of a transmembrane protein. Within the transmembrane domain is a "leucine zipper," suggesting that Evi-2 is involved in either homopolymer or heteropolymer formation, which may play an important role in the normal functioning of Evi-2. Interestingly, the human homolog of Evi-2 has recently been shown to be tightly linked to the von Recklinghausen neurofibromatosis locus, suggesting a role for Evi-2 in human disease as well. Images PMID:2167436

  17. Weak Palindromic Consensus Sequences Are a Common Feature Found at the Integration Target Sites of Many Retroviruses

    PubMed Central

    Wu, Xiaolin; Li, Yuan; Crise, Bruce; Burgess, Shawn M.; Munroe, David J.

    2005-01-01

    Integration into the host genome is one of the hallmarks of the retroviral life cycle and is catalyzed by virus-encoded integrases. While integrase has strict sequence requirements for the viral DNA ends, target site sequences have been shown to be very diverse. We carefully examined a large number of integration target site sequences from several retroviruses, including human immunodeficiency virus type 1, simian immunodeficiency virus, murine leukemia virus, and avian sarcoma-leukosis virus, and found that a statistical palindromic consensus, centered on the virus-specific duplicated target site sequence, was a common feature at integration target sites for these retroviruses. PMID:15795304

  18. Subsurface Temperature Modeling using Integrated Modeling for Nuclear Reactor Site Assessment in Volcanic Zone

    NASA Astrophysics Data System (ADS)

    Nurhandoko, Bagus Endar B.; Kurniadi, Rizal; Fatiah, Elfa; Rizal Abda, Muhammad; Martha, Rio; Widowati, Sri

    2017-01-01

    Indonesia has giant vulcanic arc that almost the largest vulcanic arc in the world. Therefore, one of the main risk for nuclear site plan is the vulcanic area. Therefore to reduce the risk, one of most safety nuclear site plant is old vulcanic area. In this paper, we propose to predict subsurface temperature profile to ensure the condition of subsurface of vulcanic zone. Geothermal heat flow is important parameter in modeling of subsurface temperature. The subsurface temperature is one of vulcanic activity parameter which very important for nuclear site plant risk assesment. The integrated modeling for predicting subsurface temperature profile is carried out by combining geothermal heat flow and subsurface profiles resulted from either seismic or gravity measurement. The finite difference of Fourier’s law is applied to surface temperature, temperature gradient, geothermal heat flow and thermal conductivity profile for producing subsurface temperature distribution accurately. This subsurface temperature profile is essential to characterize the vulcanic zone whether it is still active or inactive. Characterization of vulcanic activity is very useful to ensure or to minimize the risk of nuclear site plant in vulcanic zone. One of interesting case study of nuclear site plan in Indonesia is mount Muriah site plan, this method is useful to ensure whether mount Muriah is still active or inactive now

  19. The Site of Spontaneous Ectopic Spike Initiation Facilitates Signal Integration in a Sensory Neuron.

    PubMed

    Städele, Carola; Stein, Wolfgang

    2016-06-22

    Essential to understanding the process of neuronal signal integration is the knowledge of where within a neuron action potentials (APs) are generated. Recent studies support the idea that the precise location where APs are initiated and the properties of spike initiation zones define the cell's information processing capabilities. Notably, the location of spike initiation can be modified homeostatically within neurons to adjust neuronal activity. Here we show that this potential mechanism for neuronal plasticity can also be exploited in a rapid and dynamic fashion. We tested whether dislocation of the spike initiation zone affects signal integration by studying ectopic spike initiation in the anterior gastric receptor neuron (AGR) of the stomatogastric nervous system of Cancer borealis Like many other vertebrate and invertebrate neurons, AGR can generate ectopic APs in regions distinct from the axon initial segment. Using voltage-sensitive dyes and electrophysiology, we determined that AGR's ectopic spike activity was consistently initiated in the neuropil region of the stomatogastric ganglion motor circuits. At least one neurite branched off the AGR axon in this area; and indeed, we found that AGR's ectopic spike activity was influenced by local motor neurons. This sensorimotor interaction was state-dependent in that focal axon modulation with the biogenic amine octopamine, abolished signal integration at the primary spike initiation zone by dislocating spike initiation to a distant region of the axon. We demonstrate that the site of ectopic spike initiation is important for signal integration and that axonal neuromodulation allows for a dynamic adjustment of signal integration. Although it is known that action potentials are initiated at specific sites in the axon, it remains to be determined how the precise location of action potential initiation affects neuronal activity and signal integration. We addressed this issue by studying ectopic spiking in the axon of

  20. Technology needs for remediation: Hanford and other DOE sites. Buried Waste Integrated Demonstration Program

    SciTech Connect

    Stapp, D.C.

    1993-01-01

    Technologies are being developed under the Buried Waste Integrated Demonstration (BWID) program to facilitate remediation of the US Department of Energy`s (DOE) buried and stored low-level radioactive, transuranic (TRU), and mixed radioactive and hazardous buried wastes. The BWID program is being coordinated by the Idaho National Engineering Laboratory (INEL) in southeastern Idaho, a DOE site that has large volumes of buried radioactive wastes. The program is currently focusing its efforts on the problems at INEL`s Subsurface Disposal Area (SDA) of the Radioactive Waste Management Complex (RWMC). As specific technologies are successfully demonstrated, they will be available for transfer to applications at other DOE buried waste sites. The purpose of this study is to present buried waste technology needs that have been identified for DOE sites other than INEL.

  1. Integration Site and Clonal Expansion in Human Chronic Retroviral Infection and Gene Therapy

    PubMed Central

    Niederer, Heather A.; Bangham, Charles R. M.

    2014-01-01

    Retroviral vectors have been successfully used therapeutically to restore expression of genes in a range of single-gene diseases, including several primary immunodeficiency disorders. Although clinical trials have shown remarkable results, there have also been a number of severe adverse events involving malignant outgrowth of a transformed clonal population. This clonal expansion is influenced by the integration site profile of the viral integrase, the transgene expressed, and the effect of the viral promoters on the neighbouring host genome. Infection with the pathogenic human retrovirus HTLV-1 also causes clonal expansion of cells containing an integrated HTLV-1 provirus. Although the majority of HTLV-1-infected people remain asymptomatic, up to 5% develop an aggressive T cell malignancy. In this review we discuss recent findings on the role of the genomic integration site in determining the clonality and the potential for malignant transformation of cells carrying integrated HTLV-1 or gene therapy vectors, and how these results have contributed to the understanding of HTLV-1 pathogenesis and to improvements in gene therapy vector safety. PMID:25365582

  2. Analysis and interpretation of geophysical surveys in archaeological sites employing different integrated approach.

    NASA Astrophysics Data System (ADS)

    Piro, Salvatore; Papale, Enrico; Kucukdemirci, Melda; Zamuner, Daniela

    2017-04-01

    Non-destructive ground surface geophysical prospecting methods are frequently used for the investigation of archaeological sites, where a detailed physical and geometrical reconstructions of hidden volumes is required prior to any excavation work. All methods measure the variations of single physical parameters, therefore if these are used singularly, they could not permit a complete location and characterization of anomalous bodies. The probability of a successful result rapidly increases if a multhimethodological approach is adopted, according to the logic of objective complementarity of information and of global convergence toward a high quality multiparametric imaging of the buried structures. The representation of the static configuration of the bodies in the subsoil and of the space-time evolution of the interaction processes between targets and hosting materials have to be actually considered fundamental elements of primary knowledge in archaeological prospecting. The main effort in geophysical prospecting for archaeology is therefore the integration of different, absolutely non-invasive techniques, especially if managed in view of a ultra-high resolution three-dimensional (3D) tomographic representation mode. Following the above outlined approach, we have integrated geophysical methods which measure the variations of potential field (gradiometric methods) with active methods which measure the variations of physical properties due to the body's geometry and volume (GPR and ERT). In this work, the results obtained during the surveys of three archaeological sites, employing Ground Penetrating Radar (GPR), Electrical Resistivity Tomography (ERT) and Fluxgate Differential Magnetic (FDM) to obtain precise and detailed maps of subsurface bodies, are presented and discussed. The first site, situated in a suburban area between Itri and Fondi, in the Aurunci Natural Regional Park (Central Italy), is characterized by the presence of remains of past human activity

  3. Risks Associated With Lentiviral Vector Exposures and Prevention Strategies

    PubMed Central

    Schlimgen, Ryan; Howard, John; Wooley, Dawn; Thompson, Maureen; Baden, Lindsey R.; Yang, Otto O.; Christiani, David C.; Mostoslavsky, Gustavo; Diamond, David V.; Duane, Elizabeth Gilman; Byers, Karen; Winters, Thomas; Gelfand, Jeffrey A.; Fujimoto, Gary; Hudson, T. Warner; Vyas, Jatin M.

    2016-01-01

    Lentiviral vectors (LVVs) are powerful genetic tools that are being used with greater frequency in biomedical laboratories and clinical trials. Adverse events reported from initial clinical studies provide a basis for risk assessment of occupational exposures, yet many questions remain about the potential harm that LVVs may cause. We review those risks and provide a framework for principal investigators, Institutional Biosafety Committees, and occupational health professionals to assess and communicate the risks of exposure to staff. We also provide recommendations to federal research and regulatory agencies for tracking LVV exposures to evaluate long-term outcomes. U.S. Food and Drug Administration approved antiviral drugs for HIV have theoretical benefits in LVV exposures, although evidence to support their use is currently limited. If treatment is appropriate, we recommend a 7-day treatment with an integrase inhibitor with or without a reverse transcriptase inhibitor within 72 hours of exposure. PMID:27930472

  4. Risks Associated With Lentiviral Vector Exposures and Prevention Strategies.

    PubMed

    Schlimgen, Ryan; Howard, John; Wooley, Dawn; Thompson, Maureen; Baden, Lindsey R; Yang, Otto O; Christiani, David C; Mostoslavsky, Gustavo; Diamond, David V; Duane, Elizabeth Gilman; Byers, Karen; Winters, Thomas; Gelfand, Jeffrey A; Fujimoto, Gary; Hudson, T Warner; Vyas, Jatin M

    2016-12-01

    Lentiviral vectors (LVVs) are powerful genetic tools that are being used with greater frequency in biomedical laboratories and clinical trials. Adverse events reported from initial clinical studies provide a basis for risk assessment of occupational exposures, yet many questions remain about the potential harm that LVVs may cause. We review those risks and provide a framework for principal investigators, Institutional Biosafety Committees, and occupational health professionals to assess and communicate the risks of exposure to staff. We also provide recommendations to federal research and regulatory agencies for tracking LVV exposures to evaluate long-term outcomes. U.S. Food and Drug Administration approved antiviral drugs for HIV have theoretical benefits in LVV exposures, although evidence to support their use is currently limited. If treatment is appropriate, we recommend a 7-day treatment with an integrase inhibitor with or without a reverse transcriptase inhibitor within 72 hours of exposure.

  5. Site Selection for Ground Based Free Electron Laser Technology Integration Experiment (GBFEL-TIE), Volume 3: Preferred Site Evaluation

    DTIC Science & Technology

    1987-04-01

    established that the GBFEL-TIE could be accomplished at any of the three candidate sites. However, based on differences identified during site investigations...construction and operation of the GBFEL-TIE, including environmental mitigation costs, differed only slightly among the three sites, with the Orogrande site...the differences in the findings that would affect site selection, and established a relative ranking of the three sites under consideration. The

  6. Determining the DNA sequence elements required for binding integration host factor to two different target sites.

    PubMed Central

    Hales, L M; Gumport, R I; Gardner, J F

    1994-01-01

    Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system. IHF was unable to act as a repressor in the challenge-phage assay at H' sites containing the core consensus element but lacking the dA+dT-rich element. This indicates that both elements are required for IHF to bind to the H' site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site, which lacks an upstream dA+dT-rich region. Fifty mutants that decreased or eliminated IHF binding to the H1 site were isolated. Sequence analysis showed changes in the bases in the core consensus element only, further indicating that this determinant is sufficient for IHF binding to the H1 site. We found that placement of a dA+dT-rich element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT-rich element enhances IHF binding. PMID:8188600

  7. Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection.

    PubMed

    Garson, Jeremy A; Kellam, Paul; Towers, Greg J

    2011-02-25

    XMRV is a gammaretrovirus associated in some studies with human prostate cancer and chronic fatigue syndrome. Central to the hypothesis of XMRV as a human pathogen is the description of integration sites in DNA from prostate tumour tissues. Here we demonstrate that 2 of 14 patient-derived sites are identical to sites cloned in the same laboratory from experimentally infected DU145 cells. Identical integration sites have never previously been described in any retrovirus infection. We propose that the patient-derived sites are the result of PCR contamination. This observation further undermines the notion that XMRV is a genuine human pathogen.

  8. Integrated Weed Control for Land Stewardship at Legacy Management's Rocky Flats Site in Colorado - 13086

    SciTech Connect

    Nelson, Jody K.

    2013-07-01

    Land stewardship is one of nine sustainability programs in the U.S. Department of Energy's Environmental Management System. Land stewardship includes maintaining and improving ecosystem health. At the Rocky Flats Site near Westminster, Colorado, land stewardship is an integral component of the Office of Legacy Management's post-closure monitoring and management at the site. Nearly 263 hectares (650 acres) were disturbed and re-vegetated during site cleanup and closure operations. Proactive management of revegetation areas is critical to the successful reestablishment of native grasslands, wetlands, and riparian communities. The undisturbed native plant communities that occur at the site also require active management to maintain the high-quality wetlands and other habitats that are home to numerous species of birds and other wildlife such as elk and deer, rare plant communities, and the federally listed threatened Preble's meadow jumping mouse. Over the past several decades, an increase of Noxious weeds has impacted much of Colorado's Front Range. As a result, weed control is a key component of the land stewardship program at Rocky Flats. Thirty-three species of state-listed Noxious weeds are known to occur in the Central and Peripheral Operable Units at Rocky Flats, along with another five species that are considered invasive at the site. Early detection and rapid response to control new invasive species is crucial to the program. An integrated weed control/vegetation management approach is key to maintaining healthy, sustainable plant communities that are able to resist Noxious weed invasions. Weed mapping, field surveys, and field-staff training sessions (to learn how to identify new potential problem species) are conducted to help detect and prevent new weed problems. The integrated approach at Rocky Flats includes administrative and cultural techniques (prevention), mechanical controls, biological controls, and chemical controls. Several species of biocontrol

  9. Refinement of lentiviral vector for improved RNA processing and reduced rates of self inactivation repair

    PubMed Central

    Koldej, Rachel M; Anson, Donald S

    2009-01-01

    Background Lentiviral gene therapy vectors are now finding clinical application. In order to fully exploit their potential it is important that vectors are made as efficient and as safe as possible. Accordingly, we have modified a previously reported vector to improve RNA processing, minimise Human Immunodeficiency Virus Type-1 (HIV-1) sequence content and reduce repair of the self inactivating (SIN) deletion. Results HIV-1 sequence in the vector was reduced by substituting the polyadenylation signal with a heterologous signal. Mutation of splice donor sites was undertaken to prevent the majority of splicing within the vector genomic RNA. In addition, a number of other sequences within the vector were deleted. The combination of these modifications was able to significantly reduce the rates of both vector mobilisation and repair of the self inactivating deletion after two rounds of marker rescue. Conclusion RNA processing can be improved by mutation of the major and minor HIV-1 splice donor sites in the vector. In addition the rate of vector mobilisation and repair of SIN vectors can be successfully reduced by careful vector design that reduces homology between the 5' and 3' long terminal repeats (LTRs) to a minimum. PMID:19811661

  10. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

  11. Testing an Integrated Ground-Water Monitoring Strategy for Nuclear Waste and Decommissioning Sites

    NASA Astrophysics Data System (ADS)

    Price, V.; Dai, Z.; Heffner, D.; Temples, T. J.; Nicholson, T. J.

    2005-05-01

    This talk discusses a Nuclear Regulatory Commission-sponsored research project designed to develop an integrated and systematic strategy for monitoring ground-water flow and transport through the unsaturated zone to the underlying water-table aquifer at waste disposal sites. The goal is to provide scientifically-based guidance for monitoring across a wide range of geologic settings, waste compositions, and site designs. The monitoring will specifically support performance assessment studies and modeling. The research objectives include: (1) the strategy will couple performance confirmation monitoring to site characterization and performance assessment, and will consist of an ordered and logical sequence of procedures; (2) the research will develop the technical bases as citable references, identified guidance and analytical tools, and test case applications of the developed integrated ground-water monitoring strategy for confirming performance of nuclear waste and decommissioning sites; (3) this strategy will focus on identifying and monitoring critical performance indicators (e.g., water contents over time in the unsaturated zone, and ground-water potentials in the saturated zone) of the hydrologic system; and (4) the strategy will demonstrate the connection between performance indicators and site performance. The monitoring strategy has been developed in draft form, and the testing phase of this work is beginning. The test plan includes: 1. develop testing objectives; 2. develop success criteria based on objectives; 3. select test datasets from field sites; 4. apply the draft strategy to field data; 5. feed-back for strategy improvement. Testing objectives will include: 1. develop rules for selection of performance indicators; 2. evaluate efficient methods to develop conceptual site models; 3. develop rules for selection of monitoring points (in the spatial and temporal domain) and rules for identification of monitoring approaches (e.g., geophysical methods) and

  12. Different integrated geophysical approaches to investigate archaeological sites in urban and suburban area.

    NASA Astrophysics Data System (ADS)

    Piro, Salvatore; Papale, Enrico; Zamuner, Daniela

    2016-04-01

    Geophysical methods are frequently used in archaeological prospection in order to provide detailed information about the presence of structures in the subsurface as well as their position and their geometrical reconstruction, by measuring variations of some physical properties. Often, due to the limited size and depth of an archaeological structure, it may be rather difficult to single out its position and extent because of the generally low signal-to-noise ratio. This problem can be overcome by improving data acquisition, processing techniques and by integrating different geophysical methods. In this work, two sites of archaeological interest, were investigated employing several methods (Ground Penetrating Radar (GPR), Electrical Resistivity Tomography (ERT), Fluxgate Differential Magnetic) to obtain precise and detailed maps of subsurface bodies. The first site, situated in a suburban area between Itri and Fondi, in the Aurunci Natural Regional Park (Central Italy), is characterized by the presence of remains of past human activity dating from the third century B.C. The second site, is instead situated in an urban area in the city of Rome (Basilica di Santa Balbina), where historical evidence is also present. The methods employed, allowed to determine the position and the geometry of some structures in the subsurface related to this past human activity. To have a better understanding of the subsurface, we then performed a qualitative and quantitative integration of this data, which consists in fusing the data from all the methods used, to have a complete visualization of the investigated area. Qualitative integration consists in graphically overlaying the maps obtained by the single methods; this method yields only images, not new data that may be subsequently analyzed. Quantitative integration is instead performed by mathematical and statistical solutions, which allows to have a more accurate reconstruction of the subsurface and generates new data with high

  13. A machine-learning approach for predicting palmitoylation sites from integrated sequence-based features.

    PubMed

    Li, Liqi; Luo, Qifa; Xiao, Weidong; Li, Jinhui; Zhou, Shiwen; Li, Yongsheng; Zheng, Xiaoqi; Yang, Hua

    2017-02-01

    Palmitoylation is the covalent attachment of lipids to amino acid residues in proteins. As an important form of protein posttranslational modification, it increases the hydrophobicity of proteins, which contributes to the protein transportation, organelle localization, and functions, therefore plays an important role in a variety of cell biological processes. Identification of palmitoylation sites is necessary for understanding protein-protein interaction, protein stability, and activity. Since conventional experimental techniques to determine palmitoylation sites in proteins are both labor intensive and costly, a fast and accurate computational approach to predict palmitoylation sites from protein sequences is in urgent need. In this study, a support vector machine (SVM)-based method was proposed through integrating PSI-BLAST profile, physicochemical properties, [Formula: see text]-mer amino acid compositions (AACs), and [Formula: see text]-mer pseudo AACs into the principal feature vector. A recursive feature selection scheme was subsequently implemented to single out the most discriminative features. Finally, an SVM method was implemented to predict palmitoylation sites in proteins based on the optimal features. The proposed method achieved an accuracy of 99.41% and Matthews Correlation Coefficient of 0.9773 for a benchmark dataset. The result indicates the efficiency and accuracy of our method in prediction of palmitoylation sites based on protein sequences.

  14. The use of an optimized chimeric envelope glycoprotein enhances the efficiency of retrograde gene transfer of a pseudotyped lentiviral vector in the primate brain.

    PubMed

    Tanabe, Soshi; Inoue, Ken-Ichi; Tsuge, Hitomi; Uezono, Shiori; Nagaya, Kiyomi; Fijiwara, Maki; Kato, Shigeki; Kobayashi, Kazuto; Takada, Masahiko

    2017-02-28

    Lentiviral vectors have been used not only for various basic research experiments, but also for a wide range of gene therapy trials in animal models. The development of a pseudotyped lentiviral vector with the property of retrograde infection allows us to introduce foreign genes into neurons that are localized in regions innervating the site of vector injection. Here, we report the efficiency of retrograde gene transfer of a recently developed FuG-E pseudotyped lentiviral vector in the primate brain by comparing its transduction pattern with that of the parental FuG-C pseudotyped vector. After injection of the FuG-E vector encoding green fluorescent protein (GFP) into the striatum of macaque monkeys, many GFP-immunoreactive neurons were found in regions projecting to the striatum, such as the cerebral cortex, thalamus, and substantia nigra. Quantitative analysis revealed that in all regions, the number of neurons retrogradely transduced with the FuG-E vector was larger than in the FuG-C vector injection case. It was also confirmed that the FuG-E vector displayed explicit neuronal specificity to the same extent as the FuG-C vector. This vector might promote approaches to pathway-selective gene manipulation and provide a powerful tool for effective gene therapy trials against neurological disorders through enhanced retrograde delivery.

  15. Antiviral effects of autologous CD4 T cells genetically modified with a conditionally replicating lentiviral vector expressing long antisense to HIV.

    PubMed

    Tebas, Pablo; Stein, David; Binder-Scholl, Gwendolyn; Mukherjee, Rithun; Brady, Troy; Rebello, Tessio; Humeau, Laurent; Kalos, Michael; Papasavvas, Emmanouil; Montaner, Luis J; Schullery, Daniel; Shaheen, Farida; Brennan, Andrea L; Zheng, Zhaohui; Cotte, Julio; Slepushkin, Vladimir; Veloso, Elizabeth; Mackley, Adonna; Hwang, Wei-Ting; Aberra, Faten; Zhan, Jenny; Boyer, Jean; Collman, Ronald G; Bushman, Frederic D; Levine, Bruce L; June, Carl H

    2013-02-28

    We report the safety and tolerability of 87 infusions of lentiviral vector–modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vector–transduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells.

  16. Antiviral effects of autologous CD4 T cells genetically modified with a conditionally replicating lentiviral vector expressing long antisense to HIV

    PubMed Central

    Tebas, Pablo; Stein, David; Binder-Scholl, Gwendolyn; Mukherjee, Rithun; Brady, Troy; Rebello, Tessio; Humeau, Laurent; Kalos, Michael; Papasavvas, Emmanouil; Montaner, Luis J.; Schullery, Daniel; Shaheen, Farida; Brennan, Andrea L.; Zheng, Zhaohui; Cotte, Julio; Slepushkin, Vladimir; Veloso, Elizabeth; Mackley, Adonna; Hwang, Wei-Ting; Aberra, Faten; Zhan, Jenny; Boyer, Jean; Collman, Ronald G.; Bushman, Frederic D.; Levine, Bruce L.

    2013-01-01

    We report the safety and tolerability of 87 infusions of lentiviral vector–modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vector–transduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells. This study is registered at www.clinicaltrials.gov as number NCT00295477. PMID:23264589

  17. Glycoprotein Ibalpha promoter drives megakaryocytic lineage-restricted expression after hematopoietic stem cell transduction using a self-inactivating lentiviral vector.

    PubMed

    Lavenu-Bombled, Cécile; Izac, Brigitte; Legrand, Faézeh; Cambot, Marie; Vigier, Agathe; Massé, Jean-Marc; Dubart-Kupperschmitt, Anne

    2007-06-01

    Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short-lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral-blood mobilized CD34(+) cells were transduced with a human immunodeficiency virus-derived self-inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIbalpha, GpIIb, or EF1alpha gene regulatory sequences. Both GpIbalpha and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIbalpha promoter was strictly MK-specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328-base-pair fragment of the human GpIbalpha gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells. Disclosure of potential conflicts of interest is found at the end of this article.

  18. Co-transduction of lentiviral and adenoviral vectors for co-delivery of growth factor and shRNA genes in mesenchymal stem cells-based chondrogenic system.

    PubMed

    Zhang, Feng; Yao, Yongchang; Su, Kai; Fang, Yu; Citra, Fudiman; Wang, Dong-An

    2015-09-01

    Gene delivery takes advantage of cellular mechanisms to express gene products and is an efficient way to deliver them into cells, influencing cellular behaviours and expression patterns. Among the delivery methods, viral vectors are applied due to their high efficiency. Two typical viral vectors for gene delivery include lentiviral vector for integrative transduction and adenoviral vector for transient episomal transduction, respectively. The selection and formulation of proper viral vectors applied to cells can modulate gene expression profiles and further impact the downstream pathways. In this study, recombinant lentiviral and adenoviral vectors were co-transduced in a synovial mesenchymal stem cells (SMSCs)-based articular chondrogenic system by which two transgenes were co-delivered - the gene for transforming growth factor (TGF)β3, to facilitate SMSC chondrogenesis, and the gene for small hairpin RNA (shRNA), targeting the mRNA of type I collagen (Col I) α1 chain to silence Col I expression and minimize fibrocartilage formation. Delivery of either gene could be achieved with either lentiviral or adenoviral vectors. Therefore, co-delivery of the two transgenes via the two types of vectors was performed to determine which combination was optimal for three-dimensional (3D) articular chondrogenesis to construct articular hyaline cartilage tissue. Suppression of Col I and expression of cartilage markers, including type II collagen, aggrecan and cartilage oligomeric matrix protein (COMP), were assessed at both the transcriptome and protein phenotypic levels. It was concluded that the combination of lentiviral-mediated TGFβ3 release and adenoviral-mediated shRNA expression (LV-T + Ad-sh) generally demonstrated optimal efficacy in engineered articular cartilage with SMSCs.

  19. PROSPER: An Integrated Feature-Based Tool for Predicting Protease Substrate Cleavage Sites

    PubMed Central

    Perry, Andrew J.; Akutsu, Tatsuya; Webb, Geoffrey I.; Whisstock, James C.; Pike, Robert N.

    2012-01-01

    The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s). Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database) with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate sequence using

  20. An integrated building demolition and waste planning model for the Fernald Site

    SciTech Connect

    Hampshire, L.H.; Clark, T.R.; Frost, M.L.; Reising, J.W.

    1995-04-01

    The Fernald DOE site will begin full-scale remediation of buildings under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) during the 1995 fiscal year pursuant to a signed Record of Decision. This effort is currently estimated to cost $350 million and span a minimum duration of 8 years, if funding is not a constraint. The identification of the most viable sequence and schedule for the effort involved the development of an integrated planning model and the commissioning of a sitewide planning team. The resulting work product represents the best combination of assumptions and calculations possible at this time and provides information necessary for compliance with the CERCLA Remedial Design documentation requirements for the over 230 component structures governed by the decision. Sequence and integrated schedule development for the decontamination and dismantlement (D&D) of Fernald structures has involved evaluation of current and future utilization of structures, availability of waste storage and staging space, the needs and impacts of other on-going Fernald projects, Resource Conservation and Recovery Act (RCRA) waste management and remediation projects, the layout of site utilities, site hydrology, and the potential sizing, location, and construction rates for an on-property disposal cell.

  1. Hanford Integrated Planning Process: 1993 Hanford Site-specific science and technology plan

    SciTech Connect

    Not Available

    1993-12-01

    This document is the FY 1993 report on Hanford Site-specific science and technology (S&T) needs for cleanup of the Site as developed via the Hanford Integrated Planning Process (HIPP). It identifies cleanup problems that lack demonstrated technology solutions and technologies that require additional development. Recommendations are provided regarding allocation of funding to address Hanford`s highest-priority technology improvement needs, technology development needs, and scientific research needs, all compiled from a Sitewide perspective. In the past, the S&T agenda for Hanford Site cleanup was sometimes driven by scientists and technologists, with minimal input from the ``problem owners`` (i.e., Westinghouse Hanford Company [WHC] staff who are responsible for cleanup activities). At other times, the problem-owners made decisions to proceed with cleanup without adequate scientific and technological inputs. Under both of these scenarios, there was no significant stakeholder involvement in the decision-making process. One of the key objectives of HIPP is to develop an understanding of the integrated S&T requirements to support the cleanup mission, (a) as defined by the needs of the problem owners, the values of the stakeholders, and the technology development expertise that exists at Hanford and elsewhere. This requires a periodic, systematic assessment of these needs and values to appropriately define a comprehensive technology development program and a complementary scientific research program. Basic to our success is a methodology that is defensible from a technical perspective and acceptable to the stakeholders.

  2. Development of a Site-Directed Integration Plasmid for Heterologous Gene Expression in Mycoplasma gallisepticum

    PubMed Central

    Indikova, Ivana; Szostak, Michael P.

    2013-01-01

    Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M. gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M. gallisepticum origin or replication (oriCMG). We failed to create a disruption vector, although by adding a further short fragment of the M. gallisepticum tufB upstream region we created a “Trojan horse” plasmid. This is fully integrated into the genomic DNA of M. gallisepticum, always at the same site, oriCMG, and is able to carry and express any gene of interest in the genetic background of M. gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M. gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes. PMID:24278444

  3. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin.

    PubMed Central

    Lee, M H; Pascopella, L; Jacobs, W R; Hatfull, G F

    1991-01-01

    Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guérin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified. Images PMID:1901654

  4. Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions.

    PubMed

    ter Brake, Olivier; Berkhout, Ben

    2007-09-01

    HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production.

  5. Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

    PubMed

    Bennett, Geoffrey R; Peters, Ryan; Wang, Xiao-hong; Hanne, Jeungphill; Sobol, Robert W; Bundschuh, Ralf; Fishel, Richard; Yoder, Kristine E

    2014-01-01

    Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

  6. Icebreaker-3 Drill Integration and Testing at Two Mars-Analog Sites

    NASA Technical Reports Server (NTRS)

    Glass, B.; Bergman, D.; Yaggi, B.; Dave, A.; Zacny, K.

    2016-01-01

    A decade of evolutionary development of integrated automated drilling and sample handling at analog sites and in test chambers has made it possible to go 1 meter through hard rocks and ice layers on Mars. The latest Icebreaker-3 drill has been field tested in 2014 at the Haughton Crater Marsanalog site in the Arctic and in 2015 with a Mars lander mockup in Rio Tinto, Spain, (with sample transfer arm and with a prototype life-detection instrument). Tests in Rio Tinto in 2015 successfully demonstrated that the drill sample (cuttings) was handed-off from the drill to the sample transfer arm and thence to the on-deck instrument inlet where it was taken in and analyzed ("dirt-to-data").

  7. Analysis of a fuel cell on-site integrated energy system for a residential complex

    NASA Technical Reports Server (NTRS)

    Simons, S. N.; Maag, W. L.

    1979-01-01

    Declining supplies of domestic oil and gas and the increased cost of energy resulted in a renewed emphasis in utilizing available resources in the most efficient manner possible. This, in turn, brought about a reassessment of a number of methods for converting fossil fuels to end uses at the highest practical efficiency. One of these is the on-site integrated energy system (OS/IES). This system provides electric power from an on-site power plant and recovers heat from the power plant that would normally be rejected to the environment. An OS/IES is potentially useful in any application that requires both electricity and heat. Several OS/IES are analyzed for a residential complex. The paper is divided into two sections; the first compares three energy supply systems, the second compares various designs for fuel cell OS/IES.

  8. 300 Area Integrated Field-Scale Subsurface Research Challenge (IFRC) Field Site Management Plan

    SciTech Connect

    Freshley, Mark D.

    2008-12-31

    Pacific Northwest National Laboratory (PNNL) has established the 300 Area Integrated Field-Scale Subsurface Research Challenge (300 Area IFRC) on the Hanford Site in southeastern Washington State for the U.S. Department of Energy’s (DOE) Office of Biological and Environmental Research (BER) within the Office of Science. The project is funded by the Environmental Remediation Sciences Division (ERSD). The purpose of the project is to conduct research at the 300 IFRC to investigate multi-scale mass transfer processes associated with a subsurface uranium plume impacting both the vadose zone and groundwater. The management approach for the 300 Area IFRC requires that a Field Site Management Plan be developed. This is an update of the plan to reflect the installation of the well network and other changes.

  9. The explosion sites of nearby supernovae seen with integral field spectroscopy

    NASA Astrophysics Data System (ADS)

    Kuncarayakti, Hanindyo

    Integral field spectroscopy of nearby supernova sites within ~30 Mpc have been obtained using multiple IFU spectrographs in Hawaii and Chile. This technique enables both spatial and spectral information of the explosion sites to be acquired simultaneously, thus providing the identification of the parent stellar population of the supernova progenitor and the estimates for its physical parameters including age and metallicity via the spectrum. While this work has mainly been done in the optical wavelengths using instruments such as VIMOS, GMOS, and MUSE, a near-infrared approach has also been carried out using the AO-assisted SINFONI. By studying the supernova parent stellar population, we aim to characterize the mass and metallicity of the progenitors of different types of supernovae.

  10. Site-specific integration of bacterial artificial chromosomes into human cells.

    PubMed

    McColl, Bradley; Howden, Sara; Vadolas, Jim

    2015-01-01

    Gene therapy of inherited diseases requires long-term maintenance of the corrective transgene. Stable integration of the introduced DNA molecule into one of the host cell chromosomes is the simplest strategy for achieving this. However, genotoxicity resulting from random insertion of the transgene raises serious safety concerns that must be addressed if gene therapy is to enter the clinical mainstream. The following method makes use of the Rep integrase of adeno-associated virus to insert a transgene into the human AAVS1 site, a known "safe harbor" region within the human genome. This approach has the potential for application to novel gene therapy strategies for improved safety. In addition, with this method it is also possible to create cell lines carrying BAC transgenes in the AAVS1 site.

  11. MAC Europe 1991 campaign: AIRSAR/AVIRIS data integration for agricultural test site classification

    NASA Technical Reports Server (NTRS)

    Sangiovanni, S.; Buongiorno, M. F.; Ferrarini, M.; Fiumara, A.

    1993-01-01

    During summer 1991, multi-sensor data were acquired over the Italian test site 'Otrepo Pavese', an agricultural flat area in Northern Italy. This area has been the Telespazio pilot test site for experimental activities related to agriculture applications. The aim of the investigation described in the following paper is to assess the amount of information contained in the AIRSAR (Airborne Synthetic Aperture Radar) and AVIRIS (Airborne Visible/Infrared Imaging Spectrometer) data, and to evaluate classification results obtained from each sensor data separately and from the combined dataset. All classifications are examined by means of the resulting confusion matrices and Khat coefficients. Improvements of the classification results obtained by using the integrated dataset are finally evaluated.

  12. Site-controlled Ag nanocrystals grown by molecular beam epitaxy-Towards plasmonic integration technology

    SciTech Connect

    Urbanczyk, Adam; Noetzel, Richard

    2012-12-15

    We demonstrate site-controlled growth of epitaxial Ag nanocrystals on patterned GaAs substrates by molecular beam epitaxy with high degree of long-range uniformity. The alignment is based on lithographically defined holes in which position controlled InAs quantum dots are grown. The Ag nanocrystals self-align preferentially on top of the InAs quantum dots. No such ordering is observed in the absence of InAs quantum dots, proving that the ordering is strain-driven. The presented technique facilitates the placement of active plasmonic nanostructures at arbitrarily defined positions enabling their integration into complex devices and plasmonic circuits.

  13. Current DOT research on the effect of multiple site damage on structural integrity

    NASA Astrophysics Data System (ADS)

    Tong, P.; Arin, Kemal; Jeong, David Y.; Greif, R.; Brewer, John C.; Bobo, Stephan N.; Sampath, Sam N.

    1992-07-01

    Multiple site damage (MSD) is a type of cracking that may be found in aging airplanes and which may adversely affect their continuing airworthiness. The Volpe National Transportation Systems Center has supported the Federal Aviation Administration Technical Center on structural integrity research for the past two and half years. The work has focused on understanding the behavior of MSD, detection of MSD during airframe inspection, and the avoidance of MSD in future designs. These three elements of the MSD problem are addressed and a summary of the completed work, the current status, and requirements for future research is provided.

  14. Fuel cell on-site integrated energy system parametric analysis of a residential complex

    NASA Technical Reports Server (NTRS)

    Simons, S. N.

    1977-01-01

    A parametric energy-use analysis was performed for a large apartment complex served by a fuel cell on-site integrated energy system (OS/IES). The variables parameterized include operating characteristics for four phosphoric acid fuel cells, eight OS/IES energy recovery systems, and four climatic locations. The annual fuel consumption for selected parametric combinations are presented and a breakeven economic analysis is presented for one parametric combination. The results show fuel cell electrical efficiency and system component choice have the greatest effect on annual fuel consumption; fuel cell thermal efficiency and geographic location have less of an effect.

  15. D Integrated Methodologies for the Documentation and the Virtual Reconstruction of AN Archaeological Site

    NASA Astrophysics Data System (ADS)

    Balletti, C.; Guerra, F.; Scocca, V.; Gottardi, C.

    2015-02-01

    Highly accurate documentation and 3D reconstructions are fundamental for analyses and further interpretations in archaeology. In the last years the integrated digital survey (ground-based survey methods and UAV photogrammetry) has confirmed its main role in the documentation and comprehension of excavation contexts, thanks to instrumental and methodological development concerning the on site data acquisition. The specific aim of the project, reported in this paper and realized by the Laboratory of Photogrammetry of the IUAV University of Venice, is to check different acquisition systems and their effectiveness test, considering each methodology individually or integrated. This research focuses on the awareness that the integration of different survey's methodologies can as a matter of fact increase the representative efficacy of the final representations; these are based on a wider and verified set of georeferenced metric data. Particularly the methods' integration allows reducing or neutralizing issues related to composite and complex objects' survey, since the most appropriate tools and techniques can be chosen considering the characteristics of each part of an archaeological site (i.e. urban structures, architectural monuments, small findings). This paper describes the experience in several sites of the municipality of Sepino (Molise, Italy), where the 3d digital acquisition of cities and structure of monuments, sometimes hard to reach, was realized using active and passive techniques (rage-based and image based methods). This acquisition was planned in order to obtain not only the basic support for interpretation analysis, but also to achieve models of the actual state of conservation of the site on which some reconstructive hypotheses can be based on. Laser scanning data were merged with Structure from Motion techniques' clouds into the same reference system, given by a topographical and GPS survey. These 3d models are not only the final results of the metric

  16. INTEGRATED PROCESS GAS MODELING FOR TRITIUM SYSTEMS AT THE SAVANNAH RIVER SITE

    SciTech Connect

    Hang, T; Anita Poore, A

    2007-08-30

    Significant savings are being realized from the consolidated tritium gas-processing operations at the Savannah River Site. However, the trade-off is some reduction of operational flexibility due to decreased storage capacity for process and waste gases. Savannah River National Laboratory researchers are developing an integrated process gas model for tritium processing using Aspen Custom Modeler{trademark} (ACM) software. The modeling involves fully characterizing process flow streams (gas composition, quantity), frequency of batch transfers, and availability of equipment in the flow stream. The model provides a valuable engineering tool to identify flow bottlenecks, thereby enabling adjustments to be made to improve process operations.

  17. Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions.

    PubMed

    Romano, Oriana; Cifola, Ingrid; Poletti, Valentina; Severgnini, Marco; Peano, Clelia; De Bellis, Gianluca; Mavilio, Fulvio; Miccio, Annarita

    2017-05-28

    Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs. The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

  18. The EuroSITES network: Integrating and enhancing fixed-point open ocean observatories around Europe

    NASA Astrophysics Data System (ADS)

    Lampitt, Richard S.; Larkin, Kate E.; EuroSITES Consortium

    2010-05-01

    EuroSITES is a 3 year (2008-2011) EU collaborative project (3.5MEuro) with the objective to integrate and enhance the nine existing open ocean fixed point observatories around Europe (www.eurosites.info). These observatories are primarily composed of full depth moorings and make multidisciplinary in situ observations within the water column as the European contribution to the global array OceanSITES (www.oceansites.org). In the first 18 months, all 9 observatories have been active and integration has been significant through the maintenance and enhancement of observatory hardware. Highlights include the enhancement of observatories with sensors to measure O2, pCO2, chlorophyll, and nitrate in near real-time from the upper 1000 m. In addition, some seafloor missions are also actively supported. These include seafloor platforms currently deployed in the Mediterranean, one for tsunami detection and one to monitor fluid flow related to seismic activity and slope stability. Upcoming seafloor science missions in 2010 include monitoring benthic biological communities and associated biogeochemistry as indicators of climate change in both the Northeast Atlantic and Mediterranean. EuroSITES also promotes the development of innovative sensors and samplers in order to progress capability to measure climate-relevant properties of the ocean. These include further developing current technologies for autonomous long-term monitoring of oxygen consumption in the mesopelagic, pH and mesozooplankton abundance. Many of these science missions are directly related to complementary activities in other European projects such as EPOCA, HYPOX and ESONET. In 2010 a direct collaboration including in situ field work will take place between ESONET and EuroSITES. The demonstration mission MODOO (funded by ESONET) will be implemented in 2010 at the EuroSITES PAP observatory. Field work will include deployment of a seafloor lander system with various sensors which will send data to shore in real

  19. 2001 Columbia River Recreation Survey -- Implications for Hanford Site Integrated Assessment

    SciTech Connect

    Anderson, Dave M.; Scott, Michael J.; Bunn, Amoret L.; Fowler, Richard A.; Prendergast, Ellen L.; Miley, Terri B.; Eschbach, Tara O.; Jaksch, John A.

    2002-05-16

    This report presents the results from the Columbia River Recreation Survey conducted in the summer of 2001. The survey combined on-site personal interviews with parties engaged in river recreation with on-site field observations to develop a picture of summer river recreation on the Columbia. The study area stretched from just below Priest Rapids Dam in the north to McNary Dam in the south, and was divided into four "Areas" that correspond to the river areas used by the Groundwater/Vadose Zone Integration Project. This study is part of the Groundwater/Vadose Zone Integration Project and was commissioned specifically to document the current recreation use levels in these areas of the river, and to elicit recreation-related expenditure information from visitors. This information informs economic and environmental models used to measure the economic risk posed by possible, but unlikely, releases of contaminants from the Hanford site into the Columbia River. During the study period, researchers collected 256 survey responses and 396 field observations from recreation sites up and down both shores of the river in the study area. Results presented include analysis of trip duration by river activity, trip frequency, and visitor place of origin. Economics-related results include trip expenditure profiles by activity and place of origin. Data also were collected on fishing effort. Visitors also were asked to indicate what activity or destination substitution they would choose in the hypothetical example that the river could not be accessed. The report also highlights some limitations in the approach. Principally, by doing this research in the summer, the recreational use of the river in the other seasons was not documented. The report provides data that suggest the significance of the other seasons - particularly spring and fall - for salmon and steelhead fishing. This stretch of the Columbia is also well known for waterfowl hunting.

  20. Development of a genomic site for gene integration and expression in Enterococcus faecalis

    PubMed Central

    DebRoy, Sruti; van der Hoeven, Ransome; Singh, Kavindra V.; Gao, Peng; Harvey, Barrett R.; Murray, Barbara E.; Garsin, Danielle A.

    2012-01-01

    Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems. PMID:22542850

  1. Integrated observation and analysis of pre-earthquake related signals over major geohazard sites

    NASA Astrophysics Data System (ADS)

    Ouzounov, Dimitar; Pulinets, Sergey; Tramutoli, Valerio; Lee, Lou; Liu, Tiger; Hayakawa, Masashi; Hattori, Katsumi; Kafatos, Menas; Taylor, Patrick

    2013-04-01

    We are conducting an integrated study involving multi-parameter observations in our investigation of phenomena proceeding major earthquakes. Our approach is based on a systematic analysis of several parameters namely: foreshock seismic activities; gas discharge; thermal infrared radiation; outgoing radiation flux; ionospheric electron density; VLF sub-ionospheric propagation anomalies; and atmospheric temperature and humidity which we propose are associated with earthquakes, For the first time we intend to have a similar set of integrated geophysical measurements (in-situ and satellite) over regions of active earthquakes and volcanoes. We are in the process of establishing an iSITES (Integrated sites) framework for coordinate measurements, data sharing and validation. So far we include six regions: Southern California (USA), Eastern Honshu (Japan), Central and Southern Italy, Taiwan (ROC), Corinth rift (Greece), Kamchatka and Sakhalin (Russia). We are continuing the same approach of near real-time information sharing established under the EU-FP7 PRE-EARTHQUAKE project. In the future we may well coordinate this effort with GEOSS supersite site project. This provides a new opportunity to cross validate our results with the dense networks of in-situ and space measurements. Initially we tested the iSITES observational data in two different tectonic regions, first with recent large earthquakes, viz.- Tohoku-oki (M9, 2011, Japan) and Emilia (M5.9, 2012, N. Italy), and L'Aquia (2009, Central Italy) regions. Our retrospective analyses of these satellite data have shown the presence of anomalies in the atmosphere. Second, we did a retrospective analysis to check the re-occurrence of similar anomalous behavior over Taiwan, Japan and Kamchatka, which include 40 major earthquakes (M>5.9) for the period of 2005-2009. We found anomalous behavior before all of these events with no false negatives; false positives were less then 20%. Our initial results suggest that iSITES

  2. Improved lentiviral transduction of ALS motoneurons in vivo via dual targeting.

    PubMed

    O'Leary, Valerie B; Ovsepian, Saak V; Bodeker, Macdara; Dolly, J Oliver

    2013-11-04

    Treatment of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, is hampered by its complex etiology and lack of efficient means for targeted transfer of therapeutics into motoneurons. The objective of this research was engineering of a versatile motoneuron targeting adapter--a full-length atoxic tetanus toxin fused to core-streptavidin (CS-TeTIM)--for retro-axonal transduction of viral vectors; validation of the targeting efficiency of CS-TeTIM in vivo, by expression of green fluorescence protein (GFP) reporter in motoneurons of presymptomatic and symptomatic ALS-like SOD1(G93A) mice, and comparison with age-matched controls; and appraisal of lentiviral transduction with CS-TeTIM relative to (1) a HC binding fragment of tetanus toxin CS-TeTx(HC), (2) rabies glycoprotein (RG), and (3) a CS-TeTIM-RG dual targeting approach. CS-TeTIM and CS-TeTx(HC) were engineered using recombinant technology and site-directed mutagenesis. Biotinylated vectors, pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or RG, were linked to these adaptors and injected intraperitoneally (ip) into presymptomatic (12 weeks old), symptomatic SOD1(G93A) (22 weeks old) or wild type control mice, followed by monitoring of GFP expression in the spinal cord and supraspinal motor structures with quantitative PCR and immuno-histochemistry. Transcripts were detected in the spinal cord and supraspinal motor structures of all mice 2 weeks after receiving a single ip injection, although in symptomatic SOD1(G93A) animals reporter RNA levels were lower compared to presymptomatic and wild-type controls irrespective of the targeting approach. GFP transduction with CS-TeTIM proved more efficient than CS-TeTx(HC) across all groups while CS-TeTIM-RG dual-targeted vectors yielded the highest transcript numbers. Importantly, in both wild-type and presymptomatic SOD1(G93A) mice strong colabeling of choline-acetyltransferase (ChAT) and GFP was visualized in neurons of the

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

    PubMed

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

    2007-07-30

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

  4. Durable, safe, multi-gene lentiviral vector expression in feline trabecular meshwork.

    PubMed

    Khare, Pranay D; Loewen, Nils; Teo, Wulin; Barraza, Román A; Saenz, Dyana T; Johnson, Douglas H; Poeschla, Eric M

    2008-01-01

    Multiple disease-specific considerations have led to interest in the potential of gene therapy to permanently correct elevated intraocular pressure (IOP), the main causal risk factor for primary open angle glaucoma (POAG). Since IOP elevation results from abnormal resistance to aqueous humor outflow from the eye through the trabecular meshwork (TM), a means to genetically modify this specialized outflow organ permanently and safely is a prioritized goal. Here we tested different lentiviral vector designs and doses for long-term transgene expression in a large animal model, and investigated whether exogenously introduced myocilin proteins influenced IOP. The anterior chambers of 18 domestic cats (36 eyes) were injected with dual-gene feline immunodeficiency virus (FIV) vectors. Substantial, well-tolerated green fluorescent protein (GFP) expression was achieved in TM and monitored non-invasively in vivo for 1.2-2.3 years, using both 5' cap-translation and internal ribosome entry site (IRES)-translation. In all 36 eyes, post-mortem examination revealed substantial TM transgene expression (which often greatly exceeded that observable non-invasively during life). However, co-expression with enhanced GFP of myocilin or a juvenile glaucoma-associated mutant myocilin did not elevate IOP. These results demonstrate a safe, long-term single and dual gene expression in TM and establish an experimental system for testing candidate therapeutic transgenes for POAG.

  5. Stable genetic modification of human embryonic stem cells by lentiviral vectors.

    PubMed

    Gropp, Michal; Itsykson, Pavel; Singer, Orna; Ben-Hur, Tamir; Reinhartz, Etti; Galun, Eithan; Reubinoff, Benjamin E

    2003-02-01

    Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of the early preimplantation embryo. An efficient strategy for stable genetic modification of hES cells may be highly valuable for manipulating the cells in vitro and may promote the study of hES cell biology, human embryogenesis, and the development of cell-based therapies. Here, we demonstrate that vectors derived from self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1) are efficient tools for stable genetic modification of hES cells. Transduction of hES cells by a modified vector derived from SIN HIV-1 and containing the woodchuck hepatitis regulatory element (WPRE) and the central polypurine tract (cPPT) sequence facilitated stable transgene expression during prolonged (38 weeks) undifferentiated proliferation in vitro. Southern blot analysis revealed that the viral vector had integrated into the host cells' DNA. Transgene expression was maintained throughout differentiation into progeny of all three germ layers both in vitro and in vivo in teratomas. Thus, the transduced hES cells retained the capability for self-renewal and their pluripotent potential. Genetic modification of hES cells by lentiviral vectors provides a powerful tool for basic and applied research in the area of human ES cells.

  6. Shedding of clinical-grade lentiviral vectors is not detected in a gene therapy setting.

    PubMed

    Cesani, M; Plati, T; Lorioli, L; Benedicenti, F; Redaelli, D; Dionisio, F; Biasco, L; Montini, E; Naldini, L; Biffi, A

    2015-06-01

    Gene therapy using viral vectors that stably integrate into ex vivo cultured cells holds great promises for the treatment of monogenic diseases as well as cancer. However, carry-over of infectious vector particles has been described to occur upon ex vivo transduction of target cells. This, in turn, may lead to inadvertent spreading of viral particles to off-target cells in vivo, raising concerns for potential adverse effects, such as toxicity of ectopic transgene expression, immunogenicity from in vivo transduced antigen-presenting cells and, possibly, gene transfer to germline cells. Here, we have investigated factors influencing the extent of lentiviral vector (LV) shedding upon ex vivo transduction of human hematopoietic stem and progenitor cells. Our results indicate that, although vector carry-over is detectable when using laboratory-grade vector stocks, the use of clinical-grade vector stocks strongly decreases the extent of inadvertent transduction of secondary targets, likely because of the higher degree of purification. These data provide supportive evidence for the safe use of the LV platform in clinical settings.

  7. Quantitative dynamic imaging of immune cell signalling using lentiviral gene transfer.

    PubMed

    Bagnall, J; Boddington, C; Boyd, J; Brignall, R; Rowe, W; Jones, N A; Schmidt, L; Spiller, D G; White, M R H; Paszek, P

    2015-06-01

    Live-cell imaging of fluorescent fusion proteins has transformed our understanding of mammalian cell signalling and function. However, some cellular systems such as immune cells are unsuitable or refractory to many existing transgene delivery methods thus limiting systematic analyses. Here, a flexible lentiviral gene transfer platform for dynamic time-lapse imaging has been developed and validated with single-molecule spectroscopy, mathematical modelling and transcriptomics and used for analysis of a set of inflammation-related signalling networks. Time-lapse imaging of nuclear factor kappa B (NF-κB), signal transducer and activator of transcription (STATs) and nuclear factor of activated T-cells (NFAT) in mammalian immune cell lines provided evidence for heterogeneous temporal encoding of inflammatory signals. In particular, the absolute quantification of single-cell responses over time via fluorescent correlation spectroscopy (FCS) showed that NF-κB p65 activation in response to tumour necrosis factor α (TNFα) was differentially encoded in variable amplitude of nuclear translocation between immune and non-immune cells. The absolute number of activated molecules was dictated in part by the cell size, suggesting a morphology-dependent regulatory mechanism. The developed platform will enable further absolute quantitative analyses of the dynamic interactions between signalling networks, in and between individual cells, allowing better integration with mathematical models of signalling networks.

  8. Simian immunodeficiency virus-Vpx for improving integrase defective lentiviral vector-based vaccines

    PubMed Central

    2012-01-01

    Background Integrase defective lentiviral vectors (IDLV) represent a promising delivery system for immunization purposes. Human dendritic cells (DC) are the main cell types mediating the immune response and are readily transduced by IDLV, allowing effective triggering of in vitro expansion of antigen-specific primed CD8+ T cells. However, IDLV expression in transduced DC is at lower levels than those of the integrase (IN) competent counterpart, thus requiring further improvement of IDLV for future use in the clinic. Results In this paper we show that the addition of simian immunodeficiency (SIV)-Vpx protein in the vector preparation greatly improves transduction of human and simian DC, but not of murine DC, thus increasing the ability of transduced DC to act as functional antigen presenting cells, in the absence of integrated vector sequences. Importantly, the presence of SIV-Vpx allows for using lower dose of input IDLV during in vitro transduction, thus further improving the IDLV safety profile. Conclusions These results have significant implications for the development of IDLV-based vaccines. PMID:22913641

  9. Targeted expression of two proteins in neural retina using self-inactivating, insulated lentiviral vectors carrying two internal independent promoters.

    PubMed

    Semple-Rowland, Susan L; Eccles, Kristofer S; Humberstone, Elizabeth J

    2007-10-18

    There is increasing interest in developing viral vectors capable of reliably delivering multiple therapeutic genes to targeted cell populations. Currently, bicistronic vectors carrying two transgenes linked by an internal ribosomal entry site (IRES) are the most commonly employed vectors to accomplish this goal. We and others have found that the protein encoded downstream of the IRES in these vectors is not reliably expressed. The purpose of this study was to determine if replacement of the IRES in our self-inactivating, insulated, lentiviral vectors with a second, independent, cell-specific promoter would produce a vector that reliably expressed two proteins in targeted retinal cells in vivo. Five dual promoter lentiviral vectors were constructed using our self-inactivating (SIN), insulated, lentiviral backbone. Each vector carried two independent transgenes encoding a fluorescent protein (GFP or tdTomato) whose expression was driven by three photoreceptor promoters (interphotoreceptor retinoid binding protein-IRPB1783; guanylate cyclase activating protein 1-GCAP292; rhodopsin-mOP500) and one ubiquitously expressed promoter (elongation factor 1alpha-EF1alpha). Constructs were packaged and injected into the optic vesicles of developing chicken embryos. The day before hatching, the retinas were removed and examined as whole mount tissues and as frozen sections using fluorescent microscopy. In our first experiment, we characterized the expression of the three photoreceptor promoters in chicken retina. The activities of GCAP292 and IRBP1783 were restricted to cone cells. GCAP292 was also active in a small sub-group of inner nuclear cells. The activity of mOP500 was restricted to rod cells. In our second experiment, we characterized the activity of three dual promoter vectors: GCAP292-GFP-IRBP1783-tdTomato, IRBP-tdTomato-GCAP292-GFP, and IRBP1783-tdTomato-mOP500-GFP. All three vectors produced easily detectable levels of GFP and tdTomato in transduced retinas, a result

  10. Prediction of phosphorylation sites based on the integration of multiple classifiers.

    PubMed

    Han, R Z; Wang, D; Chen, Y H; Dong, L K; Fan, Y L

    2017-02-23

    Phosphorylation is an important part of post-translational modifications of proteins, and is essential for many biological activities. Phosphorylation and dephosphorylation can regulate signal transduction, gene expression, and cell cycle regulation in many cellular processes. Phosphorylation is extremely important for both basic research and drug discovery to rapidly and correctly identify the attributes of a new protein phosphorylation sites. Moreover, abnormal phosphorylation can be used as a key medical feature related to a disease in some cases. The using of computational methods could improve the accuracy of detection of phosphorylation sites, which can provide predictive guidance for the prevention of the occurrence and/or the best course of treatment for certain diseases. Furthermore, this approach can effectively reduce the costs of biological experiments. In this study, a flexible neural tree (FNT), particle swarm optimization, and support vector machine algorithms were used to classify data with secondary encoding according to the physical and chemical properties of amino acids for feature extraction. Comparison of the classification results obtained from the three classifiers showed that the classification of the FNT was the best. The three classifiers were then integrated in the form of a minority subordinate to the majority vote to obtain the results. The performance of the integrated model showed improvement in sensitivity (87.41%), specificity (87.60%), and accuracy (87.50%).

  11. HIV Integration Site Analysis of Cellular Models of HIV Latency with a Probe-Enriched Next-Generation Sequencing Assay

    PubMed Central

    Sunshine, Sara; Kirchner, Rory; Amr, Sami S.; Mansur, Leandra; Shakhbatyan, Rimma; Kim, Michelle; Bosque, Alberto; Siliciano, Robert F.; Planelles, Vicente; Hofmann, Oliver; Ho Sui, Shannan

    2016-01-01

    ABSTRACT Antiretroviral therapy (ART) is successful in the suppression of HIV but cannot target and eradicate the latent proviral reservoir. The location of retroviral integration into the human genome is thought to play a role in the clonal expansion of infected cells and HIV persistence. We developed a high-throughput targeted sequence capture assay that uses a pool of HIV-specific probes to enrich Illumina libraries prior to deep sequencing. Using an expanded clonal population of ACH-2 cells, we demonstrate that this sequence capture assay has an extremely low false-positive rate. This assay assessed four cellular models commonly used to study HIV latency and latency-reversing agents: ACH-2 cells, J-Lat cells, the Bcl-2-transduced primary CD4+ model, and the cultured TCM (central memory) CD4+ model. HIV integration site characteristics and genes were compared between these cellular models and to previously reported patient data sets. Across these cellular models, there were significant differences in integration site characteristics, including orientation relative to that of the host gene, the proportion of clonally expanded sites, and the proportion located within genic regions and exons. Despite a greater diversity of minority integration sites than expected in ACH-2 cells, their integration site characteristics consistently differed from those of the other models and from the patient samples. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. These findings show that integration site differences exist among the commonly used cellular models of HIV latency and in comparison to integration sites found in patient samples. IMPORTANCE Despite the success of ART, currently there is no successful therapy to eradicate integrated proviruses. Cellular models of HIV latency are used to test the efficacy of latency-reversing agents, but it is unclear how well these models reflect

  12. HIV Integration Site Analysis of Cellular Models of HIV Latency with a Probe-Enriched Next-Generation Sequencing Assay.

    PubMed

    Sunshine, Sara; Kirchner, Rory; Amr, Sami S; Mansur, Leandra; Shakhbatyan, Rimma; Kim, Michelle; Bosque, Alberto; Siliciano, Robert F; Planelles, Vicente; Hofmann, Oliver; Ho Sui, Shannan; Li, Jonathan Z

    2016-05-01

    Antiretroviral therapy (ART) is successful in the suppression of HIV but cannot target and eradicate the latent proviral reservoir. The location of retroviral integration into the human genome is thought to play a role in the clonal expansion of infected cells and HIV persistence. We developed a high-throughput targeted sequence capture assay that uses a pool of HIV-specific probes to enrich Illumina libraries prior to deep sequencing. Using an expanded clonal population of ACH-2 cells, we demonstrate that this sequence capture assay has an extremely low false-positive rate. This assay assessed four cellular models commonly used to study HIV latency and latency-reversing agents: ACH-2 cells, J-Lat cells, the Bcl-2-transduced primary CD4(+)model, and the cultured TCM(central memory) CD4(+)model. HIV integration site characteristics and genes were compared between these cellular models and to previously reported patient data sets. Across these cellular models, there were significant differences in integration site characteristics, including orientation relative to that of the host gene, the proportion of clonally expanded sites, and the proportion located within genic regions and exons. Despite a greater diversity of minority integration sites than expected in ACH-2 cells, their integration site characteristics consistently differed from those of the other models and from the patient samples. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. These findings show that integration site differences exist among the commonly used cellular models of HIV latency and in comparison to integration sites found in patient samples. Despite the success of ART, currently there is no successful therapy to eradicate integrated proviruses. Cellular models of HIV latency are used to test the efficacy of latency-reversing agents, but it is unclear how well these models reflect HIV integration

  13. Integrated Geophysycal Prospecting in Late Antiquity and Early Medieval Sites in Italy

    NASA Astrophysics Data System (ADS)

    Giannotta, Maria Teresa; Leucci, Giovanni; De Giorgi, Lara; Matera, Loredana; Persico, Raffaele; Muci, Giuseppe

    2016-04-01

    In this contribution, the results of some integrated geophysical prospecting (magnetometric and GPR) are exposed. This work has been performed in collaboration between archaeologists and geophysicists within the research project "History and Global Archaeology of the Rural Landascapes in Italy, between Late Antiquity and Medieval period. Integrated systems of sources, methodologies, and technologies for a sustainable development", financed by the Italian Ministry for Instruction, University and Research MIUR. In particular, the archaeological sites of Badia and San Giovanni in Malcantone, both in the Apulia Region (eastern-southern Italy) have been prospect. The sites have been identified on the basis of available documents, archaeological surveys and testimonies. In particular, we know that in Badia [1] it was probable the presence of an ancient roman villa of the late ancient period (strongly damaged by the subsequent ploughing activities). Whereas in San Giovanni there is still, today, a small chapel (deconsecrated) that was likely to be part of a previous larger church (probably a basilica of the early Christian period) restricted in the subsequent centuries (probably in more phases). The Saracen raids of the XVI centuries made the site ruined and abandoned. In both sites integrated prospecting have been performed [2-6] with a the integration of archaeological, magnetometer and a GPR data have provided some interesting results, allowing to overcome the difficulties relative to an extensive GPR prospecting, that could not be performed because of the intrinsic superficial roughness and/or the intensive ploughing activities. The prospecting activities, in particular, have added elements that seem to confirm the main archaeological hypothesis that motivate their performing, as it will be show at the conference. References [1] M. T, Giannotta, G. Leucci, R. Persico, M. Leo Imperiale, The archaeological site of Badia in terra d'Otranto: contribution of the

  14. An integrated observational site for monitoring pre-earthquake processes in Peloponnese, Greece. Preliminary results.

    NASA Astrophysics Data System (ADS)

    Tsinganos, Kanaris; Karastathis, Vassilios K.; Kafatos, Menas; Ouzounov, Dimitar; Tselentis, Gerassimos; Papadopoulos, Gerassimos A.; Voulgaris, Nikolaos; Eleftheriou, Georgios; Mouzakiotis, Evangellos; Liakopoulos, Spyridon; Aspiotis, Theodoros; Gika, Fevronia; E Psiloglou, Basil

    2017-04-01

    We are presenting the first results of developing a new integrated observational site in Greece to study pre-earthquake processes in Peloponnese, lead by the National Observatory of Athens. We have developed a prototype of multiparameter network approach using an integrated system aimed at monitoring and thorough studies of pre-earthquake processes at the high seismicity area of the Western Hellenic Arc (SW Peloponnese, Greece). The initial prototype of the new observational systems consists of: (1) continuous real-time monitoring of Radon accumulation in the ground through a network of radon sensors, consisting of three gamma radiation detectors [NaI(Tl) scintillators], (2) nine-station seismic array installed to detect and locate events of low magnitude (less than 1.0 R) in the offshore area of the Hellenic arc, (3) real-time weather monitoring systems (air temperature, relative humidity, precipitation, pressure) and (4) satellite thermal radiation from AVHRR/NOAA-18 polar orbit sensing. The first few moths of operations revealed a number of pre-seismic radon variation anomalies before several earthquakes (M>3.6). The radon increases systematically before the larger events. For example a radon anomaly was predominant before the event of Sep 28, M 5.0 (36.73°N, 21.87°E), 18 km ESE of Methoni. The seismic array assists in the evaluation of current seismicity and may yield identification of foreshock activity. Thermal anomalies in satellite images are also examined as an additional tool for evaluation and verification of the Radon increase. According to the Lithosphere-Atmosphere-Ionosphere Coupling (LAIC) concept, atmospheric thermal anomalies observed before large seismic events are associated with the increase of Radon concentration on the ground. Details about the integrating ground and space observations, overall performance of the observational sites, future plans in advancing the cooperation in observations will be discussed.

  15. The Cherenkov Telescope array on-site integral sensitivity: observing the Crab

    NASA Astrophysics Data System (ADS)

    Fioretti, Valentina; Bulgarelli, Andrea; Schüssler, Fabian

    2016-07-01

    The Cherenkov Telescope Array (CTA) is the future large observatory in the very high energy (VHE) domain. Operating from 20 GeV to 300 TeV, it will be composed of tens of Imaging Air Cherenkov Telescopes (IACTs) displaced in a large area of a few square kilometers in both the southern and northern hemispheres. Thanks to the wide energy coverage and the tremendous boost in effective area (10 times better than the current IACTs), for the first time a VHE observatory will be able to detect transient phenomena in short exposures. The CTA/DATA On-Site Analysis (OSA) is the system devoted to the development of dedicated pipelines and algorithms to be used at the CTA site for the reconstruction, data quality monitoring, science monitoring and realtime science alerting during observations. The minimum exposure required to issue a science alert is not a general requirement of the observatory but is a function of the astrophysical object under study, because the ability to detect a given source is determined by the integral sensitivity which, in addition to the CTA Monte Carlo simulations, providing the energy-dependent instrument response (e.g. the effective area and the background rate), requires the spectral distribution of the science target. The OSA integral sensitivity is computed here for the most studied source at Gamma-rays, the Crab Nebula, for a set of exposures ranging from 1000 seconds to 50 hours, using the full CTA Southern array. The reason for the Crab Nebula selection as the first example of OSA integral sensitivity is twofold: (i) this source is characterized by a broad spectrum covering the entire CTA energy range; (ii) it represents, at the time of writing, the standard candle in VHE and it is often used as unit for the IACTs sensitivity. The effect of different Crab Nebula emission models on the CTA integral sensitivity is evaluated, to emphasize the need for representative spectra of the CTA science targets in the evaluation of the OSA use cases. Using

  16. Collaboration for the integration of HIV prevention at Title X family planning service delivery sites.

    PubMed

    Tran, Nhan T; Hallerdin, Jule M; Flowers-Maple, Charon; Moskosky, Susan B

    2010-01-01

    Since 2001, the U.S. Department of Health and Human Services' Office of Family Planning (OFP), in collaboration with the Minority AIDS Initiative, has provided supplemental grant funds to Title X-funded family planning service delivery sites to expand the availability of human immunodeficiency virus (HIV) prevention services. This work has resulted in three major outcomes: (1) increased institutional capacity for the delivery of HIV-prevention services at Title X family planning service delivery sites, (2) the successful implementation of HIV-prevention services at these sites, and (3) the identification of HIV-positive individuals who were referred to care services. These efforts resulted in a total of 539,667 unduplicated individuals being tested for HIV. These tests resulted in the identification of 1,692 HIV-positive individuals who otherwise may not have been tested for HIV. More than 85% of the HIV-positive cases were detected among clients who self-identified as members of racial/ethnic minority groups. The integration of HIV-prevention services is a feasible and effective strategy for detecting HIV infection among women, including women in racial/ethnic minority groups.

  17. LASAGNA-Search: an integrated web tool for transcription factor binding site search and visualization.

    PubMed

    Lee, Chic; Huang, Chun-Hsi

    2013-03-01

    The release of ChIP-seq data from the ENCyclopedia Of DNA Elements (ENCODE) and Model Organism ENCyclopedia Of DNA Elements (modENCODE) projects has significantly increased the amount of transcription factor (TF) binding affinity information available to researchers. However, scientists still routinely use TF binding site (TFBS) search tools to scan unannotated sequences for TFBSs, particularly when searching for lesser-known TFs or TFs in organisms for which ChIP-seq data are unavailable. The sequence analysis often involves multiple steps such as TF model collection, promoter sequence retrieval, and visualization; thus, several different tools are required. We have developed a novel integrated web tool named LASAGNA-Search that allows users to perform TFBS searches without leaving the web site. LASAGNA-Search uses the LASAGNA (Length-Aware Site Alignment Guided by Nucleotide Association) algorithm for TFBS alignment. Important features of LASAGNA-Search include (i) acceptance of unaligned variable-length TFBSs, (ii) a collection of 1726 TF models, (iii) automatic promoter sequence retrieval, (iv) visualization in the UCSC Genome Browser, and (v) gene regulatory network inference and visualization based on binding specificities. LASAGNA-Search is freely available at http://biogrid.engr.uconn.edu/lasagna_search/.

  18. Site-specific integration and tailoring of cassette design for sustainable gene transfer.

    PubMed

    Lombardo, Angelo; Cesana, Daniela; Genovese, Pietro; Di Stefano, Bruno; Provasi, Elena; Colombo, Daniele F; Neri, Margherita; Magnani, Zulma; Cantore, Alessio; Lo Riso, Pietro; Damo, Martina; Pello, Oscar M; Holmes, Michael C; Gregory, Philip D; Gritti, Angela; Broccoli, Vania; Bonini, Chiara; Naldini, Luigi

    2011-08-21

    Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

  19. Integrating basic research and long-term management: a case study using avian research at the Savannah River Site

    Treesearch

    John B. Dunning; John C. Kilgo

    2000-01-01

    In this introduction to Avian Research at the Savannah River Site: A Model for Integrating Basic Research and Long-term Management, editors Dunning and Kilgo discuss the interrelationship between research and management at the Savannah River Site. They emphasize the increasing need for collaboration between scientists and land managers....

  20. Interstitial telomeric sequences in human chromosomes cluster with common fragile sites, mutagen sensitive sites, viral integration sites, cancer breakpoints, proto-oncogenes and breakpoints involved in primate evolution

    SciTech Connect

    Adekunle, S.S.A.; Wyandt, H.; Mark, H.F.L.

    1994-09-01

    Recently we mapped the telomeric repeat sequences to 111 interstitial sites in the human genome and to sites of gaps and breaks induced by aphidicolin and sister chromatid exchange sites detected by BrdU. Many of these sites correspond to conserved fragile sites in man, gorilla and chimpazee, to sites of conserved sister chromatid exchange in the mammalian X chromosome, to mutagenic sensitive sites, mapped locations of proto-oncogenes, breakpoints implicated in primate evolution and to breakpoints indicated as the sole anomaly in neoplasia. This observation prompted us to investigate if the interstitial telomeric sites cluster with these sites. An extensive literature search was carried out to find all the available published sites mentioned above. For comparison, we also carried out a statistical analysis of the clustering of the sites of the telomeric repeats with the gene locations where only nucleotide mutations have been observed as the only chromosomal abnormality. Our results indicate that the telomeric repeats cluster most with fragile sites, mutagenic sensitive sites and breakpoints implicated in primate evolution and least with cancer breakpoints, mapped locations of proto-oncogenes and other genes with nucleotide mutations.

  1. An Integrated Approach to Quantify Groundwater - Surface Water Interactions: the Norman Research Site, Oklahoma

    NASA Astrophysics Data System (ADS)

    Mendoza-Sanchez, I.; Phanikumar, M. S.; Masoner, J.; Cozzarelli, I.; McGuire, J. T.

    2009-12-01

    An intensive investigation of hydrogeologic and biogeochemical processes controlling contaminant migration and attenuation is in progress at the Norman Landfill Research Site in Oklahoma. The site involves a wetland that overlies a landfill leachate plume. The wetland-aquifer system actively exchanges contaminants and nutrients. These chemicals move from the wetland to the aquifer and vice versa depending on the groundwater-surface water (GW-SW) exchange rate and flow direction. The GW-SW flux has to be quantified to better understand the fate and transport of contaminants and nutrients. Different types of data have been collected at the site over a period of ten years including meteorological data, isotopic composition of water samples, water levels, pan evaporation rates, and seepage fluxes. This paper reports the development and application of a process-based water balance model based on long-term climate, soil, vegetation and hydrological dynamics of the system to determine the GW-SW flow rates. Our integrated approach involved model evaluation by means of the following independent measurements: (a) groundwater inflow calculation using stable isotopes of oxygen and hydrogen ( ) (b) seepage flux measurements in the wetland hyporheic sediment and c) pan evaporation measurements on land and in the wetland. The approach was found to be useful for identifying the dominant hydrological processes at the site, including recharge and subsurface flows. Recharge results from the model compared well with estimates obtained using isotope methods from previous studies and allowed us to identify specific annual signatures of this important process during the period of study. Results indicate that subsurface flow components in the system are seasonal and readily respond to rainfall events. The wetland water balance is dominated by local groundwater inputs and regional groundwater flow contributes little to the overall water balance.

  2. Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics.

    PubMed

    Doolittle-Hall, Janet M; Cunningham Glasspoole, Danielle L; Seaman, William T; Webster-Cyriaque, Jennifer

    2015-11-10

    Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures.

  3. Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

    PubMed Central

    Hüser, Daniela; Gogol-Döring, Andreas; Lutter, Timo; Weger, Stefan; Winter, Kerstin; Hammer, Eva-Maria; Cathomen, Toni; Reinert, Knut; Heilbronn, Regine

    2010-01-01

    Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome. PMID:20628575

  4. Integration preferences of wildtype AAV-2 for consensus rep-binding sites at numerous loci in the human genome.

    PubMed

    Hüser, Daniela; Gogol-Döring, Andreas; Lutter, Timo; Weger, Stefan; Winter, Kerstin; Hammer, Eva-Maria; Cathomen, Toni; Reinert, Knut; Heilbronn, Regine

    2010-07-08

    Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.

  5. The cost of integrated HIV care and buprenorphine/naloxone treatment: results of a cross-site evaluation.

    PubMed

    Schackman, Bruce R; Leff, Jared A; Botsko, Michael; Fiellin, David A; Altice, Fredrick L; Korthuis, P Todd; Sohler, Nancy; Weiss, Linda; Egan, James E; Netherland, Julie; Gass, Jonathan; Finkelstein, Ruth

    2011-03-01

    Implementing integrated HIV and buprenorphine/naloxone treatment requires cost estimates to plan and obtain funding. We identified costs incurred at HIV clinical sites participating in a cross-site evaluation of integrated care that followed patients for 1 year. Costs include labor, overhead, and urine toxicology analyses (clinic perspective), buprenorphine/naloxone (payer perspective) and patient time and transportation (patient perspective). Sites provided resource utilization quarterly, and providers estimated time required for each activity. With site as the unit of analysis, results are reported as median (range) of average site costs in 2008 US dollars. The median number of monthly provider encounters for integrated care patients was 3.2 (1.5-13.3) compared with 1.7 (1.1-4.2) for similar patients not in integrated care, but integrated care patients had fewer physician encounters. Median monthly clinic costs per integrated care patient were $136 ($67-$677) for labor and overhead and $8 ($2-$23) for toxicology analyses, $22 higher than clinic costs for patients not in integrated care. Median monthly costs for buprenorphine/naloxone were $209 ($165-$272), and monthly patient costs in integrated care were $11 ($1-$54) higher. Integrated HIV and buprenorphine/naloxone treatment requires different resources, including costs that are not third-party reimbursed. Implementing integrated care will require funding for training and for new staff such as buprenorphine coordinators, in addition to reimbursement for buprenorphine/naloxone. Further research is needed to identify potential cost offsets outside of the clinic setting.

  6. Application of lentiviral vectors for development of production cell lines and safety testing of lentiviral-derived cells or products.

    PubMed

    Plewa, Cherylene

    2010-01-01

    Lentiviral vectors (LVs) are frequently used to engineer cell lines for preclinical research purposes including assay development and target validation. Development of production cell lines for manufacturing recombinant protein therapeutics may also benefit from the use of LVs because they may reduce timelines and generate more uniform or higher expressing stable pools and clones. In addition, LVs could be advantageous for engineering new, alternative host cell substrates due to their ability to efficiently transduce most cell types. We demonstrate here that NS0 mouse myeloma cells, a host cell frequently used for protein production, can be transduced with LVs to greater than 80% efficiency and with no cytotoxic effects. The use of LVs for engineering of production cell lines will require additional testing procedures. Since LVs have previously been used in human gene therapy clinical trials, safety testing assays and procedures have been developed that could easily be applied to the development process for manufacturing cell lines to ensure the absence of unwanted viral material in cell banks and biologic products.

  7. High-throughput genomic mapping of vector integration sites in gene therapy studies.

    PubMed

    Beard, Brian C; Adair, Jennifer E; Trobridge, Grant D; Kiem, Hans-Peter

    2014-01-01

    Gene therapy has enormous potential to treat a variety of infectious and genetic diseases. To date hundreds of patients worldwide have received hematopoietic cell products that have been gene-modified with retrovirus vectors carrying therapeutic transgenes, and many patients have been cured or demonstrated disease stabilization as a result (Adair et al., Sci Transl Med 4:133ra57, 2012; Biffi et al., Science 341:1233158, 2013; Aiuti et al., Science 341:1233151, 2013; Fischer et al., Gene 525:170-173, 2013). Unfortunately, for some patients the provirus integration dysregulated the expression of nearby genes leading to clonal outgrowth and, in some cases, cancer. Thus, the unwanted side effect of insertional mutagenesis has become a major concern for retrovirus gene therapy. The careful study of retrovirus integration sites (RIS) and the contribution of individual gene-modified clones to hematopoietic repopulating cells is of crucial importance for all gene therapy studies. Supporting this, the US Food and Drug Administration (FDA) has mandated the careful monitoring of RIS in all clinical trials of gene therapy. An invaluable method was developed: linear amplification mediated-polymerase chain reaction (LAM-PCR) capable of analyzing in vitro and complex in vivo samples, capturing valuable genomic information directly flanking the site of provirus integration. Linking this method and similar methods to high-throughput sequencing has now made possible an unprecedented understanding of the integration profile of various retrovirus vectors, and allows for sensitive monitoring of their safety. It also allows for a detailed comparison of improved safety-enhanced gene therapy vectors. An important readout of safety is the relative contribution of individual gene-modified repopulating clones. One limitation of LAM-PCR is that the ability to capture the relative contribution of individual clones is compromised because of the initial linear PCR common to all current methods

  8. Integrated Closure and Monitoring Plan for the Area 3 and Area 5 Radioactive Waste Management Sites at the Nevada Test Site

    SciTech Connect

    S. E. Rawlinson

    2001-09-01

    Bechtel Nevada (BN) manages two low-level Radioactive Waste Management Sites (RWMSs) (one site is in Area 3 and the other is in Area 5) at the Nevada Test Site (NTS) for the U.S. Department of Energy's (DOE's) National Nuclear Security Administration Nevada Operations Office (NNSA/NV). The current DOE Order governing management of radioactive waste is 435.1. Associated with DOE Order 435.1 is a Manual (DOE M 435.1-1) and Guidance (DOE G 435.1-1). The Manual and Guidance specify that preliminary closure and monitoring plans for a low-level waste (LLW) management facility be developed and initially submitted with the Performance Assessment (PA) and Composite Analysis (CA) for that facility. The Manual and Guidance, and the Disposal Authorization Statement (DAS) issued for the Area 3 RWMS further specify that the preliminary closure and monitoring plans be updated within one year following issuance of a DAS. This Integrated Closure and Monitoring Plan (ICMP) fulfills both requirements. Additional updates will be conducted every third year hereafter. This document is an integrated plan for closing and monitoring both RWMSs, and is based on guidance issued in 1999 by the DOE for developing closure plans. The plan does not follow the format suggested by the DOE guidance in order to better accommodate differences between the two RWMSs, especially in terms of operations and site characteristics. The modification reduces redundancy and provides a smoother progression of the discussion. The closure and monitoring plans were integrated because much of the information that would be included in individual plans is the same, and integration provides efficient presentation and program management. The ICMP identifies the regulatory requirements, describes the disposal sites and the physical environment where they are located, and defines the approach and schedule for both closing and monitoring the sites.

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

    PubMed

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

    2017-12-01

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

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

    PubMed Central

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

    2017-01-01

    Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD. PMID:28303972

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

    PubMed

    Nanba, Daisuke; Matsushita, Natsuki; Toki, Fujio; Higashiyama, Shigeki

    2013-10-18

    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. 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. 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. 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.

  12. 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

  13. Added value for on-site fuel cells through equipment and application integration

    SciTech Connect

    Whitaker, R.

    1996-12-31

    On-site fuel cell power plants are not an exciting new electricity generating technology. They are an economic and beneficial addition to the operating systems of commercial buildings and industrial facilities. ONSI Corporation is part of International Fuel Cells Corporation and is jointly owned by United Technologies Corporation, Toshiba, and Ansaldo. ONSI has proven in the last three years that initial demand for packaged fuel cell power plants, like our 200 kW PC25{trademark} fuel cell shown in Figure 1, comes from the commercial building sector. However, this sector and the companies which service it are only tangentially interested in fuel cells as an emerging electricity generating technology. What they are most interested in is how the PC25 can integrate into their building`s system; how it can deliver energy efficient dollars to the bottom line, and how it can deliver operating benefits to their business.

  14. Interim Status of the Accelerated Site Technology Deployment Integrated Decontamination and Decommissioning Project

    SciTech Connect

    A. M Smith; G. E. Matthern; R. H. Meservey

    1998-11-01

    The Idaho National Engineering and Environmental Laboratory (INEEL), Fernald Environmental Management Project (FEMP), and Argonne National Laboratory - East (ANL-E) teamed to establish the Accelerated Site Technology Deployment (ASTD) Integrated Decontamination and Decommissioning (ID&D) project to increase the use of improved technologies in D&D operations. The project is making the technologies more readily available, providing training, putting the technologies to use, and spreading information about improved performance. The improved technologies are expected to reduce cost, schedule, radiation exposure, or waste volume over currently used baseline methods. They include some of the most successful technologies proven in the large-scale demonstrations and in private industry. The selected technologies are the Pipe Explorer, the GammaCam, the Decontamination Decommissioning and Remediation Optimal Planning System (DDROPS), the BROKK Demolition Robot, the Personal Ice Cooling System (PICS), the Oxy-Gasoline Torch, the Track-Mounted Shear, and the Hand-Held Shear.

  15. Fungal cell wall dynamics and infection site microenvironments: signal integration and infection outcome.

    PubMed

    Shepardson, Kelly M; Cramer, Robert A

    2013-08-01

    Upon entrance into the host, fungi encounter a myriad of host effector products and microenvironments that they sense and adapt to for survival. Alterations of the structure and composition of the cell wall is a major fungal adaptation mechanism to evade these environments. Here we discuss recent findings of host-microenvironmental induced fungal cell wall changes, including structure, composition, and protein content, and their effects on host immune responses. A take home message from these recent studies is an emerging understanding of how integration of multiple signals, of both fungal and host responses to dynamic infection site microenvironments, determines outcomes of infection. A challenge moving forward is to further understand these mechanisms and harness them for therapeutic benefit. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. CPP-603 Underwater Fuel Storage Facility Site Integrated Stabilization Management Plan (SISMP), Volume I

    SciTech Connect

    Denney, R.D.

    1995-10-01

    The CPP-603 Underwater Fuel Storage Facility (UFSF) Site Integrated Stabilization Management Plan (SISMP) has been constructed to describe the activities required for the relocation of spent nuclear fuel (SNF) from the CPP-603 facility. These activities are the only Idaho National Engineering Laboratory (INEL) actions identified in the Implementation Plan developed to meet the requirements of the Defense Nuclear Facilities Safety Board (DNFSB) Recommendation 94-1 to the Secretary of Energy regarding an improved schedule for remediation in the Defense Nuclear Facilities Complex. As described in the DNFSB Recommendation 94-1 Implementation Plan, issued February 28, 1995, an INEL Spent Nuclear Fuel Management Plan is currently under development to direct the placement of SNF currently in existing INEL facilities into interim storage, and to address the coordination of intrasite SNF movements with new receipts and intersite transfers that were identified in the DOE SNF Programmatic and INEL Environmental Restoration and Waste Management Environmental Impact Statement Record, of Decision. This SISMP will be a subset of the INEL Spent Nuclear Fuel Management Plan and the activities described are being coordinated with other INEL SNF management activities. The CPP-603 relocation activities have been assigned a high priority so that established milestones will be meet, but there will be some cases where other activities will take precedence in utilization of available resources. The Draft INEL Site Integrated Stabilization Management Plan (SISMP), INEL-94/0279, Draft Rev. 2, dated March 10, 1995, is being superseded by the INEL Spent Nuclear Fuel Management Plan and this CPP-603 specific SISMP.

  17. FISim: A new similarity measure between transcription factor binding sites based on the fuzzy integral

    PubMed Central

    Garcia, Fernando; Lopez, Francisco J; Cano, Carlos; Blanco, Armando

    2009-01-01

    Background Regulatory motifs describe sets of related transcription factor binding sites (TFBSs) and can be represented as position frequency matrices (PFMs). De novo identification of TFBSs is a crucial problem in computational biology which includes the issue of comparing putative motifs with one another and with motifs that are already known. The relative importance of each nucleotide within a given position in the PFMs should be considered in order to compute PFM similarities. Furthermore, biological data are inherently noisy and imprecise. Fuzzy set theory is particularly suitable for modeling imprecise data, whereas fuzzy integrals are highly appropriate for representing the interaction among different information sources. Results We propose FISim, a new similarity measure between PFMs, based on the fuzzy integral of the distance of the nucleotides with respect to the information content of the positions. Unlike existing methods, FISim is designed to consider the higher contribution of better conserved positions to the binding affinity. FISim provides excellent results when dealing with sets of randomly generated motifs, and outperforms the remaining methods when handling real datasets of related motifs. Furthermore, we propose a new cluster methodology based on kernel theory together with FISim to obtain groups of related motifs potentially bound by the same TFs, providing more robust results than existing approaches. Conclusion FISim corrects a design flaw of the most popular methods, whose measures favour similarity of low information content positions. We use our measure to successfully identify motifs that describe binding sites for the same TF and to solve real-life problems. In this study the reliability of fuzzy technology for motif comparison tasks is proven. PMID:19615102

  18. FISim: a new similarity measure between transcription factor binding sites based on the fuzzy integral.

    PubMed

    Garcia, Fernando; Lopez, Francisco J; Cano, Carlos; Blanco, Armando

    2009-07-20

    Regulatory motifs describe sets of related transcription factor binding sites (TFBSs) and can be represented as position frequency matrices (PFMs). De novo identification of TFBSs is a crucial problem in computational biology which includes the issue of comparing putative motifs with one another and with motifs that are already known. The relative importance of each nucleotide within a given position in the PFMs should be considered in order to compute PFM similarities. Furthermore, biological data are inherently noisy and imprecise. Fuzzy set theory is particularly suitable for modeling imprecise data, whereas fuzzy integrals are highly appropriate for representing the interaction among different information sources. We propose FISim, a new similarity measure between PFMs, based on the fuzzy integral of the distance of the nucleotides with respect to the information content of the positions. Unlike existing methods, FISim is designed to consider the higher contribution of better conserved positions to the binding affinity. FISim provides excellent results when dealing with sets of randomly generated motifs, and outperforms the remaining methods when handling real datasets of related motifs. Furthermore, we propose a new cluster methodology based on kernel theory together with FISim to obtain groups of related motifs potentially bound by the same TFs, providing more robust results than existing approaches. FISim corrects a design flaw of the most popular methods, whose measures favour similarity of low information content positions. We use our measure to successfully identify motifs that describe binding sites for the same TF and to solve real-life problems. In this study the reliability of fuzzy technology for motif comparison tasks is proven.

  19. A Two-site Randomized Clinical Trial of Integrated Psychosocial Treatment for ADHD-Inattentive Type

    PubMed Central

    Pfiffner, Linda J.; Hinshaw, Stephen P.; Owens, Elizabeth; Zalecki, Christine; Kaiser, Nina M.; Villodas, Miguel; McBurnett, Keith

    2014-01-01

    Objective This study evaluated the efficacy of the Child Life and Attention Skills (CLAS) program, a behavioral psychosocial treatment integrated across home and school, for youth with Attention Deficit Hyperactivity Disorder-Inattentive Type (ADHD-I). Method In a two-site randomized controlled trial, 199 children (ages 7-11) were randomized to CLAS (N=74), parent-focused treatment (PFT, N=74), or treatment as usual (TAU, N=51). We compared groups on parent and teacher ratings of inattention symptoms, organizational skills, social skills, and global improvement at post-treatment, and also at follow-up during the subsequent school year. Results CLAS resulted in greater improvements in teacher-reported inattention, organizational skills, social skills, and global functioning relative to both PFT and TAU at post-treatment. Parents of children in CLAS reported greater improvement in organizational skills than PFT and greater improvements on all outcomes relative to TAU at post-treatment. Differences between CLAS and TAU were maintained at follow-up for most parent-reported measures but were not significant for teacher-reported outcomes. Conclusions These findings extend support for CLAS across two study sites, revealing that integrating parent, teacher, and child treatment components, specifically adapted for ADHD-I, is superior to parent training alone and to usual care. Direct involvement of teachers and children in CLAS appears to amplify effects at school and home and underscores the importance of coordinating parent, teacher, and child treatment components for cross-setting effects on symptoms and impairment associated with ADHD-I. PMID:24865871

  20. Integrating intrusive and nonintrusive characterization methods to achieve a conceptual site model for the SLDA FUSRAP site - 8265.

    SciTech Connect

    Durham, L. A.; Peterson, J. M.; Frothingham, D. G.; Frederick, W. T.; Lenart, W.; Environmental Science Division; U. S. Army Corps of Engineers, Pittsburg District; U. S. Army Corps of Engineers, Buffalo District

    2008-01-01

    The US Army Corps of Engineers (USACE) is addressing radiological contamination following Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) requirements at the Shallow Land Disposal Area (SLDA) site, which is a radiologically contaminated property that is part of the Formerly utilized Sites Remedial Action Program (FUSRAP). The SLDA is an 18-hectare (44-acre) site in Parks township, Armstrong County, Pennsylvania, about 37 kilometers (23 miles) east-northeast of Pittsburgh. According to historical record, radioactive wastes were disposed of at the SLDA in a series of trenches by the Nuclear Materials and Equipment Company (NUMEC) in the 1960s. The wastes originated from the nearby Apollo nuclear fuel fabrication facility, which began operations under NUMEC in the late 1950s and fabricated enriched uranium into naval reactor fuel elements. It is believed that the waste materials were buried in a series of pits constructed adjacent to one another in accordance with an Atomic Energy Commission (AEC) regulation that has since been rescinded. A CERCLA remedial investigation/feasibility study (RI/FS) process was completed for the SLDA site, and the results of the human health risk assessment indicated that the radiologically contaminated wastes could pose a risk to human health in the future. There are no historical records that provide the exact location of these pits. However, based on geophysical survey results conducted in the 1980s, these pits were defined by geophysical anomalies and were depicted on historical site drawings as trenches. At the SLDA site, a combination of investigative methods and tools was used in the RI/FS and site characterization activities. The SLDA site provides an excellent example of how historical documents and data, historical aerial photo analysis, physical sampling, and nonintrusive geophysical and gamma walkover surveys were used in combination to reduce the uncertainty in the location of the trenches. The

  1. Packaging of HCV-RNA into lentiviral vector

    SciTech Connect

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

    2011-11-04

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

  2. High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion.

    PubMed

    Cattoglio, Claudia; Maruggi, Giulietta; Bartholomae, Cynthia; Malani, Nirav; Pellin, Danilo; Cocchiarella, Fabienne; Magnani, Zulma; Ciceri, Fabio; Ambrosi, Alessandro; von Kalle, Christof; Bushman, Frederic D; Bonini, Chiara; Schmidt, Manfred; Mavilio, Fulvio; Recchia, Alessandra

    2010-12-22

    The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

  3. High-Definition Mapping of Retroviral Integration Sites Defines the Fate of Allogeneic T Cells After Donor Lymphocyte Infusion

    PubMed Central

    Cattoglio, Claudia; Maruggi, Giulietta; Bartholomae, Cynthia; Malani, Nirav; Pellin, Danilo; Cocchiarella, Fabienne; Magnani, Zulma; Ciceri, Fabio; Ambrosi, Alessandro; von Kalle, Christof; Bushman, Frederic D.; Bonini, Chiara; Schmidt, Manfred; Mavilio, Fulvio; Recchia, Alessandra

    2010-01-01

    The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34+ hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction. PMID:21203516

  4. Analysis of a fuel cell on-site integrated energy system for a residential complex

    NASA Technical Reports Server (NTRS)

    Simons, S. N.; Maag, W. L.

    1979-01-01

    The energy use and costs of the on-site integrated energy system (OS/IES) which provides electric power from an on-site power plant and recovers heat that would normally be rejected to the environment is compared to a conventional system purchasing electricity from a utility and a phosphoric acid fuel cell powered system. The analysis showed that for a 500-unit apartment complex a fuel OS/IES would be about 10% more energy conservative in terms of total coal consumption than a diesel OS/IES system or a conventional system. The fuel cell OS/IES capital costs could be 30 to 55% greater than the diesel OS/IES capital costs for the same life cycle costs. The life cycle cost of a fuel cell OS/IES would be lower than that for a conventional system as long as the cost of electricity is greater than $0.05 to $0.065/kWh. An analysis of several parametric combinations of fuel cell power plant and state-of-art energy recovery systems and annual fuel requirement calculations for four locations were made. It was shown that OS/IES component choices are a major factor in fuel consumption, with the least efficient system using 25% more fuel than the most efficient. Central air conditioning and heat pumps result in minimum fuel consumption while individual air conditioning units increase it, and in general the fuel cell of highest electrical efficiency has the lowest fuel consumption.

  5. The explosion sites of nearby supernovae seen with integral field spectroscopy

    NASA Astrophysics Data System (ADS)

    Kuncarayakti, Hanindyo

    2015-08-01

    The progenitor stars of supernovae are still not very well constrained, despite numerous efforts in studying these objects directly or indirectly. There have been detections of the progenitor candidates in pre-explosion Hubble Space Telescope images, but these are rare and it is difficult to increase the statistics due to the limited availability of usable pre-explosion images. Alternatively, one may perform statistical studies on the supernova environments to derive useful constraints on the SN progenitor star. Integral field spectroscopy of nearby supernova sites within ~30 Mpc have been obtained using multiple IFU spectrographs in Hawaii and Chile. This technique enables both spatial and spectral information of the explosion sites to be acquired simultaneously, thus providing the identification of the parent stellar population of the supernova progenitor and the estimates for its physical parameters including age and metallicity. While this work has mainly been done in the optical wavelengths using instruments such as VIMOS, GMOS, and MUSE, a near-infrared approach has also been carried out using the AO-assisted SINFONI. By studying the supernova parent stellar population, we aim to characterize the mass and metallicity of the progenitors of different types of supernovae.

  6. Stepwise Activation of the ATR Signaling Pathway upon Increasing Replication Stress Impacts Fragile Site Integrity

    PubMed Central

    Koundrioukoff, Stéphane; Carignon, Sandra; Técher, Hervé; Letessier, Anne; Brison, Olivier; Debatisse, Michelle

    2013-01-01

    Breaks at common fragile sites (CFS) are a recognized source of genome instability in pre-neoplastic lesions, but how such checkpoint-proficient cells escape surveillance and continue cycling is unknown. Here we show, in lymphocytes and fibroblasts, that moderate replication stresses like those inducing breaks at CFSs trigger chromatin loading of sensors and mediators of the ATR pathway but fail to activate Chk1 or p53. Consistently, we found that cells depleted of ATR, but not of Chk1, accumulate single-stranded DNA upon Mre11-dependent resection of collapsed forks. Partial activation of the pathway under moderate stress thus takes steps against fork disassembly but tolerates S-phase progression and mitotic onset. We show that fork protection by ATR is crucial to CFS integrity, specifically in the cell type where a given site displays paucity in backup replication origins. Tolerance to mitotic entry with under-replicated CFSs therefore results in chromosome breaks, providing a pool of cells committed to further instability. PMID:23874235

  7. Analysis of a fuel cell on-site integrated energy system for a residential complex

    NASA Technical Reports Server (NTRS)

    Simons, S. N.; Maag, W. L.

    1979-01-01

    The energy use and costs of the on-site integrated energy system (OS/IES) which provides electric power from an on-site power plant and recovers heat that would normally be rejected to the environment is compared to a conventional system purchasing electricity from a utility and a phosphoric acid fuel cell powered system. The analysis showed that for a 500-unit apartment complex a fuel OS/IES would be about 10% more energy conservative in terms of total coal consumption than a diesel OS/IES system or a conventional system. The fuel cell OS/IES capital costs could be 30 to 55% greater than the diesel OS/IES capital costs for the same life cycle costs. The life cycle cost of a fuel cell OS/IES would be lower than that for a conventional system as long as the cost of electricity is greater than $0.05 to $0.065/kWh. An analysis of several parametric combinations of fuel cell power plant and state-of-art energy recovery systems and annual fuel requirement calculations for four locations were made. It was shown that OS/IES component choices are a major factor in fuel consumption, with the least efficient system using 25% more fuel than the most efficient. Central air conditioning and heat pumps result in minimum fuel consumption while individual air conditioning units increase it, and in general the fuel cell of highest electrical efficiency has the lowest fuel consumption.

  8. Study of component technologies for fuel cell on-site integrated energy systems

    NASA Technical Reports Server (NTRS)

    Lee, W. D.; Mathias, S.

    1980-01-01

    Heating, ventilation and air conditioning equipment are integrated with three types of fuel cells. System design and computer simulations are developed to utilize the thermal energy discharge of the fuel in the most cost effective manner. The fuel provides all of the electric needs and a loss of load probability analysis is used to ensure adequate power plant reliability. Equipment cost is estimated for each of the systems analyzed. A levelized annual cost reflecting owning and operating costs including the cost of money was used to select the most promising integrated system configurations. Cash flows are presented for the most promising 16 systems. Several systems for the 96 unit apartment complex (a retail store was also studied) were cost competitive with both gas and electric based conventional systems. Thermal storage is shown to be beneficial and the optimum absorption chiller sizing (waste heat recovery) in connection with electric chillers are developed. Battery storage was analyzed since the system is not electric grid connected. Advanced absorption chillers were analyzed as well. Recommendations covering financing, technical development, and policy issues are given to accelerate the commercialization of the fuel cell for on-site power generation in buildings.

  9. Study of component technologies for fuel cell on-site integrated energy systems

    NASA Astrophysics Data System (ADS)

    Lee, W. D.; Mathias, S.

    1980-12-01

    Heating, ventilation and air conditioning equipment are integrated with three types of fuel cells. System design and computer simulations are developed to utilize the thermal energy discharge of the fuel in the most cost effective manner. The fuel provides all of the electric needs and a loss of load probability analysis is used to ensure adequate power plant reliability. Equipment cost is estimated for each of the systems analyzed. A levelized annual cost reflecting owning and operating costs including the cost of money was used to select the most promising integrated system configurations. Cash flows are presented for the most promising 16 systems. Several systems for the 96 unit apartment complex (a retail store was also studied) were cost competitive with both gas and electric based conventional systems. Thermal storage is shown to be beneficial and the optimum absorption chiller sizing (waste heat recovery) in connection with electric chillers are developed. Battery storage was analyzed since the system is not electric grid connected. Advanced absorption chillers were analyzed as well. Recommendations covering financing, technical development, and policy issues are given to accelerate the commercialization of the fuel cell for on-site power generation in buildings.

  10. Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis

    PubMed Central

    Yamashita, Riu; Sathira, Nuankanya P.; Kanai, Akinori; Tanimoto, Kousuke; Arauchi, Takako; Tanaka, Yoshiaki; Hashimoto, Shin-ichi; Sugano, Sumio; Nakai, Kenta; Suzuki, Yutaka

    2011-01-01

    We performed a genome-wide analysis of transcriptional start sites (TSSs) in human genes by multifaceted use of a massively parallel sequencer. By analyzing 800 million sequences that were obtained from various types of transcriptome analyses, we characterized 140 million TSS tags in 12 human cell types. Despite the large number of TSS clusters (TSCs), the number of TSCs was observed to decrease sharply with increasing expression levels. Highly expressed TSCs exhibited several characteristic features: Nucleosome-seq analysis revealed highly ordered nucleosome structures, ChIP-seq analysis detected clear RNA polymerase II binding signals in their surrounding regions, evaluations of previously sequenced and newly shotgun-sequenced complete cDNA sequences showed that they encode preferable transcripts for protein translation, and RNA-seq analysis of polysome-incorporated RNAs yielded direct evidence that those transcripts are actually translated into proteins. We also demonstrate that integrative interpretation of transcriptome data is essential for the selection of putative alternative promoter TSCs, two of which also have protein consequences. Furthermore, discriminative chromatin features that separate TSCs at different expression levels were found for both genic TSCs and intergenic TSCs. The collected integrative information should provide a useful basis for future biological characterization of TSCs. PMID:21372179

  11. OC ToGo: bed site image integration into OpenClinica with mobile devices

    NASA Astrophysics Data System (ADS)

    Haak, Daniel; Gehlen, Johan; Jonas, Stephan; Deserno, Thomas M.

    2014-03-01

    Imaging and image-based measurements nowadays play an essential role in controlled clinical trials, but electronic data capture (EDC) systems insufficiently support integration of captured images by mobile devices (e.g. smartphones and tablets). The web application OpenClinica has established as one of the world's leading EDC systems and is used to collect, manage and store data of clinical trials in electronic case report forms (eCRFs). In this paper, we present a mobile application for instantaneous integration of images into OpenClinica directly during examination on patient's bed site. The communication between the Android application and OpenClinica is based on the simple object access protocol (SOAP) and representational state transfer (REST) web services for metadata, and secure file transfer protocol (SFTP) for image transfer, respectively. OpenClinica's web services are used to query context information (e.g. existing studies, events and subjects) and to import data into the eCRF, as well as export of eCRF metadata and structural information. A stable image transfer is ensured and progress information (e.g. remaining time) visualized to the user. The workflow is demonstrated for a European multi-center registry, where patients with calciphylaxis disease are included. Our approach improves the EDC workflow, saves time, and reduces costs. Furthermore, data privacy is enhanced, since storage of private health data on the imaging devices becomes obsolete.

  12. Space Borne and Ground Based InSAR Data Integration: The Åknes Test Site

    NASA Astrophysics Data System (ADS)

    Bardi, Federica; Raspini, Federico; Ciampalini, Andrea; Kristensen, Lene; Rouyet, Line; Rune Lauknes, Tom; Frauenfelder, Regula; Casagli, Nicola

    2016-04-01

    This work concerns a proposal of integration between InSAR (Interferometric Synthetic Aperture Radar) data acquired by ground based (GB) and satellite platforms. The selected test site is the Åknes rockslide, which affects the western Norwegian coast; the availability of GB-InSAR and satellite InSAR data, and the accessibility of a wide literature make the landslide suitable for testing the proposed procedure. The first step consists in the organization of a geodatabase, performed in GIS environment, containing all the available data. The second step concerns the analysis of satellite and GB-InSAR data, separately. Two datasets, acquired by RADARSAT-2 (related to a period between October 2008 and August 2013) and by a combination of TerraSAR-X and TanDEM-X (acquired between July 2010 and October 2012), both of them in ascending orbit, processed applying SBAS (Small BAseline Subset), are available. GB-InSAR data related to 5 different campaigns of measurements, referred to the summer seasons of 2006, 2008, 2009, 2010 and 2012 are available too. The third step relies on data integration, performed firstly on a qualitative point of view and lately on a semi-quantitative point of view. The results of the proposed procedure have been validated by comparing them with GPS (Global Positioning System) data.

  13. Integrating physically based simulators with Event Detection Systems: Multi-site detection approach.

    PubMed

    Housh, Mashor; Ohar, Ziv

    2017-03-01

    The Fault Detection (FD) Problem in control theory concerns of monitoring a system to identify when a fault has occurred. Two approaches can be distinguished for the FD: Signal processing based FD and Model-based FD. The former concerns of developing algorithms to directly infer faults from sensors' readings, while the latter uses a simulation model of the real-system to analyze the discrepancy between sensors' readings and expected values from the simulation model. Most contamination Event Detection Systems (EDSs) for water distribution systems have followed the signal processing based FD, which relies on analyzing the signals from monitoring stations independently of each other, rather than evaluating all stations simultaneously within an integrated network. In this study, we show that a model-based EDS which utilizes a physically based water quality and hydraulics simulation models, can outperform the signal processing based EDS. We also show that the model-based EDS can facilitate the development of a Multi-Site EDS (MSEDS), which analyzes the data from all the monitoring stations simultaneously within an integrated network. The advantage of the joint analysis in the MSEDS is expressed by increased detection accuracy (higher true positive alarms and fewer false alarms) and shorter detection time. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality*

    PubMed Central

    Žáková, Lenka; Kletvíková, Emília; Veverka, Václav; Lepšík, Martin; Watson, Christopher J.; Turkenburg, Johan P.; Jiráček, Jiří; Brzozowski, Andrzej M.

    2013-01-01

    Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant PheB24, one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to d-amino acids but not to l-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [d-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the l-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [d-HisB24]-insulin is more flexible, and the reverse chirality of the B24Cα atom swayed the d-HisB24 side chain into the solvent. Furthermore, the pocket vacated by PheB24 is filled by PheB25, which mimics the PheB24 side and main chains. The B25→B24 downshift results in a subsequent downshift of TyrB26 into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic l-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation. PMID:23447530

  15. Integrated risk and recovery monitoring of ecosystem restorations on contaminated sites.

    PubMed

    Hooper, Michael J; Glomb, Stephen J; Harper, David D; Hoelzle, Timothy B; McIntosh, Lisa M; Mulligan, David R

    2016-04-01

    resources have recovered, and if ecosystem services have been returned. Such retrospective analysis will allow better planning for future restoration goals and strengthen the evidence base for quantifying injuries and damages at other sites in the future. © 2015 The Authors. Integrated Environmental Assessment and Management Published by Wiley Periodicals, Inc. on behalf of SETAC.

  16. Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide.

    PubMed

    Maurice, Marielle; Verhoeyen, Els; Salmon, Patrick; Trono, Didier; Russell, Stephen J; Cosset, François-Loïc

    2002-04-01

    In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1-derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G(0) to G(1) phases of the cell cycle. Compared to unmodified HIV-1-based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

  17. Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding

    PubMed Central

    Serrao, Erik; Krishnan, Lavanya; Shun, Ming-Chieh; Li, Xiang; Cherepanov, Peter; Engelman, Alan; Maertens, Goedele N.

    2014-01-01

    Retroviruses favor target-DNA (tDNA) distortion and particular bases at sites of integration, but the mechanism underlying HIV-1 selectivity is unknown. Crystal structures revealed a network of prototype foamy virus (PFV) integrase residues that distort tDNA: Ala188 and Arg329 interact with tDNA bases, while Arg362 contacts the phosphodiester backbone. HIV-1 integrase residues Ser119, Arg231, and Lys258 were identified here as analogs of PFV integrase residues Ala188, Arg329 and Arg362, respectively. Thirteen integrase mutations were analyzed for effects on integrase activity in vitro and during virus infection, yielding a total of 1610 unique HIV-1 integration sites. Purine (R)/pyrimidine (Y) dinucleotide sequence analysis revealed HIV-1 prefers the tDNA signature (0)RYXRY(4), which accordingly favors overlapping flexible dinucleotides at the center of the integration site. Consistent with roles for Arg231 and Lys258 in sequence specific and non-specific binding, respectively, the R231E mutation altered integration site nucleotide preferences while K258E had no effect. S119A and S119T integrase mutations significantly altered base preferences at positions −3 and 7 from the site of viral DNA joining. The S119A preference moreover mimicked wild-type PFV selectivity at these positions. We conclude that HIV-1 IN residue Ser119 and PFV IN residue Ala188 contact analogous tDNA bases to effect virus integration. PMID:24520116

  18. Fitness Cost Implications of PhiC31-Mediated Site-Specific Integrations in Target-Site Strains of the Mexican Fruit Fly, Anastrepha ludens (Diptera: Tephritidae)

    PubMed Central

    Meza, José S.; Díaz-Fleischer, Francisco; Sánchez-Velásquez, Lázaro R.; Zepeda-Cisneros, Cristina Silvia; Handler, Alfred M.; Schetelig, Marc F.

    2014-01-01

    Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain. PMID:25303238

  19. Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

    PubMed

    Meza, José S; Díaz-Fleischer, Francisco; Sánchez-Velásquez, Lázaro R; Zepeda-Cisneros, Cristina Silvia; Handler, Alfred M; Schetelig, Marc F

    2014-01-01

    Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain.

  20. Lentiviral Transduction of Neurons in Adult Brain: Evaluation of Inflammatory Response and Cognitive Effects in Mice.

    PubMed

    Kunitsyna, T A; Ivashkina, O I; Roshchina, M A; Toropova, K A; Anokhin, K V

    2016-06-01

    We evaluated the effect of hippocampal injection of lentiviral particles p156-CMV-EGFP on behavior, learning, and microglial Iba1(+) cells activation in mice. Testing in the open field and elevated plus-maze revealed higher anxiety levels in lentiviral-injected mice in comparison with animals injected with vehicle. At the same time, lentivirus injection did not change learning and memory of mice in the hippocampal-dependent fear conditioning task. Microglia density in lentivirus-injected mice was significantly higher than in vehicle-injected mice. Thus, hippocampal injection of lentiviral particles with minimum content of transgenes produced evident inflammation process, changed anxiety level of experimental animals, but had no effect on hippocampal-dependent learning and memory.

  1. Integrated bio-magnetostratigraphy of ODP Site 709 (equatorial Indian Ocean).

    NASA Astrophysics Data System (ADS)

    Villa, Giuliana; Fioroni, Chiara; Florindo, Fabio

    2015-04-01

    Over the last decade, calcareous nannofossil biostratigraphy of the lower Eocene-Oligocene sediments has shown great potential, through identification of several new nannofossil species and bioevents (e.g. Fornaciari et al., 2010; Bown and Dunkley Jones, 2012; Toffanin et al., 2013). These studies formed the basis for higher biostratigraphic resolution leading to definition of a new nannofossil biozonation (Agnini et al., 2014). In this study, we investigate the middle Eocene-lower Oligocene sediments from ODP Hole 709C (ODP Leg 115) by means of calcareous nannofossils and magnetostratigraphy. Ocean Drilling Program (ODP) Site 709 was located in the equatorial Indian Ocean and biostratigraphy has been investigated in the nineties (Okada, 1990; Fornaciari et al., 1990) while paleomagnetic data from the Initial Report provided only a poorly constrained magnetostratigraphic interpretation, thus the cored succession was dated only by means of biostratigraphy. Our goal is to test the reliability in the Indian Ocean of the biohorizons recently identified at Site 711 (Fioroni et al., in press), by means of high resolution sampling, new taxonomic updates, quantitative analyses on calcareous nannofossils allowed to increase the number of useful bioevents and to compare their reliability and synchroneity. The new magnetostratigraphic analyses and integrated stratigraphy allow also to achieve an accurate biochronology of the time interval spanning Chrons C20 (middle Eocene) and C12 (early Oligocene). In addition, this equatorial site represents an opportunity to study the carbonate accumulation history and the large fluctuations of the carbonate compensation depth (CCD) during the Eocene (e.g. Pälike et al., 2012). The investigated interval encompasses the Middle Eocene Climatic Optimum (MECO), and the long cooling trend that leads to the Oligocene glacial state. By means of our new bio-magnetostratigraphic data and paleoecological results we provide further insights on

  2. Integrated Methods for Site Characterization and Conceptual Model Development for a Contaminated Fractured-Bedrock Aquifer

    NASA Astrophysics Data System (ADS)

    Johnson, C. D.; Kastrinos, J. R.; Haeni, F. P.

    2005-12-01

    A multi-disciplined and team-based approach was used to integrate geophysical, hydrologic, and chemical data to characterize lithology, fractures, and hydraulic properties of fractured crystalline bedrock and to determine the nature and extent of ground-water contamination from a landfill and former chemical-waste disposal pits at the University of Connecticut. Detection of volatile organic compounds (VOCs) in domestic bedrock wells in the mid-1980s led to this investigation, in which a team comprised of hydrologists, engineers, geophysicists, geologists, chemists, toxicologists, and community-involvement personnel collected, analyzed, and evaluated data; developed and refined a conceptual model of the ground-water flow and contaminant distribution at the site; and evaluated alternatives and implemented a final remediation plan. The characterization phase began in 1999 and the remediation phase is currently ongoing. An integrated and iterative approach of using multiple methods in phases was important for corroborating the interpretation of individual methods and essential for guiding the design and implementation of additional testing at the site. The use of geophysical data early in the investigation allowed the study team to obtain detailed subsurface information using a minimum of boreholes. Surface geophysical methods were used to target potential discharge of contaminants from the landfill for further investigation. Borehole geophysical methods were used to investigate the anomalies identified by surface geophysical methods, the location and orientation of fractures that intersect and surround each well, the direction and magnitude of ambient flow in the wells, and the transmissive fractures that could provide pathways for contaminant migration. Borehole geophysical and hydraulic data were used to design discrete-zone monitoring systems for the collection of hydraulic head and chemical data and to prevent cross contamination through the boreholes. The results

  3. Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

    PubMed Central

    Agúndez, Leticia; González-Prieto, Coral; Machón, Cristina; Llosa, Matxalen

    2012-01-01

    Background Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. Methodology/Principal Findings We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. Conclusions/Significance The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells. PMID:22292089

  4. Lentiviral Vector-Mediated Correction of a Mouse Model of Leukocyte Adhesion Deficiency Type I.

    PubMed

    Leon-Rico, Diego; Aldea, Montserrat; Sanchez-Baltasar, Raquel; Mesa-Nuñez, Cristina; Record, Julien; Burns, Siobhan O; Santilli, Giorgia; Thrasher, Adrian J; Bueren, Juan A; Almarza, Elena

    2016-09-01

    Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene and is characterized by recurrent and life-threatening bacterial infections. These mutations lead to defective or absent expression of β2 integrins on the leukocyte surface, compromising adhesion and extravasation at sites of infection. Three different lentiviral vectors (LVs) conferring ubiquitous or preferential expression of CD18 in myeloid cells were constructed and tested in human and mouse LAD-I cells. All three hCD18-LVs restored CD18 and CD11a membrane expression in LAD-I patient-derived lymphoblastoid cells. Corrected cells recovered the ability to aggregate and bind to sICAM-1 after stimulation. All vectors induced stable hCD18 expression in hematopoietic cells from mice with a hypomorphic Itgb2 mutation (CD18(HYP)), both in vitro and in vivo after transplantation of corrected cells into primary and secondary CD18(HYP) recipients. hCD18(+) hematopoietic cells from transplanted CD18(HYP) mice also showed restoration of mCD11a surface co-expression. The analysis of in vivo neutrophil migration in CD18(HYP) mice subjected to two different inflammation models demonstrated that the LV-mediated gene therapy completely restored neutrophil extravasation in response to inflammatory stimuli. Finally, these vectors were able to correct the phenotype of human myeloid cells derived from CD34(+) progenitors defective in ITGB2 expression. These results support for the first time the use of hCD18-LVs for the treatment of LAD-I patients in clinical trials.

  5. Lentiviral Vector-Mediated Correction of a Mouse Model of Leukocyte Adhesion Deficiency Type I

    PubMed Central

    Leon-Rico, Diego; Aldea, Montserrat; Sanchez-Baltasar, Raquel; Mesa-Nuñez, Cristina; Record, Julien; Burns, Siobhan O.; Santilli, Giorgia; Thrasher, Adrian J.; Bueren, Juan A.; Almarza, Elena

    2016-01-01

    Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene and is characterized by recurrent and life-threatening bacterial infections. These mutations lead to defective or absent expression of β2 integrins on the leukocyte surface, compromising adhesion and extravasation at sites of infection. Three different lentiviral vectors (LVs) conferring ubiquitous or preferential expression of CD18 in myeloid cells were constructed and tested in human and mouse LAD-I cells. All three hCD18-LVs restored CD18 and CD11a membrane expression in LAD-I patient-derived lymphoblastoid cells. Corrected cells recovered the ability to aggregate and bind to sICAM-1 after stimulation. All vectors induced stable hCD18 expression in hematopoietic cells from mice with a hypomorphic Itgb2 mutation (CD18HYP), both in vitro and in vivo after transplantation of corrected cells into primary and secondary CD18HYP recipients. hCD18+ hematopoietic cells from transplanted CD18HYP mice also showed restoration of mCD11a surface co-expression. The analysis of in vivo neutrophil migration in CD18HYP mice subjected to two different inflammation models demonstrated that the LV-mediated gene therapy completely restored neutrophil extravasation in response to inflammatory stimuli. Finally, these vectors were able to correct the phenotype of human myeloid cells derived from CD34+ progenitors defective in ITGB2 expression. These results support for the first time the use of hCD18-LVs for the treatment of LAD-I patients in clinical trials. PMID:27056660

  6. Lentiviral vectors for induction of self-differentiation and conditional ablation of dendritic cells

    PubMed Central

    Pincha, M; Salguero, G; Wedekind, D; Sundarasetty, B S; Lin, A; Kasahara, N; Brugman, M H; Jirmo, A C; Modlich, U; Gutzmer, R; Büsche, G; Ganser, A; Stripecke, R

    2011-01-01

    Development of lentiviral vectors (LVs) in the field of immunotherapy and immune regeneration will strongly rely on biosafety of the gene transfer. We demonstrated previously the feasibility of ex vivo genetic programming of mouse bone marrow precursors with LVs encoding granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), which induced autonomous differentiation of long-lived dendritic cells (DCs), referred to as self-differentiated myeloid-derived antigen-presenting-cells reactive against tumors (SMART-DCs). Here, LV biosafety was enhanced by using a DC-restricted and physiological promoter, the major histocompatibility complex (MHC) II promoter, and including co-expression of the herpes simplex virus-thymidine kinase (sr39HSV-TK) conditional suicide gene. Tricistronic vectors co-expressing sr39HSV-TK, GM-CSF and IL-4 transcriptionally regulated by the MHCII promoter or the ubiquitous cytomegalovirus (CMV) promoter were compared. Despite the different gene transfer effects, such as the kinetics, levels of transgene expression and persistency of integrated vector copies, both vectors induced highly viable SMART-DCs, which persisted for at least 70 days in vivo and could be ablated with the pro-drug Ganciclovir (GCV). SMART-DCs co-expressing the tyrosine-related protein 2 melanoma antigen administered subcutaneously generated antigen-specific, anti-melanoma protective and therapeutic responses in the mouse B16 melanoma model. GCV administration after immunotherapy did not abrogate DC vaccination efficacy. This demonstrates proof-of-principle of genetically programmed DCs that can be ablated pharmacologically. PMID:21412283

  7. Integrated Geophysical and Archaeological investigations to study the site of Aquinum (Frosinone, Italy)

    NASA Astrophysics Data System (ADS)

    Piro, Salvatore; Ceraudo, Giuseppe; Zamuner, Daniela

    2010-05-01

    To enhance the knowledge finalised to the location and conservation of the unknown buried structures below the actual studied levels, in the territory of the Ancient Aquinum (Frosinone, Italy) a scientific collaboration, inside the "Ager Aquinas Project" between the University of Salento (Department of Cultural Heritage - Laboratory of Ancient Topography and Photogrammetry) and the Institute of Technologies Applied to Cultural Heritage (ITABC-C.N.R.) has been developed, during 2008-2009 and it is still in progress. The site which is the subject of this paper had been identified in the past through air photo interpretation of vertical historical coverage and field - walking surveys. Ancient Aquinum is characterised by two main aspects: the first depends by the presence of a very big defence-system with mighty walls and large ditch; the second characteristic is the presence or regular but not orthogonal road - system of the town, bordered by an unusual parallelogram shape of the blocks. With the results obtained after the elaborations of the first aerial data sets and field surveys, has been possible to map the main town - planning, drawing the main road system inside and outside the town. Although the analysis of the air photo evidence allowed the global interpretation of the site, it was not possible to reconstruct the archaeological evidences in the central portion of the town. Therefore the Project, during 2008, started with new acquisition and elaboration of aerial photos, field-walking surveys and GPR surveys with the aim to better define the urban plan of the central portion of the ancient town. The location, depth, and size of the buried buildings were effectively estimated from non-destructive remote sensing with a gradiometric and ground-penetrating radar systems. Recent archaeological excavations made (by Prof. Giuseppe Ceraudo - University of Salento, Lecce) during the summer 2009, have confirmed the structures individuated with the geophysical methods

  8. Physicochemical and mineralogical characterization of uranium-contaminated soils from the Fernald Integrated Demonstration Site

    SciTech Connect

    Elless, M.P.; Lee, S.Y.; Timpson, M.E.

    1994-09-01

    An integrated approach that utilizes various characterization technologies has been developed for the Uranium Soil Integrated Demonstration program. The Fernald Environmental Restoration Management Corporation site near Cincinnati, Ohio, was selected as the host facility for this demonstration. Characterization of background, untreated contaminated, and treated contaminated soils was performed to assess the contamination and the effect of treatment efforts to remove uranium from these soils. Carbonate minerals were present in the contaminated soils (added for erosion control) but were absent in the nearby background soils. Because of the importance of the carbonate anion to uranium solubility, the occurrence of carbonate minerals in these soils will be an important factor in the development of a successful remediation technology. Uranium partitioning data among several particle-size fractions indicate that conventional soil washing will be ineffective for remediation of these soils and that chemical extraction will be necessary to lower the uranium concentration to the target level (52 mg/kg). Carbonate-based (sodium carbonate/bicarbonate) and acid-based (sulfuric and citric acids) lixiviants were employed for the selective removal of uranium from these soils. Characterization results have identified uranium phosphate minerals as the predominant uranium mineral form in both the untreated and treated soils. The low solubility associated with phosphate minerals is primarily responsible for their occurrence in the posttreated soils. Artificial weathering of the treated soils caused by the treatments, particularly acid-based lixiviants, was documented by their detrimental effects on several physicochemical characteristics of these soils (e.g., soil pH, particle-size distribution, and mineralogy).

  9. Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo

    PubMed Central

    Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L.; Stavans, Joel

    2014-01-01

    The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host. PMID:24799672

  10. Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70.

    PubMed Central

    Le Marrec, C; Moreau, S; Loury, S; Blanco, C; Trautwetter, A

    1996-01-01

    All the essential genetic determinants for site-specific integration of corynephage phi AAU2 are contained within a 1,756-bp DNA fragment, carried on the integrative plasmid p5510, and are shown to be functional in Escherichia coli. One open reading frame, ORF4, encoding a protein of 266 amino acids was shown to represent the phi AAU2 integrase. The nucleotide sequence of the phi AAU2 attachment site, attP, and the attB, attL, and attR sequences in the host "Arthrobacter aureus" C70 were determined. Identical nucleotide sequences were shown to be responsible for the integration of p5510 in the chromosomes of Corynebacterium glutamicum, Brevibacterium divaricatum, and B. lactofermentum, and a sequence almost identical to attB was found to be present in these three strains. In contrast to other phage site-specific recombination systems, a plasmid encompassing only int-attP failed to integrate into the host chromosome. This led to the identification of an 800-bp noncoding region, immediately upstream of int, absolutely required for site-specific integration of p5510. PMID:8606175

  11. Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70.

    PubMed

    Le Marrec, C; Moreau, S; Loury, S; Blanco, C; Trautwetter, A

    1996-04-01

    All the essential genetic determinants for site-specific integration of corynephage phi AAU2 are contained within a 1,756-bp DNA fragment, carried on the integrative plasmid p5510, and are shown to be functional in Escherichia coli. One open reading frame, ORF4, encoding a protein of 266 amino acids was shown to represent the phi AAU2 integrase. The nucleotide sequence of the phi AAU2 attachment site, attP, and the attB, attL, and attR sequences in the host "Arthrobacter aureus" C70 were determined. Identical nucleotide sequences were shown to be responsible for the integration of p5510 in the chromosomes of Corynebacterium glutamicum, Brevibacterium divaricatum, and B. lactofermentum, and a sequence almost identical to attB was found to be present in these three strains. In contrast to other phage site-specific recombination systems, a plasmid encompassing only int-attP failed to integrate into the host chromosome. This led to the identification of an 800-bp noncoding region, immediately upstream of int, absolutely required for site-specific integration of p5510.

  12. Fidelity of Target Site Duplication and Sequence Preference during Integration of Xenotropic Murine Leukemia Virus-Related Virus

    PubMed Central

    Kim, Sanggu; Rusmevichientong, Alice; Dong, Beihua; Remenyi, Roland; Silverman, Robert H.; Chow, Samson A.

    2010-01-01

    Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity. PMID:20421928

  13. Endeavour Segment, Juan de Fuca Ridge, Integrated Studies Site (ISS) Update and Opportunities

    NASA Astrophysics Data System (ADS)

    Butterfield, D.; Ridge Community

    2003-12-01

    The Ridge 2000 (R2K) Integrated Studies bull's eye on the Juan de Fuca Ridge is focused on the Main Endeavour hydrothermal field, located on the central portion of the Endeavour Segment. This vent field is one of the most vigorously venting systems along the global mid-ocean ridge spreading network, hosting at least 18 large sulfide structures that contains more than100 smokers. Prior to a magmatic event in 2000 some of the edifices had been venting 380C, volatile-rich fluids with extremely low chlorinities for a decade. In addition to the Main Endeavour Field there are four other known high temperature vent fields spaced approximately 2 kilometers apart along the segment (with hints of more) and abundant areas of diffuse flow, both nearby and distal to the high temperature venting. Diffuse flow from the structures and from a variety of basaltic-hosted sites provides rich habitats abundant with microbial and macrofaunal communities. There are well-developed gradients in volatile concentrations along axis that may reflect influence from a sedimentary source to the north, and high chlorinity fluids vent from the most southern (Mothra) and northern fields (Sasquatch). Twenty years of research have laid a firm base for the 5-year plans of R2K at this site, which include examining the response of this segment to perturbations induced by tectonic and magmatic events, identification of the reservoirs, fluxes, and feedbacks of mass and energy at this site, and predictive modeling coupled with field observations. Since designation as an IS site, high-resolution bathymetric mapping (EM300) and an extensive multi-channel seismic survey have been conducted along the entire segment. Smaller focused areas have also been mapped at meter resolution by SM2000 sonar. Intense field programs in 2003 established the first in-situ seismic array along a mid-ocean ridge, which includes installation of a buried broadband seismometer and 7 short-period seismometers emplaced within basaltic

  14. Significance of the quantitative measurement of the chr16: 51320015 integration site in hepatocytes of patients with chronic hepatitis B.

    PubMed

    Ruan, Peng; Dai, Xiufang; Sun, Zequn; Zhou, Chunfang; Yang, Fan

    2015-11-01

    The present study reported the presence of a hepatitis B virus (HBV) major integration site (MIS) chr16: 51320015 and discussed the significance of quantitative measurement of this site. A total of 30 hepatitis B e antigen (HBeAg) positive (+) and 30 HBeAg negative (‑) patients with chronic hepatitis B (CHB) were enrolled in the present study, and the levels of intrahepatic (IH) covalently closed circular DNA (cccDNA), serum HBV DNA and hepatitis B surface antigen (HBsAg) were detected. Conventional reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and Sanger sequencing were designed to verify the chr16: 51320015 integration site, and the copy numbers of this site were measured using molecular clone and SYBR Green I RT‑qPCR. This site was found to be present in the hepatocytes of all the enrolled patients, and the average number of copies was 1.46x10‑2 ± 4.94x10‑2 copies/cell (3.48x10‑5‑0.212 copies/cell). No significant difference in the copy numbers of this site were observed between the HBeAg (+) (1.43 ± 9.79x10‑1 copies/cell) and HBeAg (‑) patients (6.58x10‑2 ± 2.47x10‑2 copies/cell; P>0.05), which were positively correlated with the levels of serum HBsAg (P=0.0038), but were not correlated with the levels of IH cccDNA (P=0.7785). In conclusion, the chr16:51320015 integration site may be a novel site, which persists in a several patients with HBV infection, and may accumulate in the hepatocytes due to clonal expansion. The diagnostic and therapeutic values of this site require further investigation.

  15. Dietary vitamin A regulates wingless-related MMTV integration site signaling to alter the hair cycle.

    PubMed

    Suo, Liye; Sundberg, John P; Everts, Helen B

    2015-05-01

    Alopecia areata (AA) is an autoimmune hair loss disease caused by a cell-mediated immune attack of the lower portion of the cycling hair follicle. Feeding mice 3-7 times the recommended level of dietary vitamin A accelerated the progression of AA in the graft-induced C3H/HeJ mouse model of AA. In this study, we also found that dietary vitamin A, in a dose dependent manner, activated the hair follicle stem cells (SCs) to induce the development and growth phase of the hair cycle (anagen), which may have made the hair follicle more susceptible to autoimmune attack. Our purpose here is to determine the mechanism by which dietary vitamin A regulates the hair cycle. We found that vitamin A in a dose-dependent manner increased nuclear localized beta-catenin (CTNNB1; a marker of canonical wingless-type Mouse Mammary Tumor Virus integration site family (WNT) signaling) and levels of WNT7A within the hair follicle bulge in these C3H/HeJ mice. These findings suggest that feeding mice high levels of dietary vitamin A increases WNT signaling to activate hair follicle SCs. © 2014 by the Society for Experimental Biology and Medicine.

  16. Reducing the risk of surgical site infection using a multidisciplinary approach: an integrative review

    PubMed Central

    Gillespie, Brigid M; Kang, Evelyn; Roberts, Shelley; Lin, Frances; Morley, Nicola; Finigan, Tracey; Homer, Allison; Chaboyer, Wendy

    2015-01-01

    Purpose To identify and describe the strategies and processes used by multidisciplinary teams of health care professionals to reduce surgical site infections (SSIs). Materials and methods An integrative review of the research literature was undertaken. Searches were conducted in April 2015. Following review of the included studies, data were abstracted using summary tables and the methodological quality of each study assessed using the Standards for Quality Improvement Reporting Excellence guidelines by two reviewers. Discrepancies were dealt with through consensus. Inductive content analysis was used to identify and describe the strategies/processes used by multidisciplinary health care teams to prevent SSI. Results and discussion In total, 13 studies met the inclusion criteria. Of these, 12 studies used quantitative methods, while a single study used qualitative interviews. The majority of the studies were conducted in North America. All quantitative studies evaluated multifaceted quality-improvement interventions aimed at preventing SSI in patients undergoing surgery. Across the 13 studies reviewed, the following multidisciplinary team-based approaches were enacted: using a bundled approach, sharing responsibility, and, adhering to best practice. The majority of studies described team collaborations that were circumscribed by role. None of the reviewed studies used strategies that included the input of allied health professionals or patient participation in SSI prevention. Conclusion Patient-centered interventions aimed at increasing patient participation in SSI prevention and evaluating the contributions of allied health professionals in team-based SSI prevention requires future research. PMID:26508870

  17. Reducing the risk of surgical site infection using a multidisciplinary approach: an integrative review.

    PubMed

    Gillespie, Brigid M; Kang, Evelyn; Roberts, Shelley; Lin, Frances; Morley, Nicola; Finigan, Tracey; Homer, Allison; Chaboyer, Wendy

    2015-01-01

    To identify and describe the strategies and processes used by multidisciplinary teams of health care professionals to reduce surgical site infections (SSIs). An integrative review of the research literature was undertaken. Searches were conducted in April 2015. Following review of the included studies, data were abstracted using summary tables and the methodological quality of each study assessed using the Standards for Quality Improvement Reporting Excellence guidelines by two reviewers. Discrepancies were dealt with through consensus. Inductive content analysis was used to identify and describe the strategies/processes used by multidisciplinary health care teams to prevent SSI. In total, 13 studies met the inclusion criteria. Of these, 12 studies used quantitative methods, while a single study used qualitative interviews. The majority of the studies were conducted in North America. All quantitative studies evaluated multifaceted quality-improvement interventions aimed at preventing SSI in patients undergoing surgery. Across the 13 studies reviewed, the following multidisciplinary team-based approaches were enacted: using a bundled approach, sharing responsibility, and, adhering to best practice. The majority of studies described team collaborations that were circumscribed by role. None of the reviewed studies used strategies that included the input of allied health professionals or patient participation in SSI prevention. Patient-centered interventions aimed at increasing patient participation in SSI prevention and evaluating the contributions of allied health professionals in team-based SSI prevention requires future research.

  18. Integrative functions of the mitochondrial contact site and cristae organizing system.

    PubMed

    Schorr, Stefan; van der Laan, Martin

    2017-09-28

    Mitochondria are complex double-membrane-bound organelles of eukaryotic cells that function as energy-converting powerhouses, metabolic factories and signaling centers. The outer membrane controls the exchange of material and information with other cellular compartments. The inner membrane provides an extended, highly folded surface for selective transport and energy-coupling reactions. It can be divided into an inner boundary membrane and tubular or lamellar cristae membranes that accommodate the oxidative phosphorylation units. Outer membrane, inner boundary membrane and cristae come together at crista junctions, where the mitochondrial contact site and cristae organizing system (MICOS) acts as a membrane-shaping and -connecting scaffold. This peculiar architecture is of pivotal importance for multiple mitochondrial functions. Many elaborate studies in the past years have shed light on the subunit composition and organization of MICOS. In this review article, we summarize these insights and then move on to discuss exciting recent discoveries on the integrative functions of MICOS. Multi-faceted connections to other major players of mitochondrial biogenesis and physiology, like the protein import machineries, the oxidative phosphorylation system, carrier proteins and phospholipid biosynthesis enzymes, are currently emerging. Therefore, we propose that MICOS acts as a central hub in mitochondrial membrane architecture and functionality. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. From Chaos to Content: An Integrated Approach to Government Web Sites

    SciTech Connect

    Demuth, Nora H.; Knudson, Christa K.

    2005-01-03

    The web development team of the Environmental Technology Directorate (ETD) at the U.S. Department of Energy’s Pacific Northwest National Laboratory (PNNL) redesigned the ETD website as a database-driven system, powered by the newly designed ETD Common Information System (ETD-CIS). The ETD website was redesigned in response to an analysis that showed the previous ETD websites were inefficient, costly, and lacking in a consistent focus. Redesigned and newly created websites based on a new ETD template provide a consistent image, meet or exceed accessibility standards, and are linked through a common database. The protocols used in developing the ETD website support integration of further organizational sites and facilitate internal use by staff and training on ETD website development and maintenance. Other PNNL organizations have approached the ETD web development team with an interest in applying the methods established by the ETD system. The ETD system protocol could potentially be used by other DOE laboratories to improve their website efficiency and content focus. “The tools by which we share science information must be as extraordinary as the information itself.[ ]” – DOE Science Director Raymond Orbach

  20. The C1 area of rostral ventrolateral medulla: a central site integrating autonomic responses to hemorrhage.

    PubMed

    Reis, D J; Ruggiero, D A; Morrison, S F

    1989-12-01

    Activation of the sympathetic neurons and release of adrenomedullary catecholamines are the principal early reflex responses to hemorrhage. These are initiated by arterial baro- and chemoreceptors, from other cardiopulmonary receptors, and by intracerebral receptors responding to ischemia. A principal gateway for integrating the autonomic responses are a small collection of neurons in a region of the rostral ventrolateral medulla (RVL), containing a cluster of neurons of the C1 adrenergic cell group, the C1 area. Neurons in the C1 area of RVL project exclusively to autonomic nuclei of the spinal cord, are tonically active, and fire with a rhythm linked to the cardiac cycle. They are essential for maintaining resting discharge of sympathetic nerves and, consequently, arterial pressure (AP) and heart rate. They also are critical for reflex changes in AP in the baro- and chemoreceptor, somato-sympathetic (pain), and cerebral ischemic reflexes. Neurons of the C1 area are under tonic excitatory and inhibitory control by pathways from other autonomic centers. They are controlled by a range of neurotransmitters including, gamma aminobutyric acid (GABA), acetylcholine, catecholamines, enkephalin, and several neuropeptides. They also serve as a site of action for the hypotensive actions of several clinically important neurotransmitters. The C1-area of RVL may play a critical role in the autonomic responses to hemorrhage and may be an important target for drugs seeking to treat hemorrhagic shock.

  1. Transduction of Human Antigen-Presenting Cells with Integrase-Defective Lentiviral Vector Enables Functional Expansion of Primed Antigen-Specific CD8+ T Cells

    PubMed Central

    Bona, Roberta; Michelini, Zuleika; Leone, Pasqualina; Macchia, Iole; Klotman, Mary E.; Salvatore, Mirella

    2010-01-01

    Abstract Nonintegrating lentiviral vectors are being developed as a efficient and safe delivery system for both gene therapy and vaccine purposes. Several reports have demonstrated that a single immunization with integration-defective lentiviral vectors (IDLVs) delivering viral or tumor model antigens in mice was able to elicit broad and long-lasting specific immune responses in the absence of vector integration. At present, no evidence has been reported showing that IDLVs are able to expand preexisting immune responses in the human context. In the present study, we demonstrate that infection of human antigen-presenting cells (APCs), such as monocyte-derived dendritic cells (DCs) and macrophages with IDLVs expressing influenza matrix M1 protein resulted in effective induction of in vitro expansion of M1-primed CD8+ T cells, as evaluated by both pentamer staining and cytokine production. This is the first demonstration that IDLVs represent an efficient delivery system for gene transfer and expression in human APCs, useful for immunotherapeutic applications. PMID:20210625

  2. Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain

    PubMed Central

    Roos, Karen; Werner, Esther; Loessner, Holger

    2015-01-01

    Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors. Mini-Tn7 transposons have been used for the insertion of one such module into the chromosomal site attTn7, present only once in most Gram-negative bacteria. However, integration of multiple mini-Tn7 copies might be suitable for expression of appropriate amounts of antigen or combination of different modules. Here we demonstrate that integration of a 9.6 kb mini-Tn7 harbouring the luciferase luxCDABE (lux) occurs at the natural attTn7 site and simultaneously other locations of the Salmonella chromosome, which were engineered using λ-Red recombinase to contain one or two additional artificial attTn7 sites (a-attTn7). Multicopy integration even at closely spaced attTn7 sites was unexpected in light of the previously reported distance-dependent Tn7 target immunity. Integration of multiple copies of a mini-Tn7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini-Tn7 encoding lacZ and lux was achieved by initial transposition of lacZ-mini-Tn7, subsequent chromosomal insertion of a-attTn7 and a second round of transposition with lux-mini-Tn7. Mini-Tn7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of Salmonella and possibly other bacteria. PMID:25488129

  3. Characterization of Equine Infectious Anemia Virus Integration in the Horse Genome.

    PubMed

    Liu, Qiang; Wang, Xue-Feng; Ma, Jian; He, Xi-Jun; Wang, Xiao-Jun; Zhou, Jian-Hua

    2015-06-19

    Human immunodeficiency virus (HIV)-1 has a unique integration profile in the human genome relative to murine and avian retroviruses. Equine infectious anemia virus (EIAV) is another well-studied lentivirus that can also be used as a promising retro-transfection vector, but its integration into its native host has not been characterized. In this study, we mapped 477 integration sites of the EIAV strain EIAVFDDV13 in fetal equine dermal (FED) cells during in vitro infection. Published integration sites of EIAV and HIV-1 in the human genome were also analyzed as references. Our results demonstrated that EIAVFDDV13 tended to integrate into genes and AT-rich regions, and it avoided integrating into transcription start sites (TSS), which is consistent with EIAV and HIV-1 integration in the human genome. Notably, the integration of EIAVFDDV13 favored long interspersed elements (LINEs) and DNA transposons in the horse genome, whereas the integration of HIV-1 favored short interspersed elements (SINEs) in the human genome. The chromosomal environment near LINEs or DNA transposons potentially influences viral transcription and may be related to the unique EIAV latency states in equids. The data on EIAV integration in its natural host will facilitate studies on lentiviral infection and lentivirus-based therapeutic vectors.

  4. Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

    PubMed Central

    Kim, Yoon-Sang; Wielgosz, Matthew M; Hargrove, Phillip; Kepes, Steven; Gray, John; Persons, Derek A; Nienhuis, Arthur W

    2010-01-01

    Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34+ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34+ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34+ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45+ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector. PMID:20372106

  5. Transduction of human primitive repopulating hematopoietic cells with lentiviral vectors pseudotyped with various envelope proteins.

    PubMed

    Kim, Yoon-Sang; Wielgosz, Matthew M; Hargrove, Phillip; Kepes, Steven; Gray, John; Persons, Derek A; Nienhuis, Arthur W

    2010-07-01

    Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34(+) peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34(+) cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34(+) cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45(+) cells in total bone marrow were comparable to that of the control, mock-transduced group (37-45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the gamma-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the gamma-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.

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

    PubMed Central

    Sheu, Jonathan; Beltzer, Jim; Fury, Brian; Wilczek, Katarzyna; Tobin, Steve; Falconer, Danny; Nolta, Jan; Bauer, Gerhard

    2015-01-01

    Lentiviral vectors are widely used in the field of gene therapy as an effective method for permanent gene delivery. While current methods of producing small scale vector batches for research purposes depend largely on culture flasks, the emergence and popularity of lentiviral vectors in translational, preclinical and clinical research has demanded their production on a much larger scale, a task that can be difficult to manage with the numbers of producer cell culture flasks required for large volumes of vector. To generate a large scale, partially closed system method for the manufacturing of clinical grade lentiviral vector suitable for the generation of induced pluripotent stem cells (iPSCs), we developed a method employing a hollow fiber bioreactor traditionally used for cell expansion. We have demonstrated the growth, transfection, and vector-producing capability of 293T producer cells in this system. Vector particle RNA titers after subsequent vector concentration yielded values comparable to lentiviral iPSC induction vector batches produced using traditional culture methods in 225 cm2 flasks (T225s) and in 10-layer cell factories (CF10s), while yielding a volume nearly 145 times larger than the yield from a T225 flask and nearly three times larger than the yield from a CF10. Employing a closed system hollow fiber bioreactor for vector production offers the possibility of manufacturing large quantities of gene therapy vector while minimizing reagent usage, equipment footprint, and open system manipulation. PMID:26151065

  7. Polyploidization without mitosis improves in vivo liver transduction with lentiviral vectors.

    PubMed

    Pichard, Virginie; Couton, Dominique; Desdouets, Chantal; Ferry, Nicolas

    2013-02-01

    Lentiviral vectors are efficient gene delivery vehicles for therapeutic and research applications. In contrast to oncoretroviral vectors, they are able to infect most nonproliferating cells. In the liver, induction of cell proliferation dramatically improved hepatocyte transduction using all types of retroviral vectors. However, the precise relationship between hepatocyte division and transduction efficiency has not been determined yet. Here we compared gene transfer efficiency in the liver after in vivo injection of recombinant lentiviral or Moloney murine leukemia viral (MoMuLV) vectors in hepatectomized rats treated or not with retrorsine, an alkaloid that blocks hepatocyte division and induces megalocytosis. Partial hepatectomy alone resulted in a similar increase in hepatocyte transduction using either vector. In retrorsine-treated and partially hepatectomized rats, transduction with MoMuLV vectors dropped dramatically. In contrast, we observed that retrorsine treatment combined with partial hepatectomy increased lentiviral transduction to higher levels than hepatectomy alone. Analysis of nuclear ploidy in single cells showed that a high level of transduction was associated with polyploidization. In conclusion, endoreplication could be exploited to improve the efficiency of liver-directed lentiviral gene therapy.

  8. Construction of stable packaging cell lines for clinical lentiviral vector production

    PubMed Central

    Sanber, Khaled S.; Knight, Sean B.; Stephen, Sam L.; Bailey, Ranbir; Escors, David; Minshull, Jeremy; Santilli, Giorgia; Thrasher, Adrian J.; Collins, Mary K.; Takeuchi, Yasuhiro

    2015-01-01

    Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 106 transducing units/ml can be harvested from the final producer clones, which can be increased to 108 TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors. PMID:25762005

  9. Immune response to lentiviral bilirubin UDP-glucuronosyltransferase gene transfer in fetal and neonatal rats.

    PubMed

    Seppen, J; van Til, N P; van der Rijt, R; Hiralall, J K; Kunne, C; Elferink, R P J Oude

    2006-04-01

    Gene therapy for inherited disorders might cause an immune response to the therapeutic protein. A solution would be to introduce the gene in the fetal or neonatal period, which should lead to tolerization. Lentiviral vectors mediate long-term gene expression, and are well suited for gene therapy early in development. A model for fetal or neonatal gene therapy is the inherited disorder of bilirubin metabolism, Crigler-Najjar disease (CN). The absence of bilirubin UDP-glucoronyltransferase (UGT1A1) activity in CN patients causes high serum levels of unconjugated bilirubin and brain damage in infancy. CN is attractive for the development of gene therapy because the mutant Gunn rat closely mimics the human disease. Injection of UGT1A1 lentiviral vectors corrected the hyperbilirubinemia for more than a year in rats injected as fetuses and for up to 18 weeks in rats injected the day of birth. UGT1A1 gene transfer was confirmed by the presence of bilirubin glucuronides in bile. All animals injected with UGT1A1 lentiviral vectors developed antibodies to UGT1A1. Animals injected with green fluorescent protein (GFP) lentiviral vectors did not develop antibodies to GFP. Our results indicate that fetal and neonatal gene therapy with immunogenic proteins such as UGT1A1 does not necessarily lead to tolerization.

  10. Using remote sensing data for exploitation of integrated renewable energy at coastal site in South Italy

    NASA Astrophysics Data System (ADS)

    Calaudi, Rosamaria; Lo Feudo, Teresa; Calidonna, Claudia Roberta; Sempreviva, Anna Maria

    2016-04-01

    Renewable energy sources are major components of the strategy to reduce harmful emissions and to replace depleting fossil energy resources. Data from Remote Sensing can provide detailed information for analysis for sources of renewable energy and to determine the potential energy and socially acceptability of suggested location. Coastal sites of Southern Italy have the advantage of favorable climatic conditions to use renewable energy, such us cloud free days and local breeze phenomena. Many ports are located where they have opportunities for exploitation of renewable energy, by using existing port area and by taking advantage of their coastal locations. Policies of European-Committee and Global-Navigation-PIANC for a better use of energy and an efficient supply from renewable sources are also focused on the construction of port facilities in zero emissions. Using data from Remote Sensing, can reduce the financial resources currently required for finding and assessing suitable areas, we defined an integrated methodology for potential wind and solar energy in harbor areas. In this study we compared the hourly solar power energy using MSG-SEVIRI (Meteosat Second Generation Spinning Enhanced Visible and Infrared) data products DSSF (Down-welling Surface Short-wave-Flux), and PV-Plant measurements with Nominal Power Peak of 19,85 kWp. The PV Plant is situated at a coastal site in Calabrian region, located near Vibo Valentia harbor area. We estimate potential energy by using input solar radiation of Satellite data, with same characteristics of the PV-plant. The RMSE and BIAS for hourly averaged solar electrical reproducibility are estimated including clear and sky conditions. Comparison between energy reproducibility by using DSSF product and PV-plant measurements, made over the period October 2013-June 2014, showed a good agreement in our costal site and generally overestimate (RMSE(35W/m2) and BIAS(4W/m2)) electrical reproducibility from a PV-plant. For wind resource

  11. Characterization of the attP site of the integrative element pSAM2 from Streptomyces ambofaciens.

    PubMed

    Raynal, Alain; Friedmann, Annick; Tuphile, Karine; Guerineau, Michel; Pernodet, Jean-Luc

    2002-01-01

    pSAM2 is integrated into the Streptomyces ambofaciens chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) site. The 43 kDa integrase protein encoded by pSAM2 catalyses this recombination event. Tools have been developed to study site-specific recombination in Escherichia coli. In vivo studies showed that a 360 bp fragment of attP is required for efficient site-specific recombination and that int can be provided in trans. pSAM2 integrase was purified and overexpressed in E. coli and Int binding at the attP site was studied. DNaseI footprinting revealed two sites that bind integrase strongly and appear to be symmetrical with regard to the core site. These two P1/P2 arm-type sites both contain a 17 bp motif that is identical except at one position, GTCACGCAG(A/T)TAGACAC. P1 and P2 are essential for site-specific recombination.

  12. Low Impact Development Design—Integrating Suitability Analysis and Site Planning for Reduction of Post-Development Stormwater Quantity

    USDA-ARS?s Scientific Manuscript database

    A land-suitability analysis (LSA) was integrated with open-space conservation principles, based on watershed physiographic and soil characteristics, to derive a low-impact development (LID) residential plan for a three hectare site in Coshocton OH, USA. The curve number method was used to estimate t...

  13. Low-Impact Development Design—Integrating Suitability Analysis and Site Planning For Reduction Of Post-Development Stormwater Quantity

    EPA Science Inventory

    A land-suitability analysis (LSA) was integrated with open-space conservation principles, based on watershed physiographic and soil characteristics, to derive a low-impact development (LID) residential plan for a three hectare site in Coshocton OH, USA. The curve number method wa...

  14. Thermodynamic Characterization of Hydration Sites from Integral Equation-Derived Free Energy Densities: Application to Protein Binding Sites and Ligand Series.

    PubMed

    Güssregen, Stefan; Matter, Hans; Hessler, Gerhard; Lionta, Evanthia; Heil, Jochen; Kast, Stefan M

    2017-07-24

    Water molecules play an essential role for mediating interactions between ligands and protein binding sites. Displacement of specific water molecules can favorably modulate the free energy of binding of protein-ligand complexes. Here, the nature of water interactions in protein binding sites is investigated by 3D RISM (three-dimensional reference interaction site model) integral equation theory to understand and exploit local thermodynamic features of water molecules by ranking their possible displacement in structure-based design. Unlike molecular dynamics-based approaches, 3D RISM theory allows for fast and noise-free calculations using the same detailed level of solute-solvent interaction description. Here we correlate molecular water entities instead of mere site density maxima with local contributions to the solvation free energy using novel algorithms. Distinct water molecules and hydration sites are investigated in multiple protein-ligand X-ray structures, namely streptavidin, factor Xa, and factor VIIa, based on 3D RISM-derived free energy density fields. Our approach allows the semiquantitative assessment of whether a given structural water molecule can potentially be targeted for replacement in structure-based design. Finally, PLS-based regression models from free energy density fields used within a 3D-QSAR approach (CARMa - comparative analysis of 3D RISM Maps) are shown to be able to extract relevant information for the interpretation of structure-activity relationship (SAR) trends, as demonstrated for a series of serine protease inhibitors.

  15. Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

    PubMed

    Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira

    2011-02-01

    Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

  16. Charge transport in columnar stacked triphenylenes: Effects of conformational fluctuations on charge transfer integrals and site energies

    NASA Astrophysics Data System (ADS)

    Senthilkumar, K.; Grozema, F. C.; Bickelhaupt, F. M.; Siebbeles, L. D. A.

    2003-11-01

    Values of charge transfer integrals, spatial overlap integrals and site energies involved in transport of positive charges along columnar stacked triphenylene derivatives are provided. These parameters were calculated directly as the matrix elements of the Kohn-Sham Hamiltonian, defined in terms of the molecular orbitals on individual triphenylene molecules. This was realized by exploiting the unique feature of the Amsterdam density functional theory program that allows one to use molecular orbitals on individual molecules as a basis set in calculations on a system composed of two or more molecules. The charge transfer integrals obtained in this way differ significantly from values estimated from the energy splitting between the highest occupied molecular orbitals in a dimer. The difference is due to the nonzero spatial overlap between the molecular orbitals on adjacent molecules. Calculations were performed on unsubstituted and methoxy- or methylthio-substituted triphenylenes. Charge transfer integrals and site energies were computed as a function of the twist angle, stacking distance and lateral slide distance between adjacent molecules. The variation of the charge transfer integrals and site energies with these conformational degrees of freedom provide a qualitative explanation of the similarities and differences between the experimental charge carrier mobilities in different phases of alkoxy- and alkylthio-substituted triphenylenes. The data obtained from the present work can be used as input in quantitative studies of charge transport in columnar stacked triphenylene derivatives.

  17. Verification of geomechanical integrity and prediction of long-term mineral trapping for the Ketzin CO2 storage pilot site

    NASA Astrophysics Data System (ADS)

    Kempka, Thomas; De Lucia, Marco; Kühn, Michael

    2014-05-01

    Static and dynamic numerical modelling generally accompany the entire CO2 storage site life cycle. Thereto, it is required to match the employed models with field observations on a regular basis in order to predict future site behaviour. We investigated the coupled processes at the Ketzin CO2 storage pilot site [1] using a model coupling concept focusing on the temporal relevance of processes involved (hydraulic, chemical and mechanical) at given time-scales (site operation, abandonment and long-term stabilization). For that purpose, long-term dynamic multi-phase flow simulations [2], [3] established the basis for all simulations discussed in the following. Hereby, pressure changes resulting in geomechanical effects are largest during site operation, whereas geochemical reactions are governed by slow kinetics resulting in a long-term stabilization. To account for mechanical integrity, which may be mainly affected during site operation, we incorporated a regional-scale coupled hydro-mechanical model. Our simulation results show maximum ground surface displacements of about 4 mm, whereas shear and tensile failure are not observed. Consequently, the CO2 storage operation at the Ketzin pilot site does not compromise reservoir, caprock and fault integrity. Chemical processes responsible for mineral trapping are expected to mainly occur during long-term stabilization at the Ketzin pilot site [4]. Hence, our previous assessment [3] was extended by integrating two long-term mineral trapping scenarios. Thereby, mineral trapping contributes to the trapping mechanisms with 11.7 % after 16,000 years of simulation in our conservative and with 30.9 % in our maximum reactivity scenarios. Dynamic flow simulations indicate that only 0.2 % of the CO2 injected (about 67,270 t CO2 in total) is in gaseous state, but structurally trapped after 16,000 years. Depending on the studied long-term scenario, CO2 dissolution is the dominating trapping mechanism with 68.9 % and 88

  18. Use of an integrated flow model to estimate ecologically relevant hydrologic characteristics at stream biomonitoring sites

    USGS Publications Warehouse

    Kennen, J.G.; Kauffman, L.J.; Ayers, M.A.; Wolock, D.M.; Colarullo, S.J.

    2008-01-01

    We developed an integrated hydroecological model to provide a comprehensive set of hydrologic variables representing five major components of the flow regime at 856 aquatic-invertebrate monitoring sites in New Jersey. The hydroecological model simulates streamflow by routing water that moves overland and through the subsurface from atmospheric delivery to the watershed outlet. Snow accumulation and melt, evapotranspiration, precipitation, withdrawals, discharges, pervious- and impervious-area runoff, and lake storage were accounted for in the water balance. We generated more than 78 flow variables, which describe the frequency, magnitude, duration, rate of change, and timing of flow events. Highly correlated variables were filtered by principal component analysis to obtain a non-redundant subset of variables that explain the majority of the variation in the complete set. This subset of variables was used to evaluate the effect of changes in the flow regime on aquatic-invertebrate assemblage structure at 856 biomonitoring sites. We used non-metric multidimensional scaling (NMS) to evaluate variation in aquatic-invertebrate assemblage structure across a disturbance gradient. We employed multiple linear regression (MLR) analysis to build a series of MLR models that identify the most important environmental and hydrologic variables driving the differences in the aquatic-invertebrate assemblages across the disturbance gradient. The first axis of NMS ordination was significantly related to many hydrologic, habitat, and land-use/land-cover variables, including the average number of annual storms producing runoff, ratio of 25-75% exceedance flow (flashiness), diversity of natural stream substrate, and the percentage of forested land near the stream channel (forest buffer). Modifications in the hydrologic regime as the result of changes in watershed land use appear to promote the retention of highly tolerant aquatic species; in contrast, species that are sensitive to

  19. Methodology and software to detect viral integration site hot-spots

    PubMed Central

    2011-01-01

    Background Modern gene therapy methods have limited control over where a therapeutic viral vector inserts into the host genome. Vector integration can activate local gene expression, which can cause cancer if the vector inserts near an oncogene. Viral integration hot-spots or 'common insertion sites' (CIS) are scrutinized to evaluate and predict patient safety. CIS are typically defined by a minimum density of insertions (such as 2-4 within a 30-100 kb region), which unfortunately depends on the total number of observed VIS. This is problematic for comparing hot-spot distributions across data sets and patients, where the VIS numbers may vary. Results We develop two new methods for defining hot-spots that are relatively independent of data set size. Both methods operate on distributions of VIS across consecutive 1 Mb 'bins' of the genome. The first method 'z-threshold' tallies the number of VIS per bin, converts these counts to z-scores, and applies a threshold to define high density bins. The second method 'BCP' applies a Bayesian change-point model to the z-scores to define hot-spots. The novel hot-spot methods are compared with a conventional CIS method using simulated data sets and data sets from five published human studies, including the X-linked ALD (adrenoleukodystrophy), CGD (chronic granulomatous disease) and SCID-X1 (X-linked severe combined immunodeficiency) trials. The BCP analysis of the human X-linked ALD data for two patients separately (774 and 1627 VIS) and combined (2401 VIS) resulted in 5-6 hot-spots covering 0.17-0.251% of the genome and containing 5.56-7.74% of the total VIS. In comparison, the CIS analysis resulted in 12-110 hot-spots covering 0.018-0.246% of the genome and containing 5.81-22.7% of the VIS, corresponding to a greater number of hot-spots as the data set size increased. Our hot-spot methods enable one to evaluate the extent of VIS clustering, and formally compare data sets in terms of hot-spot overlap. Finally, we show that the

  20. Petrophysical properties of saprolites from the Oak Ridge Integrated Field Research Challenge site, Tennessee

    SciTech Connect

    Revil, André; Skold, Magnus; Hubbard, Susan S.; Wu, Yuxin; Watson, David B.; Karaoulis, Marios

    2013-01-01

    At the Oak Ridge Integrated Field Research Challenge site, near Oak Ridge, Tennessee, the shallow saprolitic aquifer is contaminated by nitric acid, uranium, and metals originating from the former S3 settling ponds. To interpret low-frequency geophysical methods used to image contaminant plumes, we have characterized the petrophysical properties of three representative saprolite core samples. Their hydraulic conductivity ranges from to in agreement with field data. Complex conductivity measurements, in the frequency range of 1 mHz to 45 kHz, were performed with NaCl solutions with electrical conductivities in the range to , a range representative of field conditions. The electrical conductivity data were well reproduced with a simple linear conductivity model between the saprolite conductivity and the pore water conductivity. The conductivity plots were used to estimate the formation factor (the cementation exponent was about ) and the surface conductivity ( ). The magnitude of the surface conductivity depended on the degree of weathering and therefore on the amount of smectite and mixed layer (illite-smectite) clays present in the saprolite. The chargeability of the core samples was in the range of and is strongly dependent on the salinity. We also performed streaming potential measurements with the same pore fluid composition as that used for the complex conductivity measurements. We found an excess of movable electrical charges on the order of 100 to in agreement with previous investigations connecting the movable excess charge density to permeability. The zeta potential was in the range of to independent on the salinity. The electrical measurements were consistent with an average cation exchange capacity in the range of 1.4 to and a specific surface area on the order of 4000 to about 30,000 . Read More: http://library.seg.org/doi/abs/10.1190/geo2012-0176.1

  1. Integration of woodchuck hepatitis virus (WHV) DNA at two chromosomal sites (Vk and gag-like) in a hepatocellular carcinoma.

    PubMed

    Yamazoe, M; Nakai, S; Ogasawara, N; Yoshikawa, H

    1991-04-01

    Integration of woodchuck hepatitis virus (WHV) DNA into the liver DNA of a woodchuck infected by the virus was investigated. Clonal viral integration was not detected three months before the appearance of four hepatocellular carcinomas (HCC). Integration of the viral DNA was detected in all four HCCs, of which one was chosen to determine the structure of the viral integration completely in a single tumor. The integration occurred in two sites. One part contains the viral DNA from the middle of the gene encoding surface antigen to two-thirds of the way through the gene encoding X protein (X) with no structural changes. The coding frame of the truncated X gene continues into the chromosomal sequence to make a possible fusion protein. The integration seems to have occurred by recombination within two direct repeats of the viral genome in one junction and by homologous recombination between viral DNA and chromosomal DNA in the other junction. The viral DNA is integrated into a spacer of the immunoglobulin L-chain Vk (IgVk) region without any chromosomal rearrangement accompanying the integration. The viral DNA at the second site has a complex structural rearrangement: part of the preS gene is duplicated and attached to the terminus of the gene encoding core antigen in a head-to-tail fashion, followed by three small fragments derived from other parts of the viral DNA. The integrated preS gene has its own 5' regulatory element and a coding frame consisting of the truncated preS gene, the other parts of viral DNA and the chromosomal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Monitoring of well integrity by magnetic imaging defectoscopy (MID) at the Ketzin pilot site, Germany

    NASA Astrophysics Data System (ADS)

    Zemke, Kornelia; Liebscher, Axel; Möller, Fabian

    2017-04-01

    One of the key requirements for safe CO2 storage operation is to ensure wellbore integrity. The CO2 triggered acid in-well environment may lead to pitting and/or surface corrosion and eventually to fatigue of well casings and cementation by this giving raise to wellbore leakage. Corrosion effects are conventionally monitored by measurement of inner casing surface, internal diameter and wall thickness. Caliper logging provides inner surface and internal diameter data while ultrasonic tools measure both the internal diameter and casing thickness as well as the bonding between casing and cement. However, both tools can only monitor and characterize the most inner casing and ultrasonic tools in addition can only be applied in fluid filled wells. At the Ketzin CO2 storage test site, Germany, about 67 kt of CO2 were injected between June 2008 and August 2013 and an interdisciplinary monitoring concept was developed with focus on the storage complex, the overburden, the surface and the wellbores. Four deep wells penetrate the reservoir and their integrity has been monitored by a combination of video inspection, pulsed neutron gamma logging PNG and magnetic imaging defectoscopy MID. MID is an advanced logging method for non-destructive testing and has the great advantages that it can be operated in gas filled boreholes and that it provides information also for outer casings. The MID tool generates electromagnetic pulsed transient eddy currents and records the response of the surrounding media. The distribution and strength of the eddy-currents is then converted into averaged, depth-resolved thicknesses of the individual casings. Run in time-lapse mode, MID provides a measure to detect changes in casing thickness and therefore hints to corrosion. At Ketzin, the four deep wells haven been monitored by repeat MID logging on a roughly annual basis in cooperation with VNG Gasspeicher GmbH (VGS) and GAZPROMENERGODIAGNOSTIKA, applying their in-house MID tool. The MID based depth

  3. Principles and Strategies for Monitoring Data Collection Integrity in a Multi-site Randomized Clinical Trial of a Behavioral Intervention

    PubMed Central

    Phillips-Salimi, Celeste R.; Stickler, Molly A. Donovan; Stegenga, Kristin; Lee, Melissa; Haase, Joan E.

    2011-01-01

    Although treatment fidelity strategies for enhancing the integrity of behavioral interventions have been well described, little has been written about monitoring data collection integrity. This article describes the principles and strategies developed to monitor data collection integrity of the “Stories and Music for Adolescent/Young Adult Resilience During Transplant” study (R01NR008583; U10CA098543; U10CA095861) — a multi-site Children’s Oncology Group randomized clinical trial of a music therapy intervention for adolescents and young adults undergoing stem cell transplant. The principles and strategies outlined in this article provide one model for development and evaluation of a data collection integrity monitoring plan for behavioral interventions that may be adapted by investigators and may be useful to funding agencies and grant application reviewers in evaluating proposals. PMID:21567433

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967

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

    PubMed

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

    2014-01-01

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

  7. Correction of murine SCID-X1 by lentiviral gene therapy using a codon-optimized IL2RG gene and minimal pretransplant conditioning.

    PubMed

    Huston, Marshall W; van Til, Niek P; Visser, Trudi P; Arshad, Shazia; Brugman, Martijn H; Cattoglio, Claudia; Nowrouzi, Ali; Li, Yuedan; Schambach, Axel; Schmidt, Manfred; Baum, Christopher; von Kalle, Christof; Mavilio, Fulvio; Zhang, Fang; Blundell, Mike P; Thrasher, Adrian J; Verstegen, Monique M A; Wagemaker, Gerard

    2011-10-01

    Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using γ-retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor γ gene (IL2RG) cDNA (coγc), regulated by its 1.1 kb promoter region (γcPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of Il2rg(-/-) mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and γcPr vectors. We conclude that SIN lentiviral gene therapy using coγc driven by the γcPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required.

  8. The actinophage RP3 DNA integrates site-specifically into the putative tRNA(Arg)(AGG) gene of Streptomyces rimosus.

    PubMed Central

    Gabriel, K; Schmid, H; Schmidt, U; Rausch, H

    1995-01-01

    The temperate actinophage RP3 integrates site-specifically into the chromosome of Streptomyces rimosus R6-554. The phage attachment site attP and the hybrid attachment sites of the integrated prophage--attL and attR--were cloned and sequenced. The 54nt core sequence, common to all RP3 related attachment sites, comprises the 3' terminal end of a putative tRNA(Arg)(AGG) gene. AttB bears the complete tRNA gene which is restored in attL after integration. A 7.5kb HindIII fragment, bearing attP, was used to construct an integrative plasmid to simulate the integration process in vivo and to localize the phage genes necessary for site specific integration. The int and xis genes were sequenced and compared to other recombination genes. PMID:7870591

  9. MLV integration site selection is driven by strong enhancers and active promoters

    PubMed Central

    LaFave, Matthew C.; Varshney, Gaurav K.; Gildea, Derek E.; Wolfsberg, Tyra G.; Baxevanis, Andreas D.; Burgess, Shawn M.

    2014-01-01

    Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3 700 000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors. PMID:24464997

  10. Identification and characterization of science-rich landing sites for lunar lander missions using integrated remote sensing observations

    NASA Astrophysics Data System (ADS)

    Flahaut, J.; Blanchette-Guertin, J.-F.; Jilly, C.; Sharma, P.; Souchon, A.; van Westrenen, W.; Kring, D. A.

    2012-12-01

    Despite more than 52 years of lunar exploration, a wide range of first-order scientific questions remain about the Moon's formation, temporal evolution, and current surface and interior properties. Addressing many of these questions requires obtaining new in situ analyses or return of lunar surface or shallow subsurface samples, and hence rely on the selection of optimal landing sites. Here, we present an approach to optimize science-rich lunar landing site selection studies based on the integration of remote sensing observations. Currently available remote sensing data, as well as features of interest published in the recent literature, were integrated in a Geographic Information System. This numerical database contains geographic information about all these findings, which can be consulted and used to simultaneously display multiple features and parameters of interest. To illustrate our approach, we identified the optimal landing sites to address the two top priorities (or goals) relative to Concept 3 of the National Research Council of the National Academies (2007), namely to 'Determine the extent and composition of the primary feldspathic crust, (ur)KREEP layer, and other products of differentiation' and to 'Inventory the variety, age, distribution and origin of lunar rock types'. We review site requirements and propose possible landing sites for both these goals. We identified 29 sites that best fulfill both these goals and compare them with the landing sites of planned future lunar lander missions. Finally, we detail two of these science-rich sites (Aristarchus and Theophilus craters) which are particularly accessible through their location on the nearside.

  11. Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines

    PubMed Central

    Olthof, Nadine C.; Huebbers, Christian U.; Kolligs, Jutta; Henfling, Mieke; Ramaekers, Frans C.S.; Cornet, Iris; van Lent-Albrechts, Josefa A.; Stegmann, Sander P.A.; Silling, Steffi; Wieland, Ulrike; Carey, Thomas E.; Walline, Heather M.; Gollin, Susanne M.; Hoffmann, Thomas K.; de Winter, Johan; Kremer, Bernd; Klussmann, Jens-Peter; Speel, Ernst-Jan M.

    2017-01-01

    HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analysed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines, and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression, and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies. PMID:25082736

  12. Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines.

    PubMed

    Olthof, Nadine C; Huebbers, Christian U; Kolligs, Jutta; Henfling, Mieke; Ramaekers, Frans C S; Cornet, Iris; van Lent-Albrechts, Josefa A; Stegmann, Alexander P A; Silling, Steffi; Wieland, Ulrike; Carey, Thomas E; Walline, Heather M; Gollin, Susanne M; Hoffmann, Thomas K; de Winter, Johan; Kremer, Bernd; Klussmann, Jens P; Speel, Ernst-Jan M

    2015-03-01

    HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

  13. Simulating dissolved organic carbon dynamics at the swedish integrated monitoring sites with the integrated catchments model for carbon, INCA-C.

    PubMed

    Futter, M N; Löfgren, S; Köhler, S J; Lundin, L; Moldan, F; Bringmark, L

    2011-12-01

    Surface water concentrations of dissolved organic carbon ([DOC]) are changing throughout the northern hemisphere due to changes in climate, land use and acid deposition. However, the relative importance of these drivers is unclear. Here, we use the Integrated Catchments model for Carbon (INCA-C) to simulate long-term (1996-2008) streamwater [DOC] at the four Swedish integrated monitoring (IM) sites. These are unmanaged headwater catchments with old-growth forests and no major changes in land use. Daily, seasonal and long-term variations in streamwater [DOC] driven by runoff, seasonal temperature and atmospheric sulfate (SO₄(2-)) deposition were observed at all sites. Using INCA-C, it was possible to reproduce observed patterns of variability in streamwater [DOC] at the four IM sites. Runoff was found to be the main short-term control on [DOC]. Seasonal patterns in [DOC] were controlled primarily by soil temperature. Measured SO₄(2-) deposition explained some of the long-term [DOC] variability at all sites.

  14. Human T-Cell Leukemia Virus Type 1 Integration Target Sites in the Human Genome: Comparison with Those of Other Retroviruses▿ ‡

    PubMed Central

    Derse, David; Crise, Bruce; Li, Yuan; Princler, Gerald; Lum, Nicole; Stewart, Claudia; McGrath, Connor F.; Hughes, Stephen H.; Munroe, David J.; Wu, Xiaolin

    2007-01-01

    Retroviral integration into the host genome is not entirely random, and integration site preferences vary among different retroviruses. Human immunodeficiency virus (HIV) prefers to integrate within active genes, whereas murine leukemia virus (MLV) prefers to integrate near transcription start sites and CpG islands. On the other hand, integration of avian sarcoma-leukosis virus (ASLV) shows little preference either for genes, transcription start sites, or CpG islands. While host cellular factors play important roles in target site selection, the viral integrase is probably the major viral determinant. It is reasonable to hypothesize that retroviruses with similar integrases have similar preferences for target site selection. Although integration profiles are well defined for members of the lentivirus, spumaretrovirus, alpharetrovirus, and gammaretrovirus genera, no members of the deltaretroviruses, for example, human T-cell leukemia virus type 1 (HTLV-1), have been evaluated. We have mapped 541 HTLV-1 integration sites in human HeLa cells and show that HTLV-1, like ASLV, does not specifically target transcription units and transcription start sites. Comparing the integration sites of HTLV-1 with those of ASLV, HIV, simian immunodeficiency virus, MLV, and foamy virus, we show that global and local integration site preferences correlate with the sequence/structure of virus-encoded integrases, supporting the idea that integrase is the major determinant of retroviral integration site selection. Our results suggest that the global integration profiles of other retroviruses could be predicted from phylogenetic comparisons of the integrase proteins. Our results show that retroviruses that engender different insertional mutagenesis risks can have similar integration profiles. PMID:17409138

  15. An integration strategy for the NEPA and RCRA/CERCLA programs at the Savannah River Site

    SciTech Connect

    Shedrow, C.B.; Gaughan, B.W.; Moore-Shedrow, D.B.

    1993-10-01

    Savannah River Site (SRS) environmental remediation activities are conducted according to applicable environmental laws and regulations, including the Resource Conservation and Recovery Act (RCRA), the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA) and the National Environmental Policy Act (NEPA). Waste unit cleanups are accomplished by evaluating RCRA and CERCLA requirements at the sites, then selecting and implementing the appropriate cleanup measures. All State and Federal regulations, including the NEPA, are considered for applicability to each waste site. This strategy is discussed.

  16. The piggyBac Transposon Displays Local and Distant Reintegration Preferences and Can Cause Mutations at Noncanonical Integration Sites

    PubMed Central

    Li, Meng Amy; Pettitt, Stephen J.; Eckert, Sabine; Ning, Zemin; Rice, Stephen; Cadiñanos, Juan; Yusa, Kosuke; Conte, Nathalie

    2013-01-01

    The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome. PMID:23358416

  17. Using integrated geospatial mapping and conceptual site models to guide risk-based environmental clean-up decisions.

    PubMed

    Mayer, Henry J; Greenberg, Michael R; Burger, Joanna; Gochfield, Michael; Powers, Charles; Kosson, David; Keren, Roger; Danis, Christine; Vyas, Vikram

    2005-04-01

    Government and private sector organizations are increasingly turning to the use of maps and other visual models to provide a depiction of environmental hazards and the potential risks they represent to humans and ecosystems. Frequently, the graphic presentation is tailored to address a specific contaminant, its location and possible exposure pathways, and potential receptors. Its format is usually driven by the data available, choice of graphics technology, and the audience being served. A format that is effective for displaying one contaminant at one scale at one site, however, may be ineffective in accurately portraying the circumstances surrounding a different contaminant at the same site, or the same contaminant at a different site, because of limitations in available data or the graphics technology being used. This is the daunting challenge facing the U.S. Department of Energy (DOE), which is responsible for the nation's legacy wastes from nuclear weapons research, testing, and production at over 100 sites in the United States. In this article, we discuss the development and use of integrated geospatial mapping and conceptual site models to identify hazards and evaluate alternative long-term environmental clean-up strategies at DOE sites located across the United States. While the DOE probably has the greatest need for such information, the Department of Defense and other public and private responsible parties for many large and controversial National Priority List or Superfund sites would benefit from a similar approach.

  18. Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes.

    PubMed

    Hirano, Nobutaka; Muroi, Tetsurou; Kihara, Yoshihiko; Kobayashi, Ryuichi; Takahashi, Hideo; Haruki, Mitsuru

    2011-03-01

    Phage integrases are enzymes that catalyze unidirectional site-specific recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific recombination system based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific recombination system that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the recombination efficiencies increased by a factor of ~10(1-3) in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This system thus may be beneficial to genome engineering for a wide variety of bacterial species.

  19. Subsurface profiling using integrated geophysical methods for 2D site response analysis in Bangalore city, India: a new approach

    NASA Astrophysics Data System (ADS)

    Chandran, Deepu; Anbazhagan, P.

    2017-10-01

    Recently, site response analysis has become a mandatory step for the design of important structures. Subsurface investigation is an essential step, from where the input parameters for the site response study like density, shear wave velocity (Vs), thickness and damping characteristics, etc, are obtained. Most site response studies at shallow bedrock sites are one-dimensional (1D) and are usually carried out by using Vs from multi-channel analysis of surface waves (MASW) or a standard penetration test (SPT) for N values with assumptions that soil layers are horizontal, uniform and homogeneous. These assumptions are not completely true in shallow bedrock regions as soil deposits are heterogeneous. The objective of this study is to generate the actual subsurface profiles in two-dimensions at shallow bedrock regions using integrated subsurface investigation testing. The study area selected for this work is Bangalore, India. Three survey lines were selected in Bangalore at two different locations; one at the Indian Institute of Science (IISc) Campus and the other at Whitefield. Geophysical surveys like ground penetrating radar (GPR) and 2D MASW were carried out at these survey lines. Geophysical test results are compared and validated with a conventional geotechnical SPT. At the IISc site, the soil profile is obtained from a trench excavated for a proposed pipeline used to compare the geophysical test results. Test results show that GPR is very useful to delineate subsurface layers, especially for shallow depths at both sites (IISc Campus and Whitefield). MASW survey results show variation of Vs values and layer thickness comparatively at deeper depths for both sites. They also show higher density soil strata with high Vs value obtained at the IISc Campus site, whereas at the Whitefield site weaker soil with low shear velocity is observed. Combining these two geophysical methods helped to generate representative 2D subsurface profiles. These subsurface profiles can be

  20. A multi-site intercomparison of integrated water vapour observations for climate change analysis

    NASA Astrophysics Data System (ADS)

    Van Malderen, R.; Brenot, H.; Pottiaux, E.; Beirle, S.; Hermans, C.; De Mazière, M.; Wagner, T.; De Backer, H.; Bruyninx, C.

    2014-08-01

    Water vapour plays a dominant role in the climate change debate. However, observing water vapour over a climatological time period in a consistent and homogeneous manner is challenging. On one hand, networks of ground-based instruments able to retrieve homogeneous integrated water vapour (IWV) data sets are being set up. Typical examples are Global Navigation Satellite System (GNSS) observation networks such as the International GNSS Service (IGS), with continuous GPS (Global Positioning System) observations spanning over the last 15+ years, and the AErosol RObotic NETwork (AERONET), providing long-term observations performed with standardized and well-calibrated sun photometers. On the other hand, satellite-based measurements of IWV already have a time span of over 10 years (e.g. AIRS) or are being merged to create long-term time series (e.g. GOME, SCIAMACHY, and GOME-2). This study performs an intercomparison of IWV measurements from satellite devices (in the visible, GOME/SCIAMACHY/GOME-2, and in the thermal infrared, AIRS), in situ measurements (radiosondes) and ground-based instruments (GPS, sun photometer), to assess their use in water vapour trends analysis. To this end, we selected 28 sites world-wide for which GPS observations can directly be compared with coincident satellite IWV observations, together with sun photometer and/or radiosonde measurements. The mean biases of the different techniques compared to the GPS estimates vary only between -0.3 to 0.5 mm of IWV. Nevertheless these small biases are accompanied by large standard deviations (SD), especially for the satellite instruments. In particular, we analysed the impact of clouds on the IWV agreement. The influence of specific issues for each instrument on the intercomparison is also investigated (e.g. the distance between the satellite ground pixel centre and the co-located ground-based station, the satellite scan angle, daytime/nighttime differences). Furthermore, we checked if the properties of

  1. A multi-site techniques intercomparison of integrated water vapour observations for climate change analysis

    NASA Astrophysics Data System (ADS)

    Van Malderen, R.; Brenot, H.; Pottiaux, E.; Beirle, S.; Hermans, C.; De Mazière, M.; Wagner, T.; De Backer, H.; Bruyninx, C.

    2014-02-01

    Water vapour plays a dominant role in the climate change debate. However, observing water vapour over a climatological time period in a consistent and homogeneous manner is challenging. At one hand, networks of ground-based instruments allowing to retrieve homogeneous Integrated Water Vapour (IWV) datasets are being set up. Typical examples are Global Navigation Satellite System (GNSS) observation networks such as the International GNSS Service (IGS), with continuous GPS (Global Positioning System) observations spanning over the last 15+ yr, and the AErosol RObotic NETwork (AERONET), providing long-term observations performed with standardized and well-calibrated sun photometers. On the other hand, satellite-based measurements of IWV already have a time span of over 10 yr (e.g. AIRS) or are being merged in order to create long-term time series (e.g. GOME, SCIAMACHY, and GOME-2). The present study aims at setting up a techniques intercomparison of IWV measurements from satellite devices (in the visible, GOME/SCIAMACHY/GOME-2, and in the thermal infrared, AIRS), in-situ measurements (radiosondes) and ground-based instruments (GPS, sun photometer), to assess the applicability of either dataset for water vapour trends analysis. To this end, we selected 28 sites worldwide at which GPS observations can directly be compared with coincident satellite IWV observations, together with sun photometer and/or radiosonde measurements. We found that the mean biases of the different techniques w.r.t. the GPS estimates vary only between -0.3 to 0.5 mm of IWV, but the small bias is accompanied by large Root Mean Square (RMS) values, especially for the satellite instruments. In particular, we analysed the impact of the presence of clouds on the techniques IWV agreement. Also, the influence of specific issues for each instrument on the intercomparison is investigated, e.g. the distance between the satellite ground pixel centre and the co-located ground-based station, the satellite scan

  2. An integrated multi criteria approach for landfill siting in a conflicting environmental, economical and socio-cultural area.

    PubMed

    Eskandari, Mahnaz; Homaee, Mehdi; Mahmodi, Shahla

    2012-08-01

    Landfill site selection is a complicated multi criteria land use planning that should convince all related stakeholders with different insights. This paper addresses an integrating approach for landfill siting based on conflicting opinions among environmental, economical and socio-cultural expertise. In order to gain optimized siting decision, the issue was investigated in different viewpoints. At first step based on opinion sampling and questionnaire results of 35 experts familiar with local situations, the national environmental legislations and international practices, 13 constraints and 15 factors were built in hierarchical structure. Factors divided into three environmental, economical and socio-cultural groups. In the next step, the GIS-database was developed based on the designated criteria. In the third stage, the criteria standardization and criteria weighting were accomplished. The relative importance weights of criteria and subcriteria were estimated, respectively, using analytical hierarchy process and rank ordering methods based on different experts opinions. Thereafter, by using simple additive weighting method, the suitability maps for landfill siting in Marvdasht, Iran, was evaluated in environmental, economical and socio-cultural visions. The importance of each group of criteria in its own vision was assigned to be higher than two other groups. In the fourth stage, the final suitability map was obtained after crossing three resulted maps in different visions and reported in five suitability classes for landfill construction. This map indicated that almost 1224 ha of the study area can be considered as best suitable class for landfill siting considering all visions. In the last stage, a comprehensive field visit was performed to verify the selected site obtained from the proposed model. This field inspection has confirmed the proposed integrating approach for the landfill siting. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Resetting the Site: Redirecting Integration of an Insertion Sequence in a Predictable Way

    PubMed Central

    Guynet, Catherine; Achard, Adeline; Hoang, Bao Ton; Barabas, Orsolya; Hickman, Alison Burgess; Dyda, Frederick; Chandler, Michael

    2013-01-01

    Target site choice is a complex and poorly understood aspect of DNA transposition despite its importance in rational transposon-mediated gene delivery. Thoughmost transposons choose target sites essentially randomly or with some slight sequence or structural preferences, insertion sequence IS608 from Helicobacter pylori, which transposes using single-stranded DNA, always inserts just 3′ of a TTAC tetranucleotide. Our results from studies on the IS608 transposition mechanism demonstrated that the transposase recognizes its target site by co-opting an internal segment of transposon DNA and utilizes it for specific recognition of the target sites through base-pairing. This suggested a way to redirect IS608 transposition to novel target sites. As we demonstrate here, we can now direct insertions in a predictable way into a variety of different chosen target sequences, both in vitro and in vivo. PMID:19524540

  4. Model coupling methodology for thermo-hydro-mechanical-chemical numerical simulations in integrated assessment of long-term site behaviour

    NASA Astrophysics Data System (ADS)

    Kempka, Thomas; De Lucia, Marco; Kühn, Michael

    2015-04-01

    The integrated assessment of long-term site behaviour taking into account a high spatial resolution at reservoir scale requires a sophisticated methodology to represent coupled thermal, hydraulic, mechanical and chemical processes of relevance. Our coupling methodology considers the time-dependent occurrence and significance of multi-phase flow processes, mechanical effects and geochemical reactions (Kempka et al., 2014). Hereby, a simplified hydro-chemical coupling procedure was developed (Klein et al., 2013) and validated against fully coupled hydro-chemical simulations (De Lucia et al., 2015). The numerical simulation results elaborated for the pilot site Ketzin demonstrate that mechanical reservoir, caprock and fault integrity are maintained during the time of operation and that after 10,000 years CO2 dissolution is the dominating trapping mechanism and mineralization occurs on the order of 10 % to 25 % with negligible changes to porosity and permeability. De Lucia, M., Kempka, T., Kühn, M. A coupling alternative to reactive transport simulations for long-term prediction of chemical reactions in heterogeneous CO2 storage systems (2014) Geosci Model Dev Discuss 7:6217-6261. doi:10.5194/gmdd-7-6217-2014. Kempka, T., De Lucia, M., Kühn, M. Geomechanical integrity verification and mineral trapping quantification for the Ketzin CO2 storage pilot site by coupled numerical simulations (2014) Energy Procedia 63:3330-3338, doi:10.1016/j.egypro.2014.11.361. Klein E, De Lucia M, Kempka T, Kühn M. Evaluation of longterm mineral trapping at the Ketzin pilot site for CO2 storage: an integrative approach using geo-chemical modelling and reservoir simulation. Int J Greenh Gas Con 2013; 19:720-730. doi:10.1016/j.ijggc.2013.05.014.

  5. Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors.

    PubMed

    Miao, Carol H

    2016-01-01

    Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable

  6. Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids.

    PubMed

    Liu, Xiaomei; Ping, Han; Zhang, Chun

    2014-09-10

    Stable human cell lines have gradually become the preferred system for large scale production of recombinant proteins for clinical applications because of their capacity of proper protein post-translational modification and low immunogenicity. However, human cell line development technologies are commonly based on random genome integration of protein expressing genes. It is required to screen large numbers of cell clones to identify stable high producer cell clones and the cell line development process usually takes 6 to 12 months. Adeno-associated virus type 2 (AAV2) Rep protein is known to induce rAAV DNA integration into a specific site (AAVS1) of the human chromosome 19 and integrated transgenes can stably express proteins. We take advantage of this AAV unique feature to develop a rapid protocol to clone a stable recombinant protein expression human cell line. We have constructed two plasmids. One plasmid, pSVAV2, contains the AAV rep gene for the synthesis of integrase; the second plasmid, pTRP5GFPFVIII-BDD, contains B-domain-deleted factor VIII (FVIII-BDD) and GFP gene flanked by AAV ITRs. Human embryonic kidney (HEK) 293 cells were co-transfected with the two plasmids and the cells were screened by green fluorescence to establish the recombinant FVIII-BDD cell line. PCR analysis showed that the FVIII-BDD gene has been integrated into the AAVS1 site of human chromosome 19. The FVIII-BDD protein secreted into the extracellular media exhibited coagulant activity. We developed a method of rapid establishment of human HEK 293 cell line expressing recombinant FVIII-BDD protein with AAV site-specific integration plasmids.

  7. Digital Reconstruction of AN Archaeological Site Based on the Integration of 3d Data and Historical Sources

    NASA Astrophysics Data System (ADS)

    Guidi, G.; Russo, M.; Angheleddu, D.

    2013-02-01

    The methodology proposed in this paper in based on an integrated approach for creating a 3D digital reconstruction of an archaeological site, using extensively the 3D documentation of the site in its current state, followed by an iterative interaction between archaeologists and digital modelers, leading to a progressive refinement of the reconstructive hypotheses. The starting point of the method is the reality-based model, which, together with ancient drawings and documents, is used for generating the first reconstructive step. Such rough approximation of a possible architectural structure can be annotated through archaeological considerations that has to be confronted with geometrical constraints, producing a reduction of the reconstructive hypotheses to a limited set, each one to be archaeologically evaluated. This refinement loop on the reconstructive choices is iterated until the result become convincing by both points of view, integrating in the best way all the available sources. The proposed method has been verified on the ruins of five temples in the My Son site, a wide archaeological area located in central Vietnam. The integration of 3D surveyed data and historical documentation has allowed to support a digital reconstruction of not existing architectures, developing their three-dimensional digital models step by step, from rough shapes to highly sophisticate virtual prototypes.

  8. TTP AL921102: An integrated geophysics program for non-intrusive characterization of mixed-Waste landfill sites. Final report

    SciTech Connect

    Hasbrouck, J.C.

    1993-09-01

    This Technical Task conducted for the US Department of Energy Office of Technology Development demonstrates the effectiveness of integrating several surface geophysical techniques to nonintrusively characterize mixed-waste landfill sites. An integrated approach enables an area to be characterized faster and cheaper because repeated access is not necessary and offers data and interpretations not attainable by a single technique. Field demonstrations using the complex galvanic resistivity, spontaneous potential (SP), ground-penetrating radar (GPR), time-domain electromagnetic (TDEM), shear-wave (S-wave) seismic and compressional-wave (P-wave) seismic geophysical techniques were conducted at the Mixed-Waste Landfill Integrated Demonstration (MWLID) test site at the Sandia National Laboratories/New Mexico in Albuquerque. Data were acquired in two areas that have both known and unknown attributes. Although data from numerous profiles were analyzed, three lines were chosen as representative of the landfill site: Line 20E that crosses both the known Chromic Acid and Organics Pits, Line 60E that transectes an essentially barren area, and Line 125E located in an area with unknown subsurface conditions.

  9. Application of service examinations to transuranic waste container integrity at the Hanford Site. Revision 1

    SciTech Connect

    Duncan, D.R.; Burbank, D.A. Jr.; Anderson, B.C.; Demiter, J.A.

    1993-09-01

    Transuranic waste containers in retrievable storage trenches at the Hanford Site and their storage environment are described. The containers are of various types, predominantly steel 0.21-m{sup 3} (55-gal) drums and boxes of many different sizes and materials. The storage environment is direct soil burial and aboveground storage under plastic tarps with earth on top of the tarps. Available data from several transuranic waste storage sites are summarized and degradation rates are projected for containers in storage at the Hanford Site.

  10. Lentiviral vector transduction of spermatozoa as a tool for the study of early development

    PubMed Central

    Chandrashekran, Anil; Isa, Ihsan; Dudhia, Jayesh; Thrasher, Adrian J.; Dibb, Nicholas; Casimir, Colin; Readhead, Carol; Winston, Robert

    2014-01-01

    Spermatozoa and lentiviruses are two of nature’s most efficient gene delivery vehicles. Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders. Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies. Lentiviral vectors encoding Green Fluorescent Protein (GFP) were shown to be efficiently processed and expressed in sperm. When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts. This simple technique of directly transducing spermatozoa has potential to be a powerful tool for the study of early and pre-implantation development and could be used as a technique in transgenic development and vertical viral transmission studies. PMID:24918038

  11. Development of Site-Specific Soil Design Basis Earthquake (DBE) Parameters for the Integrated Waste Treatment Unit (IWTU)

    SciTech Connect

    Payne, Suzette

    2008-08-01

    Horizontal and vertical PC 3 (2,500 yr) Soil Design Basis Earthquake (DBE) 5% damped spectra, corresponding time histories, and strain-compatible soil properties were developed for the Integrated Waste Treatment Unit (IWTU). The IWTU is located at the Idaho Nuclear Technology and Engineering Center (INTEC) at the Idaho National Laboratory (INL). Mean and 84th percentile horizontal DBE spectra derived from site-specific site response analyses were evaluated for the IWTU. The horizontal and vertical PC 3 (2,500 yr) Soil DBE 5% damped spectra at the 84th percentile were selected for Soil Structure Interaction (SSI) analyses at IWTU. The site response analyses were performed consistent with applicable Department of Energy (DOE) Standards, recommended guidance of the Nuclear Regulatory Commission (NRC), American Society of Civil Engineers (ASCE) Standards, and recommendations of the Blue Ribbon Panel (BRP) and Defense Nuclear Facilities Safety Board (DNFSB).

  12. Integrated test plan ResonantSonic drilling system technology demonstration-1995, at the Hanford Site: Revision 1

    SciTech Connect

    McLellan, G.W.

    1994-11-17

    This integrated test plan describes the demonstration test of the ResonantSonic drilling system. This demonstration is part of the Office of Technology Development`s Volatile Organic Compound Arid Integrated Demonstration (VOC-Arid ID). Two main purposes of this demonstration are (1) to continue testing the ResonantSonic drilling system compatibility with the Hanford Site waste characterization programs, and (2) to transfer this method for use at the Hanford Site, other government sites, and the private sector. The ResonantSonic method is a dry drilling technique. Field testing of this method began in July 1993. During the next four months, nine holes were drilled, and continuous core samples were retrieved. Penetration rates were 2 to 3 times the baseline, and the operational downtime rate was less than 10%. Successfully demonstrated equipment refinements included a prototype 300 series ResonantSonic head, a new drill rod design for 18-centimeter diameter pipe, and an automated pipe handling system. Various configurations of sampling equipment and drill bits were tested, depending on geologic conditions. The principal objective of the VOC-Arid ID is to determine the viability of emerging technologies that can be used to characterize, remediate, and/or monitor arid or semiarid sites containing VOCs (e.g., carbon tetrachloride) with or without associated metal and radionuclide contamination.

  13. Development of an Integrated Natural Barrier Database System for Site Evaluation of a Deep Geologic Repository in Korea - 13527

    SciTech Connect

    Jung, Haeryong; Lee, Eunyong; Jeong, YiYeong; Lee, Jeong-Hwan

    2013-07-01

    Korea Radioactive-waste Management Corporation (KRMC) established in 2009 has started a new project to collect information on long-term stability of deep geological environments on the Korean Peninsula. The information has been built up in the integrated natural barrier database system available on web (www.deepgeodisposal.kr). The database system also includes socially and economically important information, such as land use, mining area, natural conservation area, population density, and industrial complex, because some of this information is used as exclusionary criteria during the site selection process for a deep geological repository for safe and secure containment and isolation of spent nuclear fuel and other long-lived radioactive waste in Korea. Although the official site selection process has not been started yet in Korea, current integrated natural barrier database system and socio-economic database is believed that the database system will be effectively utilized to narrow down the number of sites where future investigation is most promising in the site selection process for a deep geological repository and to enhance public acceptance by providing readily-available relevant scientific information on deep geological environments in Korea. (authors)

  14. Distribution and integrated assessment of lead in an abandoned lead-acid battery site in Southwest China before redevelopment.

    PubMed

    Wang, Mei; Zhang, Chao; Zhang, Zhuo; Li, Fasheng; Guo, Guanlin

    2016-06-01

    Lead-acid battery sites have contributed enormous amounts of lead to the environment, significantly affecting its global biogeochemical cycle and leaving the potential risks to human health. An abandoned lead-acid battery site prepared for redevelopment was selected in order to study the distribution of lead in soils, plants, rhizosphere soils and soil solutions. In total, 197 samples from 77 boreholes were collected and analyzed. Single extractions by acetic acid (HOAc) were conducted to assess the bioavailability and speciation of lead in soils for comparison with the parts of the plants that are aboveground. Health risks for future residential development were evaluated by the integrated exposure uptake biokinetic (IEUBK) model. The results indicated that lead concentrations in 83% of the soil samples exceeded the Chinese Environmental Quality Standard for soil (350 mg/kg for Pb) and mainly occurred at depths between 0 and 1.5 m while accumulating at the surface of demolished construction waste and miscellaneous fill. Lead concentrations in soil solutions and HOAc extraction leachates were linked closely to the contents of aboveground Broussonetia papyrifera and Artemisia annua, two main types of local plants that were found at the site. The probability density of lead in blood (PbB) in excess of 10 µg/dL could overtake the 99% mark in the residential scenario. The findings provided a relatively integrated method to illustrate the onsite investigations and assessment for similar sites before remediation and future development from more comprehensive aspects.

  15. Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo

    PubMed Central

    Schwarzwaelder, Kerstin; Howe, Steven J.; Schmidt, Manfred; Brugman, Martijn H.; Deichmann, Annette; Glimm, Hanno; Schmidt, Sonja; Prinz, Claudia; Wissler, Manuela; King, Douglas J.S.; Zhang, Fang; Parsley, Kathryn L.; Gilmour, Kimberly C.; Sinclair, Joanna; Bayford, Jinhua; Peraj, Rachel; Pike-Overzet, Karin; Staal, Frank J.T.; de Ridder, Dick; Kinnon, Christine; Abel, Ulrich; Wagemaker, Gerard; Gaspar, H. Bobby; Thrasher, Adrian J.; von Kalle, Christof

    2007-01-01

    We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3+ T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34+ progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34+ progenitors during transduction. In contrast to those in CD34+ cells, RISs recovered from engrafted CD3+ T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation. PMID:17671654

  16. Green Remediation Best Management Practices: Integrating Renewable Energy into Site Cleanup

    EPA Pesticide Factsheets

    The U.S. Environmental Protection Agency (EPA) Principles for Greener Cleanups outline the Agency's policy for evaluating and minimizing the environmental 'footprint' of activities undertaken when cleaning up a contaminated site.

  17. Integration of Thirty Years of Hydrogeological Investigations at the Waste Isolation Pilot Plant Site

    NASA Astrophysics Data System (ADS)

    Beauheim, R. L.; Domski, P. S.; Holt, R. M.; Powers, D. W.

    2008-12-01

    strength CaSO4 waters, thought to represent relatively recent recharge through gypsum karst, to brines contaminated with potash-processing effluent. Geophysical logs from an abundance of oil and gas wells around the WIPP site have been used to map facies boundaries within other Rustler members that can be related to Culebra hydrology. The results of these three decades of study have been integrated into a conceptual model for Culebra hydrology. Some of these studies have been carried out in collaboration with university researchers, and all of the data from these investigations are freely available. *Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy's National Nuclear Security Administration under contract DE-AC04- 94AL85000. This research is funded by WIPP programs administered by the Office of Environmental Management (EM) of the U.S DOE.

  18. Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells.

    PubMed

    Aarbiou, Jamil; Copreni, Elena; Buijs-Offerman, Ruvalic M; van der Wegen, Pascal; Castellani, Stefano; Carbone, Annalucia; Tilesi, Francesca; Fradiani, Piera; Hiemstra, Pieter S; Yueksekdag, Guelnihal; Diana, Anna; Rosenecker, Joseph; Ascenzioni, Fiorentina; Conese, Massimo; Scholte, Bob J

    2012-12-01

    Epithelial sodium channel (ENaC) hyperactivity has been implicated in the pathogenesis of cystic fibrosis (CF) by dysregulation of fluid and electrolytes in the airways. In the present study, we show proof-of-principle for ENaC inhibition by lentiviral-mediated RNA interference. Immortalized normal (H441) and CF mutant (CFBE) airway cells, and differentiated human bronchial epithelial cells in air liquid interface culture (HBEC-ALI) were transduced with a vesicular stomatitis virus G glycoprotein pseudotyped lentiviral (LV) vector expressing a short hairpin RNA (shRNA) targeting the α subunit of ENaC (ENaCα), and a marker gene. Efficacy of ENaCα down-regulation was assayed by the real-time polymerase chain reaction (PCR), membrane potential assay, western blotting, short-circuit currents and fluid absorption. Off-target effects were investigated by a lab-on-a-chip quantitative PCR array. Transduction to near one hundred percentage efficiency of H441, CFBE and HBEC-ALI was achieved by the addition of the LV vector before differentiation and polarization. Transduction resulted in the inhibition of ENaCα mRNA and antigen expression, and a proportional decrease in ENaC-dependent short circuit current and fluid transport. No effect on transepithelial resistance or cAMP-induced secretion responses was observed in HBEC-ALI. The production of interferon α and pro-inflammatory cytokine mRNA, indicating Toll-like receptor 3 or RNA-induced silencing complex mediated off-target effects, was not observed in HBEC-ALI transduced with this vector. We have established a generic method for studying the effect of RNA interference in HBEC-ALI using standard lentiviral vectors. Down-regulation of ENaCα by lentiviral shRNA expression vectors as shown in the absence off-target effects has potential therapeutic value in the treatment of cystic fibrosis. Copyright © 2012 John Wiley & Sons, Ltd.

  19. Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

    PubMed

    Crispo, M; Vilariño, M; dos Santos-Neto, P C; Núñez-Olivera, R; Cuadro, F; Barrera, N; Mulet, A P; Nguyen, T H; Anegón, I; Menchaca, A

    2015-02-01

    Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.

  20. Retroviral Integrations in Gene Therapy Trials

    PubMed Central

    Biasco, Luca; Baricordi, Cristina; Aiuti, Alessandro

    2012-01-01

    γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector–host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient's follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis. PMID:22252453

  1. Functional integrity of the interrenal tissue of yellow perch from contaminated sites tested in vivo

    SciTech Connect

    Girard, C.; Brodeur, J.C.; Hontela, A.

    1995-12-31

    The normal activation of the hypothalamo-pituitary-interrenal axis (HPI axis) in response to capture is disrupted in fish subjected to life-long exposure to heavy metals, PCBs and PAHs. The ability to increase plasma cortisol in yellow perch (Perca flavescens) from sites contaminated by heavy metals and organic compounds, and from a reference site was assessed by the Capture stress test and by the ACTH Challenge test, a new standardized in vivo method designed for field studies. The effects of seasonal factors, such as temperature and gonadal maturity on these tests were investigated. Measures of liver and muscle glycogen and histopathology were made to further characterize the biochemical and structural changes that may occur along with hormonal changes. The Capture stress test showed that an acute source of stress induced a lower cortisol response in fish from the highly contaminated site compared to the reference site, revealing a functional impairment of the HPI axis. The ACTH Challenge test showed that the hormonal responsiveness of the cortisol-secreting interrenal tissue, stimulated by a standard dose of ACTH injected i.p., was lower in fish from the highly contaminated site than the reference site. Spring is the season during which the impairment was the most evident. The possibility of using the reduced capacity of feral fish to respond to a standardized ACTH Challenge as an early bioindicator of toxic stress is discussed.

  2. The gravity of pollination: integrating at-site features into spatial analysis of contemporary pollen movement.

    PubMed

    DiLeo, Michelle F; Siu, Jenna C; Rhodes, Matthew K; López-Villalobos, Adriana; Redwine, Angela; Ksiazek, Kelly; Dyer, Rodney J

    2014-08-01

    Pollen-mediated gene flow is a major driver of spatial genetic structure in plant populations. Both individual plant characteristics and site-specific features of the landscape can modify the perceived attractiveness of plants to their pollinators and thus play an important role in shaping spatial genetic variation. Most studies of landscape-level genetic connectivity in plants have focused on the effects of interindividual distance using spatial and increasingly ecological separation, yet have not incorporated individual plant characteristics or other at-site ecological variables. Using spatially explicit simulations, we first tested the extent to which the inclusion of at-site variables influencing local pollination success improved the statistical characterization of genetic connectivity based upon examination of pollen pool genetic structure. The addition of at-site characteristics provided better models than those that only considered interindividual spatial distance (e.g. IBD). Models parameterized using conditional genetic covariance (e.g. population graphs) also outperformed those assuming panmixia. In a natural population of Cornus florida L. (Cornaceae), we showed that the addition of at-site characteristics (clumping of primary canopy opening above each maternal tree and maternal tree floral output) provided significantly better models describing gene flow than models including only between-site spatial (IBD) and ecological (isolation by resistance) variables. Overall, our results show that including interindividual and local ecological variation greatly aids in characterizing landscape-level measures of contemporary gene flow.

  3. Lentiviral vectors and cardiovascular diseases: a genetic tool for manipulating cardiomyocyte differentiation and function.

    PubMed

    Di Pasquale, E; Latronico, M V G; Jotti, G S; Condorelli, G

    2012-06-01

    Engineered recombinant viral vectors are a powerful tool for vehiculating genetic information into mammalian cells. Because of their ability to infect both dividing and non-dividing cells with high efficiency, lentiviral vectors have gained particular interest for basic research and preclinical studies in the cardiovascular field. We review here the major applications for lentiviral-vector technology in the cardiovascular field: we will discuss their use in trailing gene expression during the induction of differentiation, in protocols for the isolation of cardiac cells and in the tracking of cardiac cells after transplantation in vivo; we will also describe lentivirally-mediated gene delivery uses, such as the induction of a phenotype of interest in a target cell or the treatment of cardiovascular diseases. In addition, a section of the review will be dedicated to reprogramming approaches, focusing attention on the generation of pluripotent stem cells and on transdifferentiation, two emerging strategies for the production of cardiac myocytes from human cells and for the investigation of human diseases. Finally, in order to give a perspective on their future clinical use we will critically discuss advantages and disadvantages of lentivirus-based strategies for the treatment of cardiovascular diseases.

  4. Assessment of selected media supplements to improve F/HN lentiviral vector production yields.

    PubMed

    Gélinas, Jean-François; Davies, Lee A; Gill, Deborah R; Hyde, Stephen C

    2017-08-31

    The development of lentiviral-based therapeutics is challenged by the high cost of current Good Manufacturing Practices (cGMP) production. Lentiviruses are enveloped viruses that capture a portion of the host cell membrane during budding, which then constitutes part of the virus particle. This process might lead to lipid and protein depletion in the cell membrane and affect cell viability. Furthermore, growth in suspension also causes stresses that can affect virus production yields. To assess the impact of these issues, selected supplements (Cholesterol Lipid Concentrate, Chemically Defined Lipid Concentrate, Lipid Mixture 1, Gelatin Peptone N3, N-Acetyl-L-Cysteine and Pluronic F-68) were assayed in order to improve production yields in a transient transfection production of a Sendai virus F/HN-pseudotyped HIV-1-based third generation lentiviral vector in FreeStyle 293 (serum-free media) in suspension. None of the supplements tested had a significant positive impact on lentiviral vector yields, but small non-significant improvements could be combined to increase vector production in a cell line where other conditions have been optimised.

  5. Priming of Hepatocytes Enhances In Vivo Liver Transduction with Lentiviral Vectors in Adult Mice

    PubMed Central

    Pichard, Virginie; Boni, Sébastien; Baron, William; Nguyen, Tuan Huy

    2012-01-01

    Abstract Lentiviral vectors are promising tools for liver disease gene therapy, because they can achieve protracted expression of transgenes in hepatocytes. However, the question as to whether cell division is required for optimal hepatocyte transduction has still not been completely answered. Liver gene-transfer efficiency after in vivo administration of recombinant lentiviral vectors carrying a green fluorescent protein reporter gene under the control of a liver-specific promoter in mice that were either hepatectomized or treated with cholic acid or phenobarbital was compared. Phenobarbital is known as a weak inducer of hepatocyte proliferation, whereas cholic acid has no direct effect on the cell cycle. This study shows that cholic acid is able to prime hepatocytes without mitosis induction. Both phenobarbital and cholic acid significantly increased hepatocyte transduction six- to ninefold, although cholic acid did not modify the mitotic index or cell-cycle entry. However, the effect of either compound was weaker than that observed after partial hepatectomy. In no cases was there a correlation between the expression of cell-cycle marker and transduction efficiency. We conclude that priming of hepatocytes should be considered a clinically applicable strategy to enhance in vivo liver gene therapy with lentiviral vectors. PMID:22428976

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

    PubMed

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

    2017-06-12

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

  7. A Guide to Approaching Regulatory Considerations for Lentiviral-Mediated Gene Therapies.

    PubMed

    White, Michael; Whittaker, Roger; Gándara, Carolina; Stoll, Elizabeth A

    2017-08-01

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

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

    PubMed

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

    2016-04-20

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

  9. Rabies virus envelope glycoprotein targets lentiviral vectors to the axonal retrograde pathway in motor neurons.

    PubMed

    Hislop, James N; Islam, Tarin A; Eleftheriadou, Ioanna; Carpentier, David C J; Trabalza, Antonio; Parkinson, Michael; Schiavo, Giampietro; Mazarakis, Nicholas D

    2014-06-06

    Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.

  10. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells

    PubMed Central

    Yang, Guanghua; Kramer, M. Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P.; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-01-01

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties. PMID:26612671

  11. Biosafety in Ex Vivo Gene Therapy and Conditional Ablation of Lentivirally Transduced Hepatocytes in Nonhuman Primates

    PubMed Central

    Menzel, Olivier; Birraux, Jacques; Wildhaber, Barbara E; Jond, Caty; Lasne, Françoise; Habre, Walid; Trono, Didier; Nguyen, Tuan H; Chardot, Christophe

    2009-01-01

    Ex vivo gene therapy is an interesting alternative to orthotopic liver transplantation (OLT) for treating metabolic liver diseases. In this study, we investigated its efficacy and biosafety in nonhuman primates. Hepatocytes isolated from liver lobectomy were transduced in suspension with a bicistronic liver-specific lentiviral vector and immediately autotransplanted (SLIT) into three cynomolgus monkeys. The vector encoded cynomolgus erythropoietin (EPO) and the conditional suicide gene herpes simplex virus-thymidine kinase (HSV-TK). Survival of transduced hepatocytes and vector dissemination were evaluated by detecting transgene expression and vector DNA. SLIT was safely performed within a day in all three subjects. Serum EPO and hematocrit rapidly increased post-SLIT and their values returned to baseline within about 1 month. Isoforms of EPO detected in monkeys' sera differed from the physiological renal EPO. In liver biopsies at months 8 and 15, we detected EPO protein, vector mRNA and DNA, demonstrating long-term survival and functionality of transplanted lentivirally transduced hepatocytes. Valganciclovir administration resulted in complete ablation of the transduced hepatocytes. We demonstrated the feasibility and biosafety of SLIT, and the long term (>1 year) functionality of lentivirally transduced hepatocytes in nonhuman primates. The HSV-TK/valganciclovir suicide strategy can increase the biosafety of liver gene therapy protocols by safely and completely ablating transduced hepatocytes on demand. PMID:19568222

  12. Lentiviral Vectors for the Engineering of Implantable Cells Secreting Recombinant Antibodies.

    PubMed

    Lathuilière, Aurélien; Schneider, Bernard L

    2016-01-01

    The implantation of genetically modified cells is considered for the chronic delivery of therapeutic recombinant proteins in vivo. In the context of gene therapy, the genetic engineering of cells faces two main challenges. First, it is critical to generate expandable cell sources, which can maintain stable high productivity of the recombinant protein of interest over time, both in culture and after transplantation. In addition, gene transfer techniques need to be developed to engineer cells synthetizing complex polypeptides, such as recombinant monoclonal antibodies, to broaden the range of potential therapeutic applications. Here, we provide a workflow for the use of lentiviral vectors as a flexible tool to generate antibody-producing cells. In particular, lentiviral vectors can be used to genetically engineer the cell types compatible with encapsulation devices protecting the implanted cells from the host immune system. Detailed methods are provided for the design and production of lentiviral vectors, optimization of cell transduction, as well as for the quantification and quality control of the produced recombinant antibody.

  13. Priming of hepatocytes enhances in vivo liver transduction with lentiviral vectors in adult mice.

    PubMed

    Pichard, Virginie; Boni, Sébastien; Baron, William; Nguyen, Tuan Huy; Ferry, Nicolas

    2012-02-01

    Lentiviral vectors are promising tools for liver disease gene therapy, because they can achieve protracted expression of transgenes in hepatocytes. However, the question as to whether cell division is required for optimal hepatocyte transduction has still not been completely answered. Liver gene-transfer efficiency after in vivo administration of recombinant lentiviral vectors carrying a green fluorescent protein reporter gene under the control of a liver-specific promoter in mice that were either hepatectomized or treated with cholic acid or phenobarbital was compared. Phenobarbital is known as a weak inducer of hepatocyte proliferation, whereas cholic acid has no direct effect on the cell cycle. This study shows that cholic acid is able to prime hepatocytes without mitosis induction. Both phenobarbital and cholic acid significantly increased hepatocyte transduction six- to ninefold, although cholic acid did not modify the mitotic index or cell-cycle entry. However, the effect of either compound was weaker than that observed after partial hepatectomy. In no cases was there a correlation between the expression of cell-cycle marker and transduction efficiency. We conclude that priming of hepatocytes should be considered a clinically applicable strategy to enhance in vivo liver gene therapy with lentiviral vectors.

  14. A Guide to Approaching Regulatory Considerations for Lentiviral-Mediated Gene Therapies

    PubMed Central

    White, Michael; Whittaker, Roger; Gándara, Carolina; Stoll, Elizabeth A.

    2017-01-01

    Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is nonpathogenic, and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations and how they are administered in the United Kingdom, although many of the principles will be similar for other regions, including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarizes the extant regulatory guidance for gene therapies, categorized as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy. PMID:28817344

  15. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells.

    PubMed

    Yang, Guanghua; Kramer, M Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-11-27

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

  16. Cytotoxicity associated with artemis overexpression after lentiviral vector-mediated gene transfer.

    PubMed

    Multhaup, Megan; Karlen, Andrea D; Swanson, Debra L; Wilber, Andrew; Somia, Nikunj V; Cowan, Morton J; McIvor, R Scott

    2010-07-01

    Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1alpha promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T(-)B(-) phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A.

  17. Cytotoxicity Associated with Artemis Overexpression After Lentiviral Vector-Mediated Gene Transfer

    PubMed Central

    Multhaup, Megan; Karlen, Andrea D.; Swanson, Debra L.; Wilber, Andrew; Somia, Nikunj V.; Cowan, Morton J.

    2010-01-01

    Abstract Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1α promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T−B− phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A. PMID:20163250

  18. 49 CFR 40.43 - What steps must operators of collection sites take to protect the security and integrity of urine...

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... must operators of collection sites take to protect the security and integrity of urine collections? (a) Collectors and operators of collection sites must take the steps listed in this section to prevent..., cleaning agents, or other possible adulterants are present; (4) Inspect the site to ensure that no...

  19. 49 CFR 40.43 - What steps must operators of collection sites take to protect the security and integrity of urine...

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... must operators of collection sites take to protect the security and integrity of urine collections? (a) Collectors and operators of collection sites must take the steps listed in this section to prevent..., cleaning agents, or other possible adulterants are present; (4) Inspect the site to ensure that no...

  20. A Ubiquitous Chromatin Opening Element (UCOE) Confers Resistance to DNA Methylation–mediated Silencing of Lentiviral Vectors

    PubMed Central

    Zhang, Fang; Frost, Amy R; Blundell, Mike P; Bales, Olivia; Antoniou, Michael N; Thrasher, Adrian J

    2010-01-01

    DNA methylation may restrict the activity of gene transfer vectors due to inadvertent silencing. In P19 embryonic carcinoma cells in vitro, we found that transgene expression regulated by the SFFV LTR and EF1α promoter declined rapidly within 16 days, but for A2UCOE derived from the human HNRPA2B1-CBX3 housekeeping gene locus, remained completely stable. Silencing correlated with extensive epigenetic methylation of CpG sites, whereas the A2UCOE was almost completely resistant. Linking of the A2UCOE upstream of the SFFV LTR protected this element from both DNA methylation and silencing. Analysis of engrafted hematopoietic cells in vivo transduced with the same vectors revealed a similar pattern. The A2UCOE displayed little or no methylation in either primary or secondary graft recipients, and gene expression profiles were highly conserved between the two groups. These studies provide convincing evidence that DNA methylation plays a direct role in regulating self-inactivating (SIN) lentiviral transgene expression, and that the stability of expression from the A2UCOE is, at least in part, due to methylation resistance. The A2UCOE therefore has considerable utility for gene therapy applications where reliable and sustained gene expression is desirable. PMID:20588258

  1. Hybrid HIV/MSCV LTR enhances transgene expression of lentiviral vectors in human CD34(+) hematopoietic cells.

    PubMed

    Choi, J K; Hoang, N; Vilardi, A M; Conrad, P; Emerson, S G; Gewirtz, A M

    2001-01-01

    HIV-based lentiviral vectors can transduce nondividing cells, an important advantage over murine leukemia virus (MLV)-based vectors when transducing slowly dividing hematopoietic stem cells. However, we find that in human CD34(+) hematopoietic cells, the HIV-based vectors with an internal cytomegalovirus (CMV) promoter express transgenes 100- to 1,000-fold less than the MLV-based retroviral vector murine stem cell virus (MSCV). To increase the expression of the integrated lentivirus, we replaced CMV promoter with that of the Rous sarcoma virus or MSCV and obtained a modest augmentation in expression. A more dramatic effect was seen when the CMV enhancer/promoter was removed and the HIV long-terminal repeat (LTR) was replaced by a novel HIV/MSCV hybrid LTR. This vector retains the ability to transduce nondividing cells but now expresses its transgene (enhanced green fluorescent protein) 10- to 100-fold greater than the original HIV-based vector. When compared under identical conditions, the HIV vector with the hybrid LTR transduced a higher percentage of CD34(+) cells than the MSCV-based retroviral vector (19.4% versus 2.4%). The number of transduced cells and level of transgene expression remain constant over 5-8 weeks as determined by long-term culture-initiating cells, fluoresence-activated cell sorting, and nonobese diabetic/severe combined immunodeficiency repopulation assay.

  2. A simple and effective method to generate lentiviral vectors for ex vivo gene delivery to mature human peripheral blood lymphocytes.

    PubMed

    Yang, Shicheng; Karne, Neel K; Goff, Stephanie L; Black, Mary A; Xu, Hui; Bischof, Daniela; Cornetta, Kenneth; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

    2012-04-01

    Human ex vivo gene therapy protocols have been used successfully to treat a variety of genetic disorders, infectious diseases, and cancer. Murine oncoretroviruses (specifically, gammaretroviruses) have served as the primary gene delivery vehicles for these trials. However, in some cases, such vectors have been associated with insertional mutagenesis. As a result, alternative vector platforms such as lentiviral vectors (LVVs) are being developed. LVVs may provide advantages compared with gammaretroviral vectors, including the ability to transduce large numbers of nondividing cells, resistance to gene silencing, and a potentially safer integration profile. The aim of this study was to develop a simplified process for the rapid production of clinical-grade LVVs. To that end, we used a self-inactivating bicistronic LVV encoding an MART (melanoma antigen recognized by T cells)-1-reactive T cell receptor containing oPRE, an optimized and truncated version of woodchuck hepatitis virus posttranslational regulatory element (wPRE). Using our simplified clinical production process, 293T cells were transiently transfected in roller bottles. The LVV supernatant was collected, treated with Benzonase, and clarified by modified step filtration. LVV produced in this manner exhibited titers and a biosafety profile similar to those of cGMP (current Good Manufacturing Practices) LVVs previously manufactured at the Indiana University Vector Production Facility in support of a phase I/II clinical trial. We describe a simple, efficient, and low-cost method for the production of clinical-grade LVV for ex vivo gene therapy protocols.

  3. The Fetal Mouse Is a Sensitive Genotoxicity Model That Exposes Lentiviral-associated Mutagenesis Resulting in Liver Oncogenesis

    PubMed Central

    Nowrouzi, Ali; Cheung, Wing T; Li, Tingting; Zhang, Xuegong; Arens, Anne; Paruzynski, Anna; Waddington, Simon N; Osejindu, Emma; Reja, Safia; von Kalle, Christof; Wang, Yoahe; Al-Allaf, Faisal; Gregory, Lisa; Themis, Matthew; Holder, Maxine; Dighe, Niraja; Ruthe, Alaine; Buckley, Suzanne MK; Bigger, Brian; Montini, Eugenio; Thrasher, Adrian J; Andrews, Robert; Roberts, Terry P; Newbold, Robert F; Coutelle, Charles; Schmidt, Manfred; Themis, Mike

    2013-01-01

    Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis. PMID:23299800

  4. The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis.

    PubMed

    Nowrouzi, Ali; Cheung, Wing T; Li, Tingting; Zhang, Xuegong; Arens, Anne; Paruzynski, Anna; Waddington, Simon N; Osejindu, Emma; Reja, Safia; von Kalle, Christof; Wang, Yoahe; Al-Allaf, Faisal; Gregory, Lisa; Themis, Matthew; Holder, Maxine; Dighe, Niraja; Ruthe, Alaine; Buckley, Suzanne Mk; Bigger, Brian; Montini, Eugenio; Thrasher, Adrian J; Andrews, Robert; Roberts, Terry P; Newbold, Robert F; Coutelle, Charles; Schmidt, Manfred; Themis, Mike

    2013-02-01

    Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.

  5. [Distribution in different Salmonella serovars and integration sites of Salmonella paratyphi C phage SPC-P1].

    PubMed

    Zhao, Pan; Peng, Yi-hong; Zou, Qing-hua

    2015-12-18

    To determine the prevalence of Salmonella paratyphi C phage (SPC-P1) in different Salmonella serovars and to identify the integration sites in host genome. Based on the complete genome of SPC-P1 in S. paratyphi C RKS4594, 6 pairs of primers were designed and used to amplify the fragments of SPC-P1 in 11 S. typhi, 11 S. paratyphi A, 12 S. paratyphi B and 23 S. paratyphi C strains. At the same time, 100 complete genomes of Salmonella including 20 serovars available in National Center for Biotechnology Information (NCBI) database were downloaded and aligned by Mauve 2.3.1 to determine the prevalence of SPC-P1 in these serovars. Primers were designed according to the integration sites of SPC-P1 in the genome of RKS4594, and used to amplify ten strains having SPC-P1 in the genome. The PCR products were sequenced to investigate the integration sites of SPC-P1. SPC-P1 was widely distributed in S.paratyphi C genome. In the study, 14 strains had all 6 fragments and 2 strains had 3-5 fragments. All the amplified fragments showed expected sizes. In contrast, in the genomes of S. typhi, S. paratyphi A and S. paratyphi B, no or only 1-2 fragments could be amplified, and the sizes were smaller than expected. The results from Mauve showed that only in the genome of S.choleraesuis, which was a close relative of S. paratyphi C, there existed an almost complete genome of SPC-P1. The insertion site of SPC-P1 in all the ten S. paratyphi C strains tested was between pgtE and yfdC genes. SPC-P1 is a unique virulence factor of S. paratyphi C. It may play roles in the host range and pathogenicity of S.paratyphi C.

  6. An integration of aeromagnetic and electrical resistivity methods in dam site investigation

    SciTech Connect

    Aina, A.; Olorunfemi, M.O.; Ojo, J.S.

    1996-03-01

    Aeromagnetic map and electrical resistivity sounding data obtained along eight traverses were examined at two sites across the Katsina-Ala River. The principal goals of this exercise were to define depths to the bedrock, bedrock relief, geologic structures, define the nature of the superficial deposit, and select probable minor and major axes for hydroelectric power dams. The aeromagnetic map shows that the basement rocks trend roughly northeast-southwest, which correlates with the strike of foliation measurements made on rock outcrops along the river channel. A network of cross cutting lineaments, suspected to be faults/fractures that trend approximately northeast/southwest and northwest/southeast, was also delineated from the magnetic map. The depths to the bedrock estimated from resistivity depth sounding data at site 1 generally vary from 1--53.1 m. Depths to the bedrock estimated at site 2 range from 1.9--19.5 m. The superficial deposit varies from clay to sandy clay, to clayey sand (with boulders in places), and to sand and laterite. The bedrock relief is relatively flat and gently undulates along most of the traverses, with an overall dip towards the river channel. Traverses E-F or I-J at site 1 and K-L at site 2 are probable dame axes. These traverses are characterized by relatively thin overburden thicknesses and rock heads dipping toward the river channel, thereby reducing the likelihood of water seepages from the flanks of the proposed dam axes.

  7. Identifying 2'-O-methylationation sites by integrating nucleotide chemical properties and nucleotide compositions.

    PubMed

    Chen, Wei; Feng, Pengmian; Tang, Hua; Ding, Hui; Lin, Hao

    2016-06-01

    2'-O-methylationation is an important post-transcriptional modification and plays important roles in many biological processes. Although experimental technologies have been proposed to detect 2'-O-methylationation sites, they are cost-ineffective. As complements to experimental techniques, computational methods will facilitate the identification of 2'-O-methylationation sites. In the present study, we proposed a support vector machine-based method to identify 2'-O-methylationation sites. In this method, RNA sequences were formulated by nucleotide chemical properties and nucleotide compositions. In the jackknife cross-validation test, the proposed method obtained an accuracy of 95.58% for identifying 2'-O-methylationation sites in the human genome. Moreover, the model was also validated by identifying 2'-O-methylation sites in the Mus musculus and Saccharomyces cerevisiae genomes, and the obtained accuracies are also satisfactory. These results indicate that the proposed method will become a useful tool for the research on 2'-O-methylation.

  8. A slingram survey on the Nevada Test Site: part of an integrated geologic geophysical study of site evaluation for nuclear waste disposal

    USGS Publications Warehouse

    Flanigan, Vincent J.

    1979-01-01

    A slingram geophysical survey was made in early 1978 as part of the integrated geologlcal-geophysical study aimed at evaluating the Eleana Formation as a possible repository for nuclear waste. The slingram data were taken over an alluvial fan and pediments along the eastern flank of Syncline Ridge about 45 km north of Mercury, Nevada, on the Nevada Test Site. The data show that the more conductive argillaceous Eleana Formation varies in depth from 40 to 85 m from west to east along traverse lines. Northeast-trending linear anomalies suggest rather abrupt changes in subsurface geology that may be associated with faults and fractures. The results of the slingram survey will, when interpreted in the light of other geologic and geophysical evidence, assist in understanding the shallow parts of the geologic setting of the Eleana Formation.

  9. Multi-Criteria Decision Analysis integrated with GIS and remote sensing for astronomical observatory site selection in Antalya province, Turkey

    NASA Astrophysics Data System (ADS)

    Koc-San, D.; San, B. T.; Bakis, V.; Helvaci, M.; Eker, Z.

    2013-07-01

    Astronomical observatory site selection is a complex problem that involves evaluation of multiple factors from different sources. The aim of this study is to select the best possible candidates for astronomical observations sites using Multi-Criteria Decision Analysis integrated with Geographical Information Systems and remote sensing technologies. The study was implemented in the Antalya province of Turkey, which is convenient for astronomical site observatory facilities with its appropriate climate properties and weather conditions. Eleven factors (cloud cover, precipitable water, earthquake zones, geology, landslide inventory, active fault lines, Digital Elevation Model, city lights, mining activities, settlement areas, roads) were determined, splitting into three categories; meteorological, geographical and anthropogenic criteria. These factors were evaluated using Analytical Hierarchy Process method and the weights of criteria layers were determined. As a result, the most suitable areas were located extensively in western and eastern part of Antalya. This study offers a robust, accurate, cost and time effective procedure for preliminary site selection for astronomical observatory. However, for a final decision of the best location of astronomical observatory, site testing measurements and atmospheric seeing observations will be further required in these preliminary areas.

  10. Integrating knowledge-based multi-criteria evaluation techniques with GIS for landfill site selection: A case study using AHP

    NASA Astrophysics Data System (ADS)

    Fagbohun, B. J.; Aladejana, O. O.

    2016-09-01

    A major challenge in most growing urban areas of developing countries, without a pre-existing land use plan is the sustainable and efficient management of solid wastes. Siting a landfill is a complicated task because of several environmental regulations. This challenge gives birth to the need to develop efficient strategies for the selection of proper waste disposal sites in accordance with all existing environmental regulations. This paper presents a knowledge-based multi-crite