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Sample records for lentiviral integration sites

  1. 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

  2. Analysis of Lentiviral Vector Integration in HIV+ Study Subjects Receiving Autologous Infusions of Gene Modified CD4+ T Cells

    PubMed Central

    Wang, Gary P; Levine, Bruce L; Binder, Gwendolyn K; Berry, Charles C; Malani, Nirav; McGarrity, Gary; Tebas, Pablo; June, Carl H; Bushman, Frederic D

    2009-01-01

    Lentiviral vector-based gene therapy has been used to target the human immunodeficiency virus (HIV) using an antisense env payload. We have analyzed lentiviral-vector integration sites from three treated individuals. We compared integration sites from the ex vivo vector-transduced CD4+ cell products to sites from cells recovered at several times after infusion. Integration sites were analyzed using 454 pyrosequencing, yielding a total of 7,782 unique integration sites from the ex vivo product and 237 unique sites from cells recovered after infusion. Integrated vector copies in both data sets were found to be strongly enriched within active genes and near epigenetic marks associated with active transcription units. Analysis of integration relative to nucleosome structure on target DNA indicated favoring of integration in outward facing DNA major grooves on the nucleosome surface. There was no indication that growth of transduced cells after infusion resulted in enrichment for integration sites near proto-oncogene 5′-ends or within tumor suppressor genes. Thus, this first look at the longitudinal evolution of cells transduced with a lentiviral vector after infusion of gene modified CD4+ cells provided no evidence for abnormal expansions of cells due to vector-mediated insertional activation of proto-oncogenes. PMID:19259065

  3. Altering Entry Site Preference of Lentiviral Vectors into Neuronal Cells by Pseudotyping with Envelope Glycoproteins.

    PubMed

    Kobayashi, Kenta; Kato, Shigeki; Inoue, Ken-Ichi; Takada, Masahiko; Kobayashi, Kazuto

    2016-01-01

    A lentiviral vector system provides a powerful strategy for gene therapy trials against a variety of neurological and neurodegenerative disorders. Pseudotyping of lentiviral vectors with different envelope glycoproteins not only confers the neurotropism to the vectors, but also alters the preference of sites of vector entry into neuronal cells. One major group of lentiviral vectors is a pseudotype with vesicular stomatitis virus glycoprotein (VSV-G) that enters preferentially cell body areas (somata/dendrites) of neurons and transduces them. Another group contains lentiviral vectors pseudotyped with fusion envelope glycoproteins composed of different sets of rabies virus glycoprotein and VSV-G segments that enter predominantly axon terminals of neurons and are transported through axons retrogradely to their cell bodies, resulting in enhanced retrograde gene transfer. This retrograde gene transfer takes a considerable advantage of delivering the transgene into neuronal cell bodies situated in regions distant from the injection site of the vectors. The rational use of these two vector groups characterized by different entry mechanisms will further extend the strategy for gene therapy of neurological and neurodegenerative disorders. PMID:26611586

  4. 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. PMID:27317182

  5. Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations

    PubMed Central

    Cesana, Daniela; Sgualdino, Jacopo; Rudilosso, Laura; Merella, Stefania; Naldini, Luigi; Montini, Eugenio

    2012-01-01

    Gamma-retroviral/lentiviral vectors (γRV/LV) with self-inactivating (SIN) long terminal repeats (LTRs) and internal moderate cellular promoters pose a reduced risk of insertional mutagenesis when compared with vectors with active LTRs. Yet, in a recent LV-based clinical trial for β-thalassemia, vector integration within the HMGA2 gene induced the formation of an aberrantly spliced mRNA form that appeared to cause clonal dominance. Using a method that we developed, cDNA linear amplification-mediated PCR, in combination with high-throughput sequencing, we conducted a whole transcriptome analysis of chimeric LV-cellular fusion transcripts in transduced human lymphoblastoid cells and primary hematopoietic stem/progenitor cells. We observed a surprising abundance of read-through transcription originating outside and inside the provirus and identified the vector sequences contributing to the aberrant splicing process. We found that SIN LV has a sharply reduced propensity to engage in aberrant splicing compared with that of vectors carrying active LTRs. Moreover, by recoding the identified vector splice sites, we reduced residual read-through transcription and demonstrated an effective strategy for improving vectors. Characterization of the mechanisms and genetic features underlying vector-induced aberrant splicing will enable the generation of safer vectors, with low impact on the cellular transcriptome. PMID:22523064

  6. Virological and Preclinical Characterization of a Dendritic Cell Targeting, Integration-deficient Lentiviral Vector for Cancer Immunotherapy

    PubMed Central

    Odegard, Jared M.; Kelley-Clarke, Brenna; Tareen, Semih U.; Campbell, David J.; Flynn, Patrick A.; Nicolai, Christopher J.; Slough, Megan M.; Vin, Chintan D.; McGowan, Patrick J.; Nelson, Lisa T.; Dubensky, Thomas W.; Robbins, Scott H.

    2015-01-01

    Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and therefore attractive targets for cancer immunotherapy. We have developed an integration-deficient lentiviral vector termed ID-VP02 that is designed to deliver antigen-encoding nucleic acids selectively to human DCs in vivo. ID-VP02 utilizes a genetically and glycobiologically engineered Sindbis virus glycoprotein to target human DCs through the C-type lectin DC-SIGN (CD209) and also binds to the homologue murine receptor SIGNR1. Specificity of ID-VP02 for antigen-presenting cells in the mouse was confirmed through biodistribution studies showing that following subcutaneous administration, transgene expression was only detectable at the injection site and the draining lymph node. A single immunization with ID-VP02 induced a high level of antigen-specific, polyfunctional effector and memory CD8 T-cell responses that fully protected against vaccinia virus challenge. Upon homologous readministration, ID-VP02 induced a level of high-quality secondary effector and memory cells characterized by stable polyfunctionality and expression of IL-7Rα. Importantly, a single injection of ID-VP02 also induced robust cytotoxic responses against an endogenous rejection antigen of CT26 colon carcinoma cells and conferred both prophylactic and therapeutic antitumor efficacy. ID-VP02 is the first lentiviral vector which combines integration deficiency with DC targeting and is currently being investigated in a phase I trial in cancer patients. PMID:25658613

  7. Vaccines delivered by integration-deficient lentiviral vectors targeting dendritic cells induces strong antigen-specific immunity

    PubMed Central

    Hu, Biliang; Dai, Bingbing; Wang, Pin

    2010-01-01

    We report a study of an integration-deficient lentiviral vector (IDLV) enveloped with a Sindbis virus glycoprotein mutant (SVGmu) capable of selectively binding to dendritic cells (DCs) for its potential as a vaccine carrier. The in vitro assays showed that the D64V point mutation in the catalytic domain of HIV-1 integrase efficiently inhibited the integration of the transgene upon vector transduction, while the targeting specificity of the vector to preferentially transduce and mediate durable expression in DCs was maintained. Substantial immune responses in C57BL/6 mice and complete protection against a challenge with the C57BL/6 thymoma EG.7 tumor expressing a delivered ovalbumin (OVA) antigen in mice have been achieved through the direct injection of the DC-directed IDLV encoding OVA. Thus, this DC-directed IDLV system represents a promising and efficient vector platform with remarkably improved safety for the future development of DC-based immunotherapy. PMID:20709004

  8. 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

  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. 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. PMID:27626061

  11. 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

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

    PubMed

    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

  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. Strategies for targeting lentiviral vectors.

    PubMed

    Frecha, Cecilia; Szécsi, Judit; Cosset, Francois-Loîc; Verhoeyen, Els

    2008-12-01

    Vectors derived from retroviruses such as lentiviruses and onco-retroviruses are probably among the most suitable tools to achieve a long-term gene transfer since they allow stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors should be preferred gene delivery vehicles over vectors derived from onco-retroviruses (MLV) since in contrast to the latter they can transduce non-proliferating target cells. Moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer approaches in vivo. Here we provide an overview of innovative approaches to upgrade lentiviral vectors for tissue or cell targeting and which have potential for in vivo gene delivery. In this overview we distinguish between three types of lentiviral vector targeting strategies (Fig 1): 1) targeting of vectors at the level of vector-cell entry through lentiviral vector surface modifications; 2) targeting at the level of transgene transcription by insertion of tissue specific promoters into lentiviral vectors; 3) a novel microRNA technology that rather than targeting the 'right' cells will 'detarget' transgene expression from non-target cells while achieving high expression in the target-cell. It is clear that each strategy is of enormous value for several gene therapy approaches but combining these three layers of transgene expression control will offer tools to really overcome several drawbacks in the field such as side-effect of off-target expression, clearance of transgene modified cells by immune response to the transgene and lack of biosecurity and efficiency in in vivo approaches. PMID:19075628

  15. Insulin producing cells established using non-integrated lentiviral vector harboring PDX1 gene

    PubMed Central

    Boroujeni, Zahra Niki; Aleyasin, Ahmad

    2013-01-01

    AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene. METHODS: In this study, human adipose tissue derived stem cells (hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis. Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1 (Non-integrated LV-PDX1) were constructed using specific plasmids (pLV-HELP, pMD2G, LV-105-PDX1-1). Then, hADSCs were transduced with non-integrated LV-PDX1. After transduction, ADSCsPDX1+ were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 and insulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3 (Ngn3), glucagon, glucose transporter2 (Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+ in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+ were implanted into hyperglycemic rats. RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture. Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1 became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis. Significant expressions of PDX1, Ngn3, glucagon, Glut2 and

  16. 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. PMID:25342980

  17. 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. PMID:14978753

  18. Integration-deficient Lentiviral Vectors Expressing Codon-optimized R338L Human FIX Restore Normal Hemostasis in Hemophilia B Mice

    PubMed Central

    Suwanmanee, Thipparat; Hu, Genlin; Gui, Tong; Bartholomae, Cynthia C; Kutschera, Ina; von Kalle, Christof; Schmidt, Manfred; Monahan, Paul E; Kafri, Tal

    2014-01-01

    Integration-deficient lentiviral vectors (IDLVs) have been shown to transduce a wide spectrum of target cells and organs in vitro and in vivo and to maintain long-term transgene expression in nondividing cells. However, epigenetic silencing of episomal vector genomes reduces IDLV transgene expression levels and renders these safe vectors less efficient. In this article, we describe for the first time a complete correction of factor IX (FIX) deficiency in hemophilia B mice by IDLVs carrying a novel, highly potent human FIX cDNA. A 50-fold increase in human FIX cDNA potency was achieved by combining two mechanistically independent yet synergistic strategies: (i) optimization of the human FIX cDNA codon usage to increase human FIX protein production per vector genome and (ii) generation of a highly catalytic mutant human FIX protein in which the arginine residue at position 338 was substituted with leucine. The enhanced human FIX activity was not associated with liver damage or with the formation of human FIX-directed inhibitory antibodies and rendered IDLV-treated FIX-knockout mice resistant to a challenging tail-clipping assay. A novel S1 nuclease-based B1-quantitative polymerase chain reaction assay showed low levels of IDLV integration in mouse liver. Overall, this study demonstrates that IDLVs carrying an improved human FIX cDNA safely and efficiently cure hemophilia B in a mouse model. PMID:23941813

  19. Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors

    PubMed Central

    Trobridge, Grant D.; Beard, Brian C.; Gooch, Christina; Wohlfahrt, Martin; Olsen, Philip; Fletcher, James; Malik, Punam

    2008-01-01

    Lentiviral vectors are attractive for hematopoietic stem cell (HSC) gene therapy because they do not require mitosis for nuclear entry, they efficiently transduce hematopoietic repopulating cells, and self-inactivating (SIN) designs can be produced at high titer. Experiments to evaluate HIV-derived lentiviral vectors in nonhuman primates prior to clinical trials have been hampered by low transduction frequencies due in part to host restriction by TRIM5α. We have established conditions for efficient transduction of pigtailed macaque (Macaca nemestrina) long-term repopulating cells using VSV-G–pseudotyped HIV-based lentiviral vectors. Stable, long-term, high-level gene marking was observed in 3 macaques using relatively low MOIs (5-10) in a 48-hour ex vivo transduction protocol. All animals studied had rapid neutrophil engraftment with a median of 10.3 days to a count greater than 0.5 × 109/L (500/μL). Expression was detected in all lineages, with long-term marking levels in granulocytes at approximately 20% to 30%, and in lymphocytes at approximately 12% to 23%. All animals had polyclonal engraftment as determined by analysis of vector integration sites. These data suggest that lentiviral vectors should be highly effective for HSC gene therapy, particularly for diseases in which maintaining the engraftment potential of stem cells using short-term ex vivo transduction protocols is critical. PMID:18388180

  20. 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.

  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. PMID:23840834

  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. 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. PMID:26202078

  4. 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

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

    PubMed

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

    2015-01-01

    Fanconi anemia is a DNA repair-deficiency syndrome mainly characterized by cancer predisposition and bone marrow failure. Trying to restore the hematopoietic function in these patients, lentiviral vector-mediated gene therapy trials have recently been proposed. However, because no insertional oncogenesis studies have been conducted so far in DNA repair-deficiency syndromes such as Fanconi anemia, we have carried out a genome-wide screening of lentiviral insertion sites after the gene correction of Fanca(-/-) hematopoietic stem cells (HSCs), using LAM-PCR and 454-pyrosequencing. Our studies first demonstrated that transduction of Fanca(-/-) HSCs with a lentiviral vector designed for clinical application efficiently corrects the phenotype of Fanconi anemia repopulating cells without any sign of toxicity. The identification of more than 6,500 insertion sites in primary and secondary recipients showed a polyclonal pattern of reconstitution, as well as a continuous turnover of corrected Fanca(-/-) HSC clones, without evidences of selection towards specific common integration sites. Taken together our data show, for the first time in a DNA repair-deficiency syndrome, that lentiviral vector-mediated gene therapy efficiently corrects the phenotype of affected HSCs and promotes a healthy pattern of clonal turnover in vivo. These studies will have a particular impact in the development of new gene therapy trials in patients affected by DNA repair syndromes, particularly in Fanconi anemia. PMID:26415575

  6. Quantitative analysis of lentiviral transgene expression in mice over seven generations.

    PubMed

    Wang, Yong; Song, Yong-tao; Liu, Qin; Liu, Cang'e; Wang, Lu-lu; Liu, Yu; Zhou, Xiao-yang; Wu, Jun; Wei, Hong

    2010-10-01

    Lentiviral transgenesis is now recognized as an extremely efficient and cost-effective method to produce transgenic animals. Transgenes delivered by lentiviral vectors exhibited inheritable expression in many species including those which are refractory to genetic modification such as non-human primates. However, epigenetic modification was frequently observed in lentiviral integrants, and transgene expression found to be inversely correlated with methylation density. Recent data showed that about one-third lentiviral integrants exhibited hypermethylation and low expression, but did not demonstrate whether those integrants with high expression could remain constant expression and hypomethylated during long term germline transmission. In this study, using lentiviral eGFP transgenic mice as the experimental animals, lentiviral eGFP expression levels and its integrant numbers in genome were quantitatively analyzed by fluorescent quantitative polymerase-chain reaction (FQ-PCR), using the house-keeping gene ribosomal protein S18 (Rps18) and the single copy gene fatty acid binding protein of the intestine (Fabpi) as the internal controls respectively. The methylation densities of the integrants were quantitatively analyzed by bisulfite sequencing. We found that the lentiviral integrants with high expression exhibited a relative constant expression level per integrant over at least seven generations. Besides, the individuals containing these integrants exhibited eGFP expression levels which were positively and almost linearly correlated with the integrant numbers in their genomes, suggesting that no remarkable position effect on transgene expression of the integrants analyzed was observed. In addition, over seven generations the methylation density of these integrants did not increase, but rather decreased remarkably, indicating that these high expressing integrants were not subjected to de novo methylation during at least seven generations of germline transmission. Taken

  7. Lentiviral Vectors for Immune Cells Targeting

    PubMed Central

    Froelich, Steven; Tai, April; Wang, Pin

    2009-01-01

    Lentiviral vectors are efficient gene delivery vehicles suitable for delivering long-term transgene expression in various cell types. Engineering lentiviral vectors to have the capacity to transduce specific cell types is of great interest to advance the translation of lentiviral vectors towards the clinic. Here we provide an overview of innovative approaches to target lentiviral vectors to cells of the immune system. In this overview we distinguish between two types of lentiviral vector targeting strategies: 1) targeting of the vectors to specific cells by lentiviral vector surface modifications, and 2) targeting at the level of transgene transcription by insertion of tissue-specific promoters to drive transgene expression. It is clear that each strategy is of enormous value but ultimately combining these approaches may help reduce the effects of off-target expression and improve the efficiency and saftey of lentiviral vectors for gene therapy. PMID:20085508

  8. 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.

  9. The BET family of proteins targets Moloney Murine Leukemia Virus integration near transcription start sites

    PubMed Central

    De Rijck, Jan; de Kogel, Christine; Demeulemeester, Jonas; Vets, Sofie; Ashkar, Sara El; Malani, Nirav; Bushman, Frederic D; Landuyt, Bart; Husson, Steven J.; Busschots, Katrien; Gijsbers, Rik; Debyser, Zeger

    2014-01-01

    Summary A hallmark of retroviral replication is integration of the viral genome in the host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to Murine Leukemia Virus (MLV)-derived vector integration, hamper their application. Here we show that bromodomain and extraterminal (BET) proteins (BRD2, BRD3 and BRD4) and MLV integrase specifically interact and co-localize within the nucleus of the cell. Inhibition of the BET proteins chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75, retargets MLV integration away from TSS and into the body of actively transcribed genes, conform to the Human Immunodeficiency Virus (HIV) integration pattern. Together these data validate BET proteins as MLV integration targeting factors. PMID:24183673

  10. 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

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

    PubMed

    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. PMID:27278774

  12. 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

  13. Launch site integration for mixed fleet operations

    NASA Technical Reports Server (NTRS)

    Scott, L. P.

    1990-01-01

    Launch site impacts and integration planning issues are presented to support launch operations for a mixed vehicle fleet (manned and cargo). Proposed ground systems and launch site configurations are described. Prelaunch processing scenarios and schedules are developed for candidate launch vehicles. Earth-to-orbit (ETO) vehicle architectures are presented to meet future launch requirements, including the Space Exploration Initiative (SEI). Flight vehicle design recommendations to enhance launch processing are discussed. The significance of operational designs for future launch vehicles is shown to be a critical factor in planning for mixed fleet launch site operations.

  14. 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.

  15. 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. PMID:23845948

  16. 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. PMID:25804479

  17. 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

  18. Integrase-Deficient Lentiviral Vectors Mediate Efficient Gene Transfer to Human Vascular Smooth Muscle Cells with Minimal Genotoxic Risk

    PubMed Central

    Chick, Helen E.; Nowrouzi, Ali; Fronza, Raffaele; McDonald, Robert A.; Kane, Nicole M.; Alba, Raul; Delles, Christian; Sessa, William C.; Schmidt, Manfred; Thrasher, Adrian J.

    2012-01-01

    Abstract We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer. PMID:22931362

  19. Integration Site of Noninducible Coliphage 186

    PubMed Central

    Woods, Walter H.; Egan, J. Barry

    1972-01-01

    From conjugational data, the attachment site for noninducible coliphage 186 (att186) was located between the origins of Hfr strains KL16 and KL98, and close to the pheA gene in Escherichia coli K-12. P1 transductions indicated that att186 lies at 51 min on the standard genetic map of E. coli, with the order cysC-nalB-att186-pheA. The presence of prophage 186 in the donor destroyed linkage between nalB and pheA, which is taken as evidence for the integration of the 186 prophage between these genes. PMID:4559723

  20. 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

  1. 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. PMID:27228988

  2. Plasmid-based shRNA lentiviral particle production for RNAi applications

    PubMed Central

    Shum, David; Djaballah, Hakim

    2014-01-01

    Lentiviral vectors have become mainstream gene transfer vehicles for their ability to delivery and integrate into host cells. In RNA interference (RNAi) applications, lentiviral constructs constitutively express dsRNA molecules usually as short hairpin RNA (shRNA) enabling long-term gene silencing and when pseudotyped with a broad host glycoprotein envelope; allows a multitude of cell types to be transduced. Their successful use ultimately relies on the production of lentiviral particles in high-titer and uniformity. Typical methods require the transfection of three or more plasmids in which essential viral elements have been encoded separated so as to remain replication deficient. These transfection procedures are of critical importance; however, methods often vary among laboratories making it difficult to assess the overall efficiency of lentiviral particle production. In this report, we focused exclusively on this step and compared the overall impact of the commercial transfection reagent FuGENE 6 to FuGENE HD. We found that FuGENE HD resulted in at least 5-fold improvement in viral particle titer as assessed by the p24 standard ELISA assay. We present the complete optimized workflow and demonstrate this utility in which a single modification of this transfection step improved the lentiviral particle production. PMID:24939963

  3. A Large U3 Deletion Causes Increased In Vivo Expression from a Nonintegrating Lentiviral Vector

    PubMed Central

    Bayer, Matthew; Kantor, Boris; Cockrell, Adam; Ma, Hong; Zeithaml, Brian; Li, Xiangping; McCown, Thomas; Kafri, Tal

    2008-01-01

    The feasibility of employing nonintegrating lentiviral vectors has been demonstrated by recent studies showing the ability of nonintegrating lentiviral vectors to maintain transgene expression in vitro and in vivo. Furthermore, HIV-1 vectors packaged with a mutated integrase were able to correct retinal disease in a mouse model. Interestingly, these results differ from earlier studies in which first-generation nonintegrating lentiviral vectors yielded insignificant levels of transduction. However, to date a rigorous characterization of transgene expression from the currently used self-inactivating (SIN) nonintegrating lentiviral vectors has not been published. Here we characterize transgene expression from SIN nonintegrating lentiviral vectors. Overall, we found that nonintegrating vectors express transgenes at a significantly lower level than their integrating counterparts. Expression from nonintegrating vectors was improved upon introducing a longer deletion in the vector’s U3 region. A unique shuttle-vector assay indicated that the relative abundance of the different episomal forms was not altered by the longer U3 deletion. Interestingly, the longer U3 deletion did not enhance expression in the corpus callosum of the rat brain, suggesting that the extent of silencing of episomal transcription is influenced by tissue-specific factors. Finally, and for the first time, episomal expression in the mouse liver was potent and sustained. PMID:18797449

  4. Kinetics of lentiviral vector transduction in human CD34(+) cells.

    PubMed

    Uchida, Naoya; Green, Rashidah; Ballantine, Josiah; Skala, Luke P; Hsieh, Matthew M; Tisdale, John F

    2016-02-01

    Unlike cell lines, human hematopoietic stem cells (HSCs) are less efficiently transduced with HIV-1 vectors, potentially limiting this approach. To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, we evaluated the kinetics of lentiviral transduction in human CD34(+) cells. We transduced HeLa and CD34(+) cells with self-inactivating HIV-1 vector at low and tenfold higher multiplicity of infection (MOI) and evaluated vector amounts at various time points based on the rationale that if a given step was not limiting, tenfold greater vector amounts would be obtained at the tenfold higher MOI. We observed slower internalization (>60 min), a peak in reverse transcription at 24 hours, and completion of integration at 3 days in CD34(+) cells. In HeLa cells, there were approximately tenfold greater amounts at high MOI at all time points. When compared with HeLa cells, CD34(+) cells exhibited larger differences in vector amounts between high and low MOIs at 2-6 hours and a smaller difference at 12 hours to 10 days, revealing a limitation in human CD34(+) cell transduction around 12 hours, which corresponds to reverse transcription. In serial measurements of reverse transcription at 24 hours, vector amounts did not decrease once detected among CD34(+) cells. When using an HSC expansion medium, we observed less limitation for starting reverse transcription and more efficient transduction among CD34(+) cells in vitro and in xenografted mice. These data suggest that it is the initiation of reverse transcription that limits lentiviral transduction of human CD34(+) cells. Our findings provide an avenue for optimizing human CD34(+) cell transduction. PMID:26499040

  5. 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. PMID:26499046

  6. Learning with two sites of synaptic integration.

    PubMed

    Körding, K P; König, P

    2000-02-01

    Since the classical work of D O Hebb 1949 The Organization of Behaviour (New York: Wiley) it is assumed that synaptic plasticity solely depends on the activity of the pre- and the postsynaptic cells. Synapses influence the plasticity of other synapses exclusively via the post-synaptic activity. This confounds effects on synaptic plasticity and neuronal activation and, thus, makes it difficult to implement networks which optimize global measures of performance. Exploring solutions to this problem, inspired by recent research on the properties of apical dendrites, we examine a network of neurons with two sites of synaptic integration. These communicate in such a way that one set of synapses mainly influences the neurons' activity; the other set gates synaptic plasticity. Analysing the system with a constant set of parameters reveals: (1) the afferents that gate plasticity act as supervisors, individual to every cell. (2) While the neurons acquire specific receptive fields the net activity remains constant for different stimuli. This ensures that all stimuli are represented and, thus, contributes to information maximization. (3) Mechanisms for maximization of coherent information can easily be implemented. Neurons with non-overlapping receptive fields learn to fire correlated and preferentially transmit information that is correlated over space. (4) We demonstrate how a new measure of performance can be implemented: cells learn to represent only the part of the input that is relevant to the processing at higher stages. This criterion is termed 'relevant infomax'. PMID:10735527

  7. Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones.

    PubMed

    Dambrot, Cheryl; Buermans, Henk P J; Varga, Eszter; Kosmidis, Georgios; Langenberg, Karin; Casini, Simona; Elliott, David A; Dinnyes, Andras; Atsma, Douwe E; Mummery, Christine L; Braam, Stefan R; Davis, Richard P

    2014-10-01

    Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis. PMID:24836851

  8. 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.

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

    PubMed Central

    Murphy, Michele E; Vin, Chintan D; Slough, Megan M; Gombotz, Wayne R; Kelley-Clarke, Brenna

    2016-01-01

    Using lentiviral vector products in clinical applications requires an accurate method for measuring transduction titer. For vectors lacking a marker gene, quantitative polymerase chain reaction is used to evaluate the number of vector DNA copies in transduced target cells, from which a transduction titer is calculated. Immune Design previously described an integration-deficient lentiviral vector pseudotyped with a modified Sindbis virus envelope for use in cancer immunotherapy (VP02, of the ZVex platform). Standard protocols for titering integration-competent lentiviral vectors employ commercial spin columns to purify vector DNA from transduced cells, but such columns are not optimized for isolation of extrachromosomal (nonintegrated) DNA. Here, we describe a 96-well transduction titer assay in which DNA extraction is performed in situ in the transduction plate, yielding quantitative recovery of extrachromosomal DNA. Vector titers measured by this method were higher than when commercial spin columns were used for DNA isolation. Evaluation of the method’s specificity, linear range, and precision demonstrate that it is suitable for use as a lot release assay to support clinical trials with VP02. Finally, the method is compatible with titering both integrating and nonintegrating lentiviral vectors, suggesting that it may be used to evaluate the transduction titer for any lentiviral vector. PMID:26942209

  10. Conditional RNAi Using the Lentiviral GLTR System.

    PubMed

    Pfeiffenberger, Elisabeth; Sigl, Reinhard; Geley, Stephan

    2016-01-01

    RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success depends on the effective expression of target gene-specific RNAi-inducing small double-stranded interfering RNA molecules (siRNAs). Here, were describe the use of a recently developed lentiviral RNAi system that allows the rapid generation of stable cell lines with inducible RNAi based on conditional expression of double-stranded short hairpin RNA (shRNA). These lentiviral vectors can be generated rapidly using the GATEWAY recombination cloning technology. Conditional cell lines can be established by using either a two-vector system in which the regulator is encoded by a separate vector or by a one-vector system. The available different lentiviral vectors for conditional shRNA expression cassette delivery co-express additional genes that allow (1) the use of fluorescent proteins for color-coded combinatorial RNAi or monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one-vector system (pGLTR-X). PMID:27317178

  11. Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice.

    PubMed

    Themis, Mike; Waddington, Simon N; Schmidt, Manfred; von Kalle, Christof; Wang, Yoahe; Al-Allaf, Faisal; Gregory, Lisa G; Nivsarkar, Megha; Themis, Matthew; Holder, Maxine V; Buckley, Suzanne M K; Dighe, Niraja; Ruthe, Alaine T; Mistry, Ajay; Bigger, Brian; Rahim, Ahad; Nguyen, Tuan H; Trono, Didier; Thrasher, Adrian J; Coutelle, Charles

    2005-10-01

    Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients' chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application. PMID:16084128

  12. 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.

  13. A Graph Based Framework to Model Virus Integration Sites.

    PubMed

    Fronza, Raffaele; Vasciaveo, Alessandro; Benso, Alfredo; Schmidt, Manfred

    2016-01-01

    With next generation sequencing thousands of virus and viral vector integration genome targets are now under investigation to uncover specific integration preferences and to define clusters of integration, termed common integration sites (CIS), that may allow to assess gene therapy safety or to detect disease related genomic features such as oncogenes. Here, we addressed the challenge to: 1) define the notion of CIS on graph models, 2) demonstrate that the structure of CIS enters in the category of scale-free networks and 3) show that our network approach analyzes CIS dynamically in an integrated systems biology framework using the Retroviral Transposon Tagged Cancer Gene Database (RTCGD) as a testing dataset. PMID:27257470

  14. Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases

    PubMed Central

    Hu, Biliang; Tai, April; Wang, Pin

    2011-01-01

    Summary The increasing level of understanding of the lentivirus biology has been instrumental in shaping the design strategy of creating therapeutic lentiviral delivery vectors. As a result, lentiviral vectors have become one of the most powerful gene transfer vehicles. They are widely used for therapeutic purposes as well as in studies of basic biology, due to their unique characteristics. Lentiviral vectors have been successfully employed to mediate durable and efficient antigen expression and presentation in dendritic cells both in vitro and in vivo, leading to the activation of cellular immunity and humoral responses. This capability makes the lentiviral vector an ideal choice for immunizations that target a wide range of cancers and infectious diseases. Further advances into optimizing the vector system and understanding the relationship between the immune system and diseases pathogenesis will only augment the potential benefits and utility of lentiviral vaccines for human health. PMID:21198664

  15. Comprehensive Integrated Planning Process for the Oak Ridge Operations Sites

    SciTech Connect

    Bechtel Jacobs Company LLC; Lockheed Martin Energy Research Corporation; Lockheed Martin Energy Systems, Inc.

    1999-09-01

    This plan is intended to assist the U.S. Department of Energy (DOE) and contractor personnel in implementing a comprehensive integrated planning process consistent with DOE Order 430.1A, "Life Cycle Asset Management," and Oak Ridge Operations (ORO) Order 430 on sites under the jurisdiction of DOE-ORO. Those sites are the Oak Ridge Reservation, in Oak Ridge, Tennessee; the Paducah Gaseous Diffusion Plant, in Paducah, Kentucky; and the Portsmouth Gaseous Diffusion Plant, in Piketon, Ohio. DOE contractors at these sites are charged with developing and producing this plan, which is referred to as simply the Comprehensive Integrated Plan.

  16. Generation of Transgenic Rats Using Lentiviral Vectors.

    PubMed

    Reichardt, Holger M; Fischer, Henrike J

    2016-01-01

    Transgenesis is a valuable tool with which to study different aspects of gene function in the context of the intact organism. During the last two decades a tremendous number of transgenic animals have been generated, and the continuous improvement of technology and the development of new systems have fostered their widespread application in biomedical research. Generally, transgenic animals are generated by introducing foreign DNA into fertilized oocytes, which can be achieved either by injecting recombinant DNA into the pronucleus or by transferring lentiviral particles into the perivitelline space. While mice remain the favored species in many laboratories, there are a number of applications where the use of rats is advantageous. One such research area is multiple sclerosis. Here, several experimental models are available that are closely mimicking the human disease, and it is possible to induce neuroinflammation by transferring pathogenic T cells which can then be studied by flow cytometry and 2-photon live imaging. Unlike for mice, the development of transgenic rats has encountered some hurdles in the past, e.g., due to a complicated reproductive biology and the frailty of the fertilized oocytes in vitro. In this chapter we provide a protocol describing how we manipulate single cell embryos in our lab in order to efficiently generate transgenic rats in a variety of different strains using lentiviral gene transfer. PMID:25063498

  17. Lentiviral delivery of short hairpin RNAs

    PubMed Central

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

    2009-01-01

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

  18. Lentiviral vector gene transfer to porcine airways.

    PubMed

    Sinn, Patrick L; Cooney, Ashley L; Oakland, Mayumi; Dylla, Douglas E; Wallen, Tanner J; Pezzulo, Alejandro A; Chang, Eugene H; McCray, Paul B

    2012-01-01

    In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).Molecular Therapy - Nucleic Acids (2012) 1, e56; doi:10.1038/mtna.2012.47; published online 27 November 2012. PMID:23187455

  19. Converting Maturing Nuclear Sites to Integrated Power Production Islands

    DOE PAGESBeta

    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

  20. Methods for integration site distribution analyses in animal cell genomes

    PubMed Central

    Ciuffi, Angela; Ronen, Keshet; Brady, Troy; Malani, Nirav; Wang, Gary; Berry, Charles C.; Bushman, Frederic D.

    2014-01-01

    The question of where retroviral DNA becomes integrated in chromosomes is important for understanding (i) the mechanisms of viral growth, (ii) devising new anti-retroviral therapy, (iii) understanding how genomes evolve, and (iv) developing safer methods for gene therapy. With the completion of genome sequences for many organisms, it has become possible to study integration targeting by cloning and sequencing large numbers of host–virus DNA junctions, then mapping the host DNA segments back onto the genomic sequence. This allows statistical analysis of the distribution of integration sites relative to the myriad types of genomic features that are also being mapped onto the sequence scaffold. Here we present methods for recovering and analyzing integration site sequences. PMID:19038346

  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. 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. PMID:21822899

  3. Cellular Cofactors of Lentiviral Integrase: From Target Validation to Drug Discovery

    PubMed Central

    Taltynov, Oliver; Desimmie, Belete A.; Demeulemeester, Jonas; Christ, Frauke; Debyser, Zeger

    2012-01-01

    To accomplish their life cycle, lentiviruses make use of host proteins, the so-called cellular cofactors. Interactions between host cell and viral proteins during early stages of lentiviral infection provide attractive new antiviral targets. The insertion of lentiviral cDNA in a host cell chromosome is a step of no return in the replication cycle, after which the host cell becomes a permanent carrier of the viral genome and a producer of lentiviral progeny. Integration is carried out by integrase (IN), an enzyme playing also an important role during nuclear import. Plenty of cellular cofactors of HIV-1 IN have been proposed. To date, the lens epithelium-derived growth factor (LEDGF/p75) is the best studied cofactor of HIV-1 IN. Moreover, small molecules that block the LEDGF/p75-IN interaction have recently been developed for the treatment of HIV infection. The nuclear import factor transportin-SR2 (TRN-SR2) has been proposed as another interactor of HIV IN-mediating nuclear import of the virus. Using both proteins as examples, we will describe approaches to be taken to identify and validate novel cofactors as new antiviral targets. Finally, we will highlight recent advances in the design and the development of small-molecule inhibitors binding to the LEDGF/p75-binding pocket in IN (LEDGINs). PMID:22928108

  4. Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters.

    PubMed

    Yu, Fu-xian; Zhu, Zhi-wei; Chen, Xiao-yu; Huang, Jing; Shi, Tuan-yuan; Li, Jun-xing; Pan, Jian-zhi

    2014-11-01

    The promoter plays an important role in the regulation of gene expression. To analyze a promoter's activity, we developed a novel lentiviral T/A vector that contains two reporter genes, a luciferase (Luc2) gene and a green fluorescent protein (Venus) gene, that are linked via an internal ribosome entry site (IRES2). To test the performance of this vector, phosphoglycerate kinase-1 (PGK) and elongation factor-1α (EF1α) promoters were amplified by PCR and inserted into this lentiviral T/A vector using T4 DNA ligase, yielding two promoter-reporter vectors: pLent-T-PGK and pLent-T-EF1α. When these vectors were transfected into 293T cells, we observed a higher level of Venus expression under a fluorescence microscopy in the case of pLent-T-EF1α as compared to pLent-T-PGK. The results of the luciferase reporter assay showed that the ratio of the promoter activities of EF1α and PGK was approximately 9:1. The two promoter-reporter vectors were also packaged as lentiviral particles to conduct promoter activity assay in cultured cells. The ratio of the promoter activities of EF1α and PGK was 4.23:1 when they were infected into 293T cells at a multiplicity of infection of 1. This value is comparable to that of a parallel experiment using the commercial luciferase reporter vector pGL4.10 with an activity ratio of 5.99:1 for EF1α and PGK. These results indicate that lentiviral T/A vector will be a useful tool for analysis of promoter activity and specificity. PMID:25091945

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. 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

  7. 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. PMID:24619149

  8. Recent Advances in Lentiviral Vaccines for HIV-1 Infection

    PubMed Central

    Norton, Thomas D.; Miller, Elizabeth A.

    2016-01-01

    The development of an effective HIV vaccine to prevent and/or cure HIV remains a global health priority. Given their central role in the initiation of adaptive immune responses, dendritic cell (DC)-based vaccines are being increasingly explored as immunotherapeutic strategies to enhance HIV-specific T cells in infected individuals and, thus, promote immune responses that may help facilitate a functional cure. HIV-1-based lentiviral (LV) vectors have inherent advantages as DC vaccine vectors due to their ability to transduce non-dividing cells and integrate into the target cell genomic DNA, allowing for expression of encoded antigens over the lifespan of the cell. Moreover, LV vectors may express additional immunostimulatory and immunoregulatory proteins that enhance DC function and direct antigen-specific T cells responses. Recent basic and clinical research efforts have broadened our understanding of LV vectors as DC-based vaccines. In this review, we provide an overview of the pre-clinical and clinical LV vector vaccine studies for treating HIV to date. We also discuss advances in LV vector designs that have enhanced DC transduction efficiency, target cell specificity, and immunogenicity, and address potential safety concerns regarding LV vector-based vaccines. PMID:27446074

  9. A molecular site-site integral equation that yields the dielectric constant

    NASA Astrophysics Data System (ADS)

    Dyer, Kippi M.; Perkyns, John S.; Stell, George; Pettitt, B. Montgomery

    2008-09-01

    Our recent derivation [K. M. Dyer et al., J. Chem. Phys. 127, 194506 (2007)] of a diagrammatically proper, site-site, integral equation theory using molecular angular expansions is extended to polar fluids. With the addition of atomic site charges we take advantage of the formal long-ranged potential field cancellations before renormalization to generate a set of numerically stable equations. Results for calculations in a minimal (spherical) angular basis set are presented for the radial distribution function, the first dipolar (110) projection, and the dielectric constant for two model diatomic systems. All results, when compared to experiment and simulation, are a significant quantitative and qualitative improvement over previous site-site theories. More importantly, the dielectric constant is not trivial and close to simulation and experiment.

  10. 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. PMID:27139406

  11. 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. PMID:26020529

  12. 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

  13. Site wide integration of the Rocky Flats closure project

    SciTech Connect

    Burdge, L.F.; Golan, P.

    1998-06-01

    The prime contractor for the Rocky Flats Closure Project (RFCP), Kaiser-Hill, in concert with the Department of Energy--Rocky Flats Field Office (DOE-RFFO) has applied a fully integrated, life-cycle, critical path schedule and work planning system to manage the work that is required to close the Site. The closure of the Site is complex, in that it houses over 700 facilities, 19,600 kilograms of Special Nuclear Material (Plutonium and Uranium), and over 160,000 cubic meters of Transuranic, Low Level, and Hazardous Waste. The deactivation, decommissioning, decontaminating, and demolition of this large number of facilities, while at the same time accommodating difficult on-going activities, significantly increases the sophistication required in the planning process. The Rocky Flats team has overcome these difficulties by establishing a money oriented critical path process, to provide a least-cost avenue to supporting on-going activities and a line-of-balance process for production oriented activities. These processes, when integrated with a typical activity-based project planning system, guide the way to the shortest and most cost-effective course for the closure of the Rocky Flats Site.

  14. An Integrated Site-Wide Assessment of Nuclear Wastes to Remain at the Hanford Site, Washington

    SciTech Connect

    Morse, J.G.; Bryce, R.W.; Hildebrand, R.D.; Kincaid, C.T.

    2004-10-06

    Since its creation in 1943 until 1988, the Hanford Site, a facility in the U.S. Department of Energy (DOE) nuclear weapons complex was dedicated to the production of weapons grade plutonium and other special nuclear materials. The Hanford Site is located in eastern Washington State and is bordered on the north and east by the Columbia River. Decades of creating fuel, irradiating it in reactors, and processing it to recover nuclear material left numerous waste sites that involved the discharge of contaminated liquids and the disposal of contaminated solid waste. Today, the primary mission of the Hanford Site is to safely cleanup and manage the site's legacy waste. A site-wide risk assessment methodology has been developed to assist the DOE, as well as state and federal regulatory agencies, in making decisions regarding needed remedial actions at past waste sites, and safe disposal of future wastes. The methodology, referred to as the System Assessment Capability (SAC), utilizes an integrated set of models that track potential contaminants from inventory through vadose zone, groundwater, Columbia River and air pathways to human and ecological receptors.

  15. What Integration Sites Tell Us about HIV Persistence.

    PubMed

    Hughes, Stephen H; Coffin, John M

    2016-05-11

    Advances in technology have made it possible to analyze integration sites in cells from HIV-infected patients. A significant fraction of infected cells in patients on long-term therapy are clonally expanded; in some cases the integrated viral DNA contributes to the clonal expansion of the infected cells. Although the large majority (>95%) of the HIV proviruses in treated patients are defective, expanded clones can carry replication-competent proviruses, and cells from these clones can release infectious virus. As discussed in this Perspective, it is likely that cells that produce virus are strongly selected against in vivo, and cells with replication competent proviruses expand and survive because only a small fraction of the cells produce virus. These findings have implications for strategies that are intended to eliminate the reservoir of infected cells that has made it almost impossible to cure HIV-infected patients. PMID:27173927

  16. 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.

  17. 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.

  18. Efficient Large Volume Lentiviral Vector Production Using Flow Electroporation

    PubMed Central

    Witting, Scott R.; Li, Lin-Hong; Jasti, Aparna; Allen, Cornell; Cornetta, Kenneth; Brady, James; Shivakumar, Rama

    2012-01-01

    Abstract Lentiviral vectors are beginning to emerge as a viable choice for human gene therapy. Here, we describe a method that combines the convenience of a suspension cell line with a scalable, nonchemically based, and GMP-compliant transfection technique known as flow electroporation (EP). Flow EP parameters for serum-free adapted HEK293FT cells were optimized to limit toxicity and maximize titers. Using a third generation, HIV-based, lentiviral vector system pseudotyped with the vesicular stomatitis glycoprotein envelope, both small- and large-volume transfections produced titers over 1×108 infectious units/mL. Therefore, an excellent option for implementing large-scale, clinical lentiviral productions is flow EP of suspension cell lines. PMID:21933028

  19. Comparison of Lentiviral Packaging Mixes and Producer Cell Lines for RNAi Applications.

    PubMed

    Albrecht, Christian; Hosiner, Stefanie; Tichy, Brigitte; Aldrian, Silke; Hajdu, Stefan; Nürnberger, Sylvia

    2015-06-01

    Lentiviral transduction is a highly efficient DNA delivery method for RNA interference applications. However, obtaining high lentiviral titers of shRNA and miRNA encoding vectors is challenging, since shRNA and miRNA cassettes have been shown to reduce lentiviral titers. In this study, we compare four commercially available packaging mixes and two producer cell lines in order to optimize lentiviral production for gene silencing experiments. Lentiviral vectors encoding a miRNA sequence and emerald green fluorescence protein were co-transfected with ViraPower™, Lenti-X™ HTX, MISSION(®) Lentiviral or Trans-Lentiviral™ packaging mix in HEK-293T or 293FT cells. After transducing HeLa cells with virus-containing supernatant, lentiviral titers were determined by flow cytomerty. In both cell lines, the highest lentiviral titer was obtained with MISSION(®) Lentiviral packaging mix, followed by ViraPower™, Lenti-X™ HTX, and Trans-Lentiviral™. On average, HEK-293T cells produced 6.2-fold higher lentiviral titers than 293FT cells (p < 0.001). With the combination of MISSION(®) Lentiviral packaging mix and HEK-293T cells, an up to 48.5-fold higher lentiviral titer was reached compared to other packaging mixes and producer cell lines. The optimized selection of packaging mix and cell line described in this work should facilitate the production of high-titer lentiviruses for gene silencing experiments. PMID:25616840

  20. 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

  1. Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord.

    PubMed

    Schoderboeck, L; Riad, S; Bokor, A M; Wicky, H E; Strauss, M; Bostina, M; Oswald, M J; Empson, R M; Hughes, S M

    2015-05-01

    Lentiviral vectors have proved an effective method to deliver transgenes into the brain; however, they are often hampered by a lack of spread from the site of injection. Modifying the viral envelope with a portion of a rabies envelope glycoprotein can enhance spread in the brain by using long-range axon projections to facilitate retrograde transport. In this study, we generated two chimeric envelopes containing the extra-virion and transmembrane domain of rabies SADB19 or CVS-N2c with the intra-virion domain of vesicular stomatitis virus. Viral particles were packaged containing a green fluorescent protein reporter construct under the control of the phosphoglycerokinase promoter. Both vectors produced high-titer particles with successful integration of the glycoproteins into the particle envelope and significant transduction of neurons in vitro. Injection of the SADB19 chimeric viral vector into the lumbar spinal cord of adult mice mediated a strong preference for gene transfer to local neurons and axonal terminals, with retrograde transport to neurons in the brainstem, hypothalamus and cerebral cortex. Development of this vector provides a useful means to reliably target select populations of neurons by retrograde targeting. PMID:25630949

  2. 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)

  3. 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.

  4. 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.

  5. 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.

  6. Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein

    PubMed Central

    Mi, Michael Y; Zhang, Jiying; He, Yukai

    2005-01-01

    Background A critical step in the production of new HIV virions involves the TAT protein binding to the TAR element. The TAT protein contains in close proximity its TAR RNA binding domain and protein transduction domain (PTD). The PTD domain of TAT has been identified as being instrumental in the protein's ability to cross mammalian cell and nuclear membranes. All together, this information led us to form the hypothesis that a protein containing the TAR RNA binding domain could compete with the native full length TAT protein and effectively block the TAR RNA binding site in transduced HIV infected cells. Results We synthesized a short peptide named Tat-P, which contained the TAR RNA binding and PTD domains to examine whether the peptide has the potential of inhibiting TAT dependent HIV replication. We investigated the inhibiting effects of Tat-P in vitro using a HIV derived lentiviral vector model. We found that the TAT PTD domain not only efficiently transduced test cells, but also effectively inhibited the production of lentiviral particles in a TAT dependent manner. These results were also supported by data derived from the TAT activated LTR-luciferase expression model and RNA binding assays. Conclusion Tat-P may become part of a category of anti-HIV drugs that competes with full length TAT proteins to inhibit HIV replication. In addition, this study indicates that the HIV derived lentiviral vector system is a safe and reliable screening method for anti-HIV drugs, especially for those targeting the interaction of TAT and TAR RNAs. PMID:16293193

  7. Site-Wide Integrated Water Monitoring - Defining and Implementing Sampling Objectives to Support Site Closure - 13060

    SciTech Connect

    Wilborn, Bill; Knapp, Kathryn; Farnham, Irene; Marutzky, Sam

    2013-07-01

    The Underground Test Area (UGTA) activity is responsible for assessing and evaluating the effects of the underground nuclear weapons tests on groundwater at the Nevada National Security Site (NNSS), formerly the Nevada Test Site (NTS), and implementing a corrective action closure strategy. The UGTA strategy is based on a combination of characterization, modeling studies, monitoring, and institutional controls (i.e., monitored natural attenuation). The closure strategy verifies through appropriate monitoring activities that contaminants of concern do not exceed the SDWA at the regulatory boundary and that adequate institutional controls are established and administered to ensure protection of the public. Other programs conducted at the NNSS supporting the environmental mission include the Routine Radiological Environmental Monitoring Program (RREMP), Waste Management, and the Infrastructure Program. Given the current programmatic and operational demands for various water-monitoring activities at the same locations, and the ever-increasing resource challenges, cooperative and collaborative approaches to conducting the work are necessary. For this reason, an integrated sampling plan is being developed by the UGTA activity to define sampling and analysis objectives, reduce duplication, eliminate unnecessary activities, and minimize costs. The sampling plan will ensure the right data sets are developed to support closure and efficient transition to long-term monitoring. The plan will include an integrated reporting mechanism for communicating results and integrating process improvements within the UGTA activity as well as between other U.S. Department of Energy (DOE) Programs. (authors)

  8. Site-Wide Integrated Water Monitoring -- Defining and Implementing Sampling Objectives to Support Site Closure

    SciTech Connect

    Wilborn, Bill; Marutzky, Sam; Knapp, Kathryn

    2013-02-24

    The Underground Test Area (UGTA) activity is responsible for assessing and evaluating the effects of the underground nuclear weapons tests on groundwater at the Nevada National Security Site (NNSS), formerly the Nevada Test Site (NTS), and implementing a corrective action closure strategy. The UGTA strategy is based on a combination of characterization, modeling studies, monitoring, and institutional controls (i.e., monitored natural attenuation). The closure strategy verifies through appropriate monitoring activities that contaminants of concern do not exceed the SDWA at the regulatory boundary and that adequate institutional controls are established and administered to ensure protection of the public. Other programs conducted at the NNSS supporting the environmental mission include the Routine Radiological Environmental Monitoring Program (RREMP), Waste Management, and the Infrastructure Program. Given the current programmatic and operational demands for various water-monitoring activities at the same locations, and the ever-increasing resource challenges, cooperative and collaborative approaches to conducting the work are necessary. For this reason, an integrated sampling plan is being developed by the UGTA activity to define sampling and analysis objectives, reduce duplication, eliminate unnecessary activities, and minimize costs. The sampling plan will ensure the right data sets are developed to support closure and efficient transition to long-term monitoring. The plan will include an integrated reporting mechanism for communicating results and integrating process improvements within the UGTA activity as well as between other U.S. Department of Energy (DOE) Programs.

  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. Lentiviral vectors for treating and modeling human CNS disorders.

    PubMed

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

    2004-09-01

    Vectors based on lentiviruses efficiently deliver genes into many different types of primary neurons from a broad range of species including man and the resulting gene expression is long term. These vectors are opening up new approaches for the treatment of neurological diseases such as Parkinson's disease (PD), Huntington's disease (HD), and motor neuron diseases (MNDs). Numerous animal studies have now been undertaken with these vectors and correction of disease models has been obtained. Lentiviral vectors also provide a new strategy for in vivo modeling of human diseases; for example, the lentiviral-mediated overexpression of mutated human alpha-synuclein or huntingtin genes in basal ganglia induces neuronal pathology in animals resembling PD and HD in man. These vectors have been refined to a very high level and can be produced safely for the clinic. This review will describe the general features of lentiviral vectors with particular emphasis on vectors derived from the non-primate lentivirus, equine infectious anemia virus (EIAV). It will then describe some key examples of genetic correction and generation of genetic animal models of neurological diseases. The prospects for clinical application of lentiviral vectors for the treatment of PD and MNDs will also be outlined. PMID:15352068

  12. Transduction patterns of pseudotyped lentiviral vectors in the nervous system.

    PubMed

    Wong, Liang-Fong; Azzouz, Mimoun; Walmsley, Lucy E; Askham, Zoe; Wilkes, Fraser J; Mitrophanous, Kyriacos A; Kingsman, Susan M; Mazarakis, Nicholas D

    2004-01-01

    We have developed a non-primate-based lentiviral vector based on the equine infectious anemia virus (EIAV) for efficient gene transfer to the central and peripheral nervous systems. Previously we have demonstrated that pseudotyping lentiviral vectors with the rabies virus glycoprotein confers retrograde axonal transport to these vectors. In the present study we have successfully produced high-titer EIAV vectors pseudotyped with envelope glycoproteins from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains); rabies virus [various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)]; Lyssavirus Mokola virus, a rabies-related virus; and Arenavirus lymphocytic choriomeningitis virus (LCMV). These vectors were delivered to the striatum or spinal cord of adult rats or muscle of neonatal mice by direct injection. We report that the lentiviral vectors pseudotyped with envelopes from the VSV Indiana strain, wild-type ERA, and CVS strains resulted in strong transduction in the striatum, while Mokola- and LCMV-pseudotyped vectors exhibited moderate and weak transduction, respectively. Furthermore ERA- and CVS-pseudotyped lentiviral vectors demonstrated retrograde transport and expression in distal neurons after injection in brain, spinal cord, and muscle. The differences in transduction efficiencies and retrograde transport conferred by these envelope glycoproteins present novel opportunities in designing therapeutic strategies for different neurological diseases. PMID:14741783

  13. 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

  14. 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.

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

    PubMed Central

    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. PMID:26041987

  16. 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. PMID:26731312

  17. Use of Lentiviral Vectors to Deliver and Express Bicistronic Transgenes in Developing Chicken Embryos

    PubMed Central

    Semple-Rowland, Susan L; Berry, Jonathan

    2013-01-01

    The abilities of lentiviral vectors to carry large transgenes (~ 8Kb) and to efficiently infect and integrate these genes into the genomes of both dividing and non-dividing cells make them ideal candidates for transport of genetic material into cells and tissues. Given the properties of these vectors, it is somewhat surprising that they have seen only limited use in studies of developing tissues and in particular of the developing nervous system. Over the past several years, we have taken advantage of the large capacity of these vectors to explore the expression characteristics of several dual promoter and 2A peptide bicistronic transgenes in developing chick neural retina, with the goal of identifying transgene designs that reliably express multiple proteins in infected cells. Here we summarize the activities of several of these transgenes in neural retina and provide detailed methodologies for packaging lentivirus and delivering the virus into the developing neural tubes of chicken embryos in ovo, procedures that have been optimized over the course of several years of use in our laboratory. Conditions to hatch injected embryos are also discussed. The chicken-specific techniques will be of highest interest to investigators using avian embryos, development and packaging of lentiviral vectors that reliably express multiple proteins in infected cells should be of interest to all investigators whose experiments demand manipulation and expression of multiple proteins in developing cells and tissues. PMID:23816789

  18. 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

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

    PubMed Central

    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. PMID:25520191

  20. The Eastern Lau Basin Integrated Studies Site (ISS)

    NASA Astrophysics Data System (ADS)

    Wiens, D.

    2003-12-01

    A new venue for Ridge 2000 (R2K) Integrated Studies, the Eastern Lau Spreading Center (ELSC) adds the element of a spreading center in a back-arc basin to the R2K program. The ELSC, located in the western Pacific near Tonga, is a 390 km-long first-order ridge segment that displays a broad range of effects of the back-arc environment. At its southern end it is only 40 km from the Tonga arc volcanic front and is propagating southward into a back-arc rift. At its northern end it is 100 km from the volcanic front and terminates at a large, non-transform offset. The ELSC displays substantial and systematic changes in multiple parameters: spreading rate, magma source and lava chemistry, axial depth and morphology, melt lens characteristics, and crustal thickness and structure. A main focus of the work at the ELSC is to determine how changes in these forcing functions affect key parameters such as magma source composition, crustal structure, and characteristics of hydrothermal venting such as temperature, chemistry, and faunal composition and abundance. Prior reconnaissance investigation shows that these hydrothermal field characteristics also vary within the southernmost segments, and show distinct differences compared with well-studied mid-ocean ridge sites. Four R2K cruises are planned for the ELSC in 2004. The first cruise (PI: Martinez) will investigate interrelationships among crustal structure, volcanism, and hydrothermal activity using deep tow, CTD's, and Tow-Yo. A second cruise (PI: Langmuir) will focus on petrological and water column properties using dredging, CTD's and ABE. The next cruise (PI: Tivey) will provide an initial characterization of vent fields, fluid chemistry, mineralogy, and biodiversity using Jason II and net tows, and a final cruise (PI: Childress) will investigate community ecology using Jason II. An additional study (PI: Thurnherr) will deploy autonomous floats to investigate hydrothermal plume circulation and dispersal . An overview of the

  1. Selective pressures to maintain attachment site specificity of integrative and conjugative elements.

    PubMed

    Menard, Kayla L; Grossman, Alan D

    2013-01-01

    Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs. PMID:23874222

  2. 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.

  3. 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.

  4. Efficient production of germline transgenic chickens using lentiviral vectors.

    PubMed

    McGrew, Michael J; Sherman, Adrian; Ellard, Fiona M; Lillico, Simon G; Gilhooley, Hazel J; Kingsman, Alan J; Mitrophanous, Kyriacos A; Sang, Helen

    2004-07-01

    An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations. PMID:15192698

  5. Efficient production of germline transgenic chickens using lentiviral vectors

    PubMed Central

    McGrew, Michael J; Sherman, Adrian; Ellard, Fiona M; Lillico, Simon G; Gilhooley, Hazel J; Kingsman, Alan J; Mitrophanous, Kyriacos A; Sang, Helen

    2004-01-01

    An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations. PMID:15192698

  6. Integrated Research Methods for Applied Urban Hydrogeology of Karst Sites

    NASA Astrophysics Data System (ADS)

    Epting, J.; Romanov, D. K.; Kaufmann, G.; Huggenberger, P.

    2008-12-01

    Integrated and adaptive surface- and groundwater monitoring and management in urban areas require innovative process-oriented approaches. To accomplish this, it is necessary to develop and combine interdisciplinary instruments that facilitate adequately quantifying cumulative effects on groundwater flow regimes. While the characterization and modeling of flow in heterogeneous and fractured media has been investigated intensively, there are no well-developed long-term hydrogeological research sites for gypsum karst. Considering that infrastructures in karst regions, particularly in gypsum, are prone to subsidence, severe problems can arise in urban areas. In the 1880's, a river dam was constructed on gypsum-containing rock, Southeast of Basel, Switzerland. Over the last 30 years, subsidence of the dam and an adjacent highway has been observed. Surface water infiltrates upstream of the dam, circulates in the gravel deposits and in the weathered bedrock around and beneath the dam and exfiltrates downstream into the river. These processes enhance karstification processes in the soluble units of the gypsum. As a result an extended weathering zone within the bedrock and the development of preferential flow paths within voids and conduits can be observed. To prevent further subsidence, construction measures were conducted in two major project phases in 2006 and 2007. The highway was supported by a large number of pillars embedded in the non- weathered rock and by a sealing pile wall, to prevent infiltrating river water circulating around the dam and beneath the foundation of the highway. To safeguard surface and subsurface water resources during the construction measures, an extensive observation network was set up. Protection schemes and geotechnical investigations that are necessary for engineering projects often provide "windows of opportunity", bearing the possibility to change perceptions concerning the sustainable development of water resources and coordinate future

  7. 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

  8. Integrated Mapping and Imaging at a Legacy Test Site (Invited)

    NASA Astrophysics Data System (ADS)

    Sussman, A. J.; Schultz-Fellenz, E. S.; Kelley, R. E.; Sweeney, J. J.; Vigil, S.; DiBenedetto, J.; Chipman, V.

    2013-12-01

    A team of multi-disciplinary geoscientists was tasked to characterize and evaluate a legacy nuclear detonation site in order to develop research locations with the long-term goal of improving treaty monitoring, verification, and other national security applications. There was a test at the site of interest that was detonated on June 12, 1985 in a vertical emplacement borehole at a depth of 608m below the surface in rhyolites. With announced yield of 20-150 kt, the event did not collapse to the surface and form a crater, but rather experienced a subsurface collapse with more subtle surface expressions of deformation. This result provides the team with an opportunity to evaluate a number of surface and subsurface inspection technologies in a broad context. The team collected ground-based visual observation, ground penetrating radar, electromagnetic, ground-based and airborne LiDAR, ground-based and airborne hyperspectral, gravity and magnetics, dc and induction electrical methods, and active seismic data during field campaigns in the summers of 2012 and 2013. Detection of features was performed using various approaches that were assessed for accuracy, efficiency and diversity of target features. For example, whereas the primary target of the ground-based visual observation survey was to map the surface features, the target of the gravity survey was to attempt the detection of a possible subsurface collapse zone which might be located as little as 200 meters below the surface. The datasets from surveys described above are integrated into a geographical information system (GIS) database for analysis and visualization. Other presentations during this session provide further details as to some of the work conducted. Work by Los Alamos National Laboratory and Lawrence Livermore National Laboratory was sponsored by the National Nuclear Security Administration Award No. DE-AC52-06NA25946/NST10-NCNS-PD00. Work by National Security Technologies, LLC, was performed under

  9. Development of lentiviral vectors for antiangiogenic gene delivery.

    PubMed

    Shichinohe, T; Bochner, B H; Mizutani, K; Nishida, M; Hegerich-Gilliam, S; Naldini, L; Kasahara, N

    2001-11-01

    Growth and metastasis of malignant tumors requires angiogenesis. Inhibition of tumor-induced angiogenesis may represent an effective cytostatic strategy. We have constructed recombinant self-inactivating lentiviral vectors expressing angiostatin and endostatin, and have tested their antiangiogenic activities. As VSV-G-pseudotyped lentiviral vectors showed low relative transduction titers on bovine aortic and human umbilical vein endothelial cells, it was difficult to achieve significant inhibition of endothelial cell growth by lentivirus-mediated antiangiogenic gene transfer directly to endothelial cells without concomitant vector-associated cytotoxicity. However, lentivirus vectors could efficiently and stably transduce T24 human bladder cancer cells that are relatively resistant to adenovirus infection due to loss of coxsackievirus-adenovirus receptor expression. Long-term expression and secretion of angiostatin and endostatin from lentivirus-transduced T24 cells resulted in significant inhibition of cellular proliferation on coculture with endothelial cells. This report represents the first use of lentivirus-based vectors to deliver the antiangiogenic factors, angiostatin and endostatin, and suggests the potential utility of antiangiogenic gene therapy with lentiviral vectors for the treatment of cancer. PMID:11773978

  10. 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

    2016-01-01

    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. PMID

  11. Repetitive, marker-free, site-specific integration as a novel tool for multiple chromosomal integration of DNA.

    PubMed

    Petersen, Kia Vest; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

    2013-06-01

    We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attB(min)), two mutated loxP sequences (lox66 and lox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection of loxP recombination on the pKV6 vector. When transformed into L. lactis expressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked by attL and attR hybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate in L. lactis, loxP recombinants can be selected for by using 5-fluoroorotic acid. The introduced attB(min) site can subsequently be used for a second round of integration. To examine if attP recombination was specific to the attB site, integration was performed in strains containing the attB, attL, and attR sites or the attL and attR sites only. Only attP-attB recombination was observed when all three sites were present. In the absence of the attB site, a low frequency of attP-attL recombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABR and xylT) from L. lactis strain KF147 were integrated into the chromosome of L. lactis strain MG1363 in two steps. PMID:23542630

  12. 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

  13. Prioritizing functional phosphorylation sites based on multiple feature integration

    PubMed Central

    Xiao, Qingyu; Miao, Benpeng; Bi, Jie; Wang, Zhen; Li, Yixue

    2016-01-01

    Protein phosphorylation is an important type of post-translational modification that is involved in a variety of biological activities. Most phosphorylation events occur on serine, threonine and tyrosine residues in eukaryotes. In recent years, many phosphorylation sites have been identified as a result of advances in mass-spectrometric techniques. However, a large percentage of phosphorylation sites may be non-functional. Systematically prioritizing functional sites from a large number of phosphorylation sites will be increasingly important for the study of their biological roles. This study focused on exploring the intrinsic features of functional phosphorylation sites to predict whether a phosphosite is likely to be functional. We found significant differences in the distribution of evolutionary conservation, kinase association, disorder score, and secondary structure between known functional and background phosphorylation datasets. We built four different types of classifiers based on the most representative features and found that their performances were similar. We also prioritized 213,837 human phosphorylation sites from a variety of phosphorylation databases, which will be helpful for subsequent functional studies. All predicted results are available for query and download on our website (Predict Functional Phosphosites, PFP, http://pfp.biosino.org/). PMID:27090940

  14. Using Pulmozyme DNase treatment in lentiviral vector production.

    PubMed

    Shaw, Aaron; Bischof, Daniela; Jasti, Aparna; Ernstberger, Aaron; Hawkins, Troy; Cornetta, Kenneth

    2012-02-01

    In the production of lentiviral vector for clinical studies the purity of the final product is of vital importance. To remove plasmid and producer cell line DNA, investigators have incubated the vector product with Benzonase, a bacterially derived DNase. As an alternative we investigated the use of Pulmozyme, a U.S. Food and Drug Administration-approved human DNase for the treatment of cystic fibrosis, by comparing the efficiency of DNA removal from lentiviral vector preparations. A green fluorescent protein-expressing lentiviral vector was prepared by transient calcium phosphate transfection of HEK 293T cells and DNA removal was compared when treating vector after harvest or immediately after transfection. The effectiveness of DNase treatment was measured by quantitative PCR using primers for vesicular stomatitis virus glycoprotein G viral envelope plasmid. When treating the final product, 1-hr incubations (37°C) with Pulmozyme at 20 U/ml reduced plasmid DNA to undetectable levels. Longer incubations (up to 4 hr) did not improve DNA removal at lower concentrations and the effectiveness was equivalent to or better than Benzonase at 50 U/ml. Attempting to use Pulmozyme immediately after transfection, but before final medium change, as a means to decrease Pulmozyme concentration in the final product provided a 2-log reduction in DNA but was inferior to treatment at the end of production. Pulmozyme, at concentrations up to 100 U/ml, had no measurable effect on infectious titer of the final vector product. The use of Pulmozyme is likely to increase the cost of DNase treatment when preparing vector product and should be considered when generating clinical-grade vector products. PMID:22428981

  15. HIV integration site distributions in resting and activated CD4+ T cells infected in culture

    PubMed Central

    Brady, Troy; Agosto, Luis M.; Malani, Nirav; Berry, Charles C.; O'Doherty, Una; Bushman, Frederic

    2010-01-01

    Objective The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting. Design Primary resting or activated CD4+ T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined. Methods We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions. Results HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes. Conclusion These data indicate that integration site distributions show modest but significant differences between resting and activated CD4+ T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression. PMID:19550285

  16. Lentiviral vector-mediated gene transfer and RNA silencing technology in neuronal dysfunctions.

    PubMed

    Dreyer, Jean-Luc

    2011-02-01

    Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson's disease, or in investigating gene function in Huntington's disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition. PMID:20862616

  17. 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

  18. Preclinical Evaluation of Efficacy and Safety of an Improved Lentiviral Vector for the Treatment of β-Thalassemia and Sickle Cell Disease

    PubMed Central

    Negre, Olivier; Bartholomae, Cynthia; Beuzard, Yves; Cavazzana, Marina; Christiansen, Lauryn; Courne, Céline; Deichmann, Annette; Denaro, Maria; de Dreuzy, Edouard; Finer, Mitchell; Fronza, Raffaele; Béatrix, Gillet-Legrand; Joubert, Christophe; Kutner, Robert; Leboulch, Philippe; Maouche, Leïla; Paulard, Anaïs; Pierciey Jr., 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 self-inactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-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. PMID:25429463

  19. Enhancing Students' Thinking Skills: Exploring Model Technology-Integration Sites.

    ERIC Educational Resources Information Center

    Moersch, Christopher

    1998-01-01

    Examines ways to integrate technology into social studies, science, mathematics, and language arts. Describes model elementary and middle-school classrooms in which technology is used to investigate the concept of property, study soil porosity and the water cycle, run a student store, and promote environmental activism. (PEN)

  20. INTEGRATING FUEL CELL TECHNOLOGY AND AFO/CAFO WASTE SITES

    EPA Science Inventory

    This project is an effort to evaluate animal feeding operation and confined animal feeding operation sites for energy-production on a state-wide level. The project will determine the potential energy and environmental benefits of methane utilization in hydrogen fuel cells in the...

  1. Lentiviral vectors for cancer immunotherapy and clinical applications.

    PubMed

    Liechtenstein, Therese; Perez-Janices, Noemi; Escors, David

    2013-09-01

    The success of immunotherapy against infectious diseases has shown us the powerful potential that such a treatment offers, and substantial work has been done to apply this strategy in the fight against cancer. Cancer is however a fiercer opponent than pathogen-caused diseases due to natural tolerance towards tumour associated antigens and tumour-induced immunosuppression. Recent gene therapy clinical trials with viral vectors have shown clinical efficacy in the correction of genetic diseases, HIV and cancer. The first successful gene therapy clinical trials were carried out with onco(γ-)retroviral vectors but oncogenesis by insertional mutagenesis appeared as a serious complication. Lentiviral vectors have emerged as a potentially safer strategy, and recently the first clinical trial of patients with advanced leukemia using lentiviral vectors has proven successful. Additionally, therapeutic lentivectors have shown clinical efficacy for the treatment of HIV, X-linked adrenoleukodystrophy, and β-thalassaemia. This review aims at describing lentivectors and how they can be utilized to boost anti-tumour immune responses by manipulating the effector immune cells. PMID:24078865

  2. Multipurpose modular lentiviral vectors for RNA interference and transgene expression.

    PubMed

    Kesireddy, Venu; van der Ven, Peter F M; Fürst, Dieter O

    2010-07-01

    We have created a multipurpose modular lentiviral vector system for expressing both transgenes and miRNA 30-based short hairpins (shRNAmirs) for RNAi. The core of the resulting vector system, pLVmir, allows a simple two step cloning procedure for expressing shRNAmirs under the control of a Pol II promoter in both a constitutive and conditional manner. The adapted cloning method includes a PCR-free method for transferring shRNAmir based RNAi clones from a publicly available library (Open Biosystems). The addition of a Pol II promoter-driven shRNAmir cassette and broadening the choice of Pol III promoters and silencing triggers offers great flexibility to this system. The combination of several preexisting and additional modules created here caters to common needs of researchers. Our modular vector system was validated regarding functionality of promoters, inducibility and reversibility. We successfully applied the system to knockdown Xirp2 mRNA expression in H2kb-tsA58 muscle cells and determined that this had no spurious effect on the expression of a closely related protein. Finally, our set of lentiviral vectors may be used to achieve synergistic effects, for simultaneous knockdown of two genes, as a rescue plasmid and for studying mutant proteins in a physiological context. PMID:19798586

  3. [Integration sites and their characteristic analysis of piggyBac transposon in cattle genome].

    PubMed

    Du, Xin-Hua; Gao, Xue; Zhang, Lu-Pei; Gao, Hui-Jiang; Li, Jun-Ya; Xu, Shang-Zhong

    2013-06-01

    As a useful tool for genetic engineering, piggyBac (PB) transposons have been widely used in more than one species of transgenosis or generating mutation studies. At present, the studies about PB transposons in cattle were few. In order to get the PB transposon integration sites and summarize its characteristics in bovine genome, donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase were constructed and transferred into bovine fibroblasts by Amaxa basic nucleofector kit for primary mammalian fibroblasts. Cell clones stably transfected were obtained after screening by G-418. Genomic DNA of transgenic cells was extracted and the integration sites of PB transposon were detected by genome walking technology. Eight integration sites were obtained in bovine genome, although only 5 sites were mapped on chromosomes 1, 2, 11, and X chromosome. We found that PB transposon was inserted into the "TTAA" location and integrated into the intergenic non-regulatory sites between two genes. Analysis of the composition of the five bases, which was close to the side of the PB integration sites "TTAA", showed that PB 5' tended to be inserted into region rich in GC (62.5%). From the study, we got that transposition occurred in cattle genome by PB transposons and the integration site information acquired from the research will provide theoretical references for bovine study by PB transposon. PMID:23774022

  4. 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

  5. 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. PMID:26508664

  6. Impact of Nucleoporin-Mediated Chromatin Localization and Nuclear Architecture on HIV Integration Site Selection.

    PubMed

    Wong, Richard W; Mamede, João I; Hope, Thomas J

    2015-10-01

    It has been known for a number of years that integration sites of human immunodeficiency virus type 1 (HIV-1) DNA show a preference for actively expressed chromosomal locations. A number of viral and cellular proteins are implicated in this process, but the underlying mechanism is not clear. Two recent breakthrough publications advance our understanding of HIV integration site selection by focusing on the localization of the preferred target genes of integration. These studies reveal that knockdown of certain nucleoporins and components of nucleocytoplasmic trafficking alter integration site preference, not by altering the trafficking of the viral genome but by altering the chromatin subtype localization relative to the structure of the nucleus. Here, we describe the link between the nuclear basket nucleoporins (Tpr and Nup153) and chromatin organization and how altering the host environment by manipulating nuclear structure may have important implications for the preferential integration of HIV into actively transcribed genes, facilitating efficient viral replication. PMID:26136574

  7. Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.

    PubMed

    Lizée, Gregory; Aerts, Joeri L; Gonzales, Monica I; Chinnasamy, Nachimuthu; Morgan, Richard A; Topalian, Suzanne L

    2003-04-10

    The use of lentiviral vectors for basic research and potential future clinical applications requires methodologies that can accurately determine lentiviral titers and monitor viral transgene expression within target cells, beyond the context of reporter genes typically used for this purpose. Here we describe a quantitative RT-PCR (qRT-PCR) method that achieves both goals using primer sequences that are specific for the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), an enhancer contained in many retroviral vectors and that is incorporated in the 3' UTR of nascent transgene transcripts. Quantitation of titers of three recombinant lentiviruses, genetically identical except for the transgene, demonstrated consistent differences in titer that were likely due to transgene-associated toxicity in producer cells and target cells. Viruses encoding the tumor-associated antigens tyrosinase and neo-poly(A) polymerase yielded reproducibly lower titers than a virus encoding enhanced green fluorescent protein (GFP) at the viral RNA, integrated DNA, and transgene mRNA levels, as measured by WPRE qPCR. Furthermore, the magnitude of differences in expression of the three transgenes in transduced target cells could not have been predicted by measuring vector DNA integration events. Since transgene expression in target cells is the most common goal of lentiviral transduction, and since methods to quantify transgene expression on the protein level are not always readily available, qRT-PCR based on a nucleotide sequence included in the transcript provides a useful tool for titering novel recombinant lentiviruses. PMID:12718761

  8. 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.

  9. 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. PMID:22407723

  10. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

    PubMed Central

    Ranzani, Marco; Cesana, Daniela; Bartholomae, Cynthia C.; Sanvito, Francesca; Pala, Mauro; Benedicenti, Fabrizio; Gallina, Pierangela; Sergi, Lucia Sergi; Merella, Stefania; Bulfone, Alessandro; Doglioni, Claudio; von Kalle, Christof; Kim, Yoon Jun; Schmidt, Manfred; Tonon, Giovanni; Naldini, Luigi; Montini, Eugenio

    2013-01-01

    Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells, hampering the identification of early cancer-driving events amongst bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVV) by which we could efficiently induce hepatocellular carcinoma (HCC) in 3 different mouse models. By virtue of LVV’s replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of 4 new liver cancer genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. Our newly identified cancer genes are likely to play a role in human disease, since they are upregulated and/or amplified/deleted in human HCCs and can predict clinical outcome of patients. PMID:23314173

  11. 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. PMID:27343048

  12. INTEGRAL RCS : Launch Site Operations and In-orbit Behavior

    NASA Astrophysics Data System (ADS)

    Pessana, M.; Ravera, F.; Poidomani, G.; Gitins, M.; Lafranconi, R.

    2004-10-01

    The INTEGRAL (INTErnational Gamma-Ray Astrophysics Laboratory) Spacecraft was launched from Baikonur on October the 17th 2002. The Spacecraft Propulsion (RCS) is a monopropellant system, using anhydrous hydrazine and 20 N thrusters. It was loaded and pressurised at the Baikonur filling facilities prior the launch. The outcomes of this activity together with a correlation with the tanks loading procedure are given. During LEOP the behaviour of the RCS was recorded via the telemetry by the three Pressure Transducers, the TCS thermistors and by the thrusters thermocouples. The above parameters allow determining the proper status of the system and its functional behaviour. During the LEOP an anomalous temperature evolution was detected on a line spot downstream the tanks.

  13. CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array.

    PubMed

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

    In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA. PMID:27315553

  14. Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas

    PubMed Central

    Xu, Bo; Chotewutmontri, Sasithorn; Wolf, Stephan; Klos, Ursula; Schmitz, Martina; Dürst, Matthias; Schwarz, Elisabeth

    2013-01-01

    Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3′-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3′-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the

  15. DNFSB Recommendation 94-1 Hanford site integrated stabilization management plan, volumes 1 and 2

    SciTech Connect

    Gerber, E.W.

    1996-03-15

    This document comprises the Hanford Site Integrated Stabilization Management Plan (SISMP). This document describes the DOE`s plans at the Hanford Site to address concerns identified in Defense Nuclear Facilites Safety Board (DNFSB) Recommendation 94-1. This document also identifies plans for other spent nuclear fuel (SNF) inventories at the Hanford Site which are not within the scope of DNFSB Recommendation 94-1 for reference purposes because of their interrelationship with plans for SNF within the scope of DNFSB Recommendation 94-1. The SISMP was also developed to assist DOE in initial formulation of the Research and Development Plan and the Integrated Facilities Plan.

  16. Practical Considerations for Using Pooled Lentiviral CRISPR Libraries.

    PubMed

    McDade, Joel R; Waxmonsky, Nicole C; Swanson, Lianna E; Fan, Melina

    2016-01-01

    CRISPR/Cas9 technology is ideally suited for genome-wide screening applications due to the ease of generating guide RNAs (gRNAs) and the versatility of Cas9 or Cas9 derivatives to knockout, repress, or activate expression of target genes. Several pooled lentiviral CRISPR libraries have been developed and are now publicly available, but while using CRISPR/Cas9 for genetic experiments has become widely adopted, genome-wide screening experiments remain technically challenging. This review covers the basics of CRISPR/Cas9, describes several publicly available CRISPR libraries, and provides a general protocol for conducting genome-wide screening experiments using CRISPR/Cas9. © 2016 by John Wiley & Sons, Inc. PMID:27366891

  17. Organics in soils and groundwater at non-arid sites (A-1) integrated demonstration

    SciTech Connect

    Steele, J.L.; Kaback, D.S.; Looney, B.B.

    1994-06-01

    One of the most common environmental problems in the United States is soils and groundwater contaminated with volatile chemical solvents classified as Volatile Organic Compounds (VOCs), which were used as degreasers and cleaning agents. Leakage of solvents (trichloroethylene and tetrachloroethylene) from an underground process sewer line has contaminated soils and underlying groundwaters at SRS. This site was chosen for DOE-OTD`s integrated demonstration program to demonstrate innovative technologies for cleanup of soils and groundwater contaminated with VOCs. The Savannah River Site was especially well suited as the test bed for this integrated demonstration project due to the presence of a pre-existing line source of soil and groundwater-based contamination, on-going environmental remediation efforts at the site, and full cooperation from the concerned environmental regulatory agencies. The Integrated Demonstration (ID) at the Savannah River Site has demonstrated systems of technologies and evaluated them with respect to performance, safety and cost effectiveness.

  18. 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.

  19. 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. PMID:25122746

  20. DNFSB recommendation 94-1 Hanford site integrated stabilization management plan - VOLUMES 1-3

    SciTech Connect

    Gerber, E.W.

    1996-09-23

    The US Department of Energy (DOE) has developed an Integrated Program Plan (IPP) to address concerns identified in Defense Nuclear Facilities Safety Board (DNFSB) 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 DNFSB Recommendation 94-1 Implementation Plan (IP), which was transmitted to the DNFSB on February 28, 1995. 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. These 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 supported formulation of the initial versions of the Integrated Facilities Plan and the Research and Development Plan. The SISMPs are periodically updated to reflect improved integration between DOE sites as identified during the IPP systems engineering evaluations. This document constitutes the Hanford SISMP. This document includes the planned work scope, costs and schedules for activities at the Hanford site to implement the DNFSB Recommendation 94-1 IPP.

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

    PubMed

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

    2016-01-01

    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. PMID:27482458

  2. 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.

  3. 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

  4. Analysis of autophagosome formation using lentiviral biosensors for live fluorescent cellular imaging.

    PubMed

    Long, Kevin; Mohan, Chandra; Anderl, Janet; Huryn-Selvar, Karyn; Liu, Haizhen; Su, Kevin; Santos, Mark; Hsu, Matthew; Armstrong, Lucas; Ma, Jun

    2015-01-01

    Autophagy, a highly regulated homeostatic degradative process, allows cells to reallocate nutrients from less important to more essential processes under extreme conditions of starvation. Autophagy also prevents the buildup of damaged proteins and organelles that cause chronic tissue damage and disease. Although a topic of great interest with involvement of multiple signaling pathways, there are limitations in real-time detection of the autophagic process. EMD Millipore has developed technologies where prepackaged, ready-to-use, high-titer lentiviral particles, "lentiviral biosensors," encoding GFP- or RFP-tagged proteins provide a convenient and robust solution for fluorescent imaging of cells undergoing autophagy. Compared to nonviral transfection methods, lentiviral transduction, in many cases, offers higher transfection efficiency and more homogeneous protein expression, particularly for traditionally hard-to-transfect primary cell types. Lentiviral biosensors are ideal for use with fixed and live cell fluorescent microscopy, and are nondisruptive towards cellular function. GFP- or RFP-protein localization matches well with antibody-based immunostaining and demonstrates altered patterns of expression upon treatment with modulators of cell function and phenotype. Lentiviral biosensors provide a broadly effective, convenient method for visualization of cell behavior under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities. In this study, we focus on lentiviral biosensors containing GFP-LC3 and RFP-LC3 to study the formation of autophagosomes. PMID:25308268

  5. 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."

  6. 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.

  7. 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.

  8. 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.

  9. Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression

    PubMed Central

    Bersten, David C.; Sullivan, Adrienne E.; Li, Dian; Bhakti, Veronica; Bent, Stephen J.; Whitelaw, Murray L.

    2015-01-01

    Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function. PMID:25768837

  10. Integrase-defective Lentiviral Vectors as a Delivery Platform for Targeted Modification of Adenosine Deaminase Locus

    PubMed Central

    Joglekar, Alok V; Hollis, Roger P; Kuftinec, Gabriela; Senadheera, Shantha; Chan, Rebecca; Kohn, Donald B

    2013-01-01

    We investigated the use of integrase-defective lentiviral vectors (IDLVs) for transient delivery of zinc finger nucleases (ZFNs) and donor templates for site-specific modification of the human adenosine deaminase (hADA) gene. Initially, we constructed IDLVs carrying ZFN monomers (Single-IDLVs) and found them to be able to deliver their gene-editing payload to K562 cells successfully upon cotransduction, with minimal cytotoxicity. To simplify delivery, we designed an IDLV construct to deliver both ZFN monomers from the same vector (Double-IDLV). However, this construct in its original state was prone to rearrangements of the vector genome, resulting in greatly reduced functionality; this was due to recombination between highly similar ZFN monomers arranged in tandem. We modified the Double-IDLV constructs to reduce recombination and restored simultaneous delivery of both ZFNs. We also tested an IDLV construct for delivery of donor templates and demonstrated its efficacy for gene modification. In summary, we highlighted the importance of modifying vector design for co-delivery of highly similar sequences inherent to genome-editing nucleases, and demonstrated significant improvement in the use of IDLVs for delivery of ZFNs and donor templates for genome modification. PMID:23857176

  11. Inhibition of storage pathology in prenatal CLN5-deficient sheep neural cultures by lentiviral gene therapy.

    PubMed

    Hughes, Stephanie M; Hope, Katie M; Xu, Janet Boyu; Mitchell, Nadia L; Palmer, David N

    2014-02-01

    The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are inherited neurodegenerative lysosomal storage diseases caused by mutations in several different genes. Mutations in CLN5 cause a variant late-infantile human disease and some cases of juvenile and adult clinical disease. NCLs also occur in animals, and a flock of New Zealand Borderdale sheep with a CLN5 splice-site mutation has been developed for model studies. Dissociated mixed neural cells from CLN5-deficient foetal sheep brains contained no obvious storage bodies at plating but these accumulated rapidly in culture, mainly in microglial cells and also in neurons and astrocytes. Accumulation was very obvious after a week, as monitored by fluorescent microscopy and immunostaining for subunit c of mitochondrial ATP synthase. Photography at intervals revealed the dynamic nature of the cultures and a flow of storage bodies between cells, specifically the phagocytosis of storage-body containing cells by microglia and incorporation of the storage bodies into the host cells. No storage was observed in cultured control cells. Transduction of cell cultures with a lentiviral vector expressing a C-terminal Myc tagged CLN5 resulted in secretion of post-translationally glycosylated and processed CLN5. Transduction of CLN5-deficient cultures with this construct rapidly reversed storage body accumulation, to less than half in only six days. These results show that storage body accumulation is reversible with enzyme correction and support the use of these cultures for testing of therapeutics prior to whole animal studies. PMID:24269732

  12. Association with PAK2 Enables Functional Interactions of Lentiviral Nef Proteins with the Exocyst Complex

    PubMed Central

    Imle, Andrea; Abraham, Libin; Tsopoulidis, Nikolaos; Hoflack, Bernard; Saksela, Kalle

    2015-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication and contributes to immune evasion in vivo, but the underlying molecular mechanisms remain incompletely defined. Nef interferes with host cell actin dynamics to restrict T lymphocyte responses to chemokine stimulation and T cell receptor engagement. This relies on the assembly of a labile multiprotein complex including the host kinase PAK2 that Nef usurps to phosphorylate and inactivate the actin-severing factor cofilin. Components of the exocyst complex (EXOC), an octameric protein complex involved in vesicular transport and actin remodeling, were recently reported to interact with Nef via the same molecular surface that mediates PAK2 association. Exploring the functional relevance of EXOC in Nef-PAK2 complex assembly/function, we found Nef-EXOC interactions to be specifically mediated by the PAK2 interface of Nef, to occur in infected human T lymphocytes, and to be conserved among lentiviral Nef proteins. In turn, EXOC was dispensable for direct downstream effector functions of Nef-associated PAK2. Surprisingly, PAK2 was essential for Nef-EXOC association, which required a functional Rac1/Cdc42 binding site but not the catalytic activity of PAK2. EXOC was dispensable for Nef functions in vesicular transport but critical for inhibition of actin remodeling and proximal signaling upon T cell receptor engagement. Thus, Nef exploits PAK2 in a stepwise mechanism in which its kinase activity cooperates with an adaptor function for EXOC to inhibit host cell actin dynamics. PMID:26350970

  13. Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites.

    PubMed

    Serrao, Erik; Cherepanov, Peter; Engelman, Alan N

    2016-01-01

    Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients. PMID:27023428

  14. 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. PMID:25350000

  15. 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

  16. Enhanced Central Nervous System Transduction with Lentiviral Vectors Pseudotyped with RVG/HIV-1gp41 Chimeric Envelope Glycoproteins

    PubMed Central

    Trabalza, Antonio; Eleftheriadou, Ioanna; Sgourou, Argyro; Liao, Ting-Yi; Patsali, Petros; Lee, Heyne

    2014-01-01

    ABSTRACT To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the “Kennedy” sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer

  17. 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

    2016-02-01

    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. 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.

  19. Multiethnic Neighbourhoods as Sites of Social Capital Formation: Examining Social to Political "Integration" in Schools

    ERIC Educational Resources Information Center

    Basu, Ranu

    2006-01-01

    In an "ideal" democratic society, publicly funded schools serve many purposes. Aside from its educational mandate, schools are places for neighbourhood integration, social capital formation and the fostering of civil society. For newly arrived immigrants, especially those with young children, schools are important sites of settlement experiences.…

  20. INTEGRATION OF BUILDING AND ENERGY TECHNOLOGY WITH ON-SITE WASTE MANAGEMENT IN THE YEAR 2000

    EPA Science Inventory

    During this study the potential feasibility of integrating waste management, water supply and on-site energy generation was examined with the objective of improving the objective of improving the overall resource efficiency of the typical residential unit. This report can be used...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. 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...

  4. 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.

  5. Retrotransposons. An RNA polymerase III subunit determines sites of retrotransposon integration.

    PubMed

    Bridier-Nahmias, Antoine; Tchalikian-Cosson, Aurélie; Baller, Joshua A; Menouni, Rachid; Fayol, Hélène; Flores, Amando; Saïb, Ali; Werner, Michel; Voytas, Daniel F; Lesage, Pascale

    2015-05-01

    Mobile genetic elements are ubiquitous. Their integration site influences genome stability and gene expression. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. Here we describe an interaction between Ty1 integrase and the AC40 subunit of Pol III and demonstrate that AC40 is the predominant determinant targeting Ty1 integration upstream of Pol III-transcribed genes. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends. The mechanism of target specificity allows Ty1 to proliferate and yet minimizes genetic damage to its host. PMID:25931562

  6. 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.

  7. Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

    PubMed

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Shirai, Makoto

    2016-06-01

    The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes. PMID:26870903

  8. Biosafety Assessment of Site-directed Transgene Integration in Human Umbilical Cord–lining Cells

    PubMed Central

    Sivalingam, Jaichandran; Krishnan, Shruti; Ng, Wai Har; Lee, Sze Sing; Phan, Toan Thang; Kon, Oi Lian

    2010-01-01

    Biosafety and efficacy considerations that impede clinical application of gene therapy could be addressed by nonviral ex vivo cell therapy, utilizing transgenic cells that have been comprehensively pre-evaluated for genotoxic potential and transgene expression. We evaluated the genotoxic potential of phiC31 bacteriophage integrase-mediated transgene integration in cord-lining epithelial cells (CLECs) readily cultured from the outer membrane of human umbilical cords, by sequencing and mapping integration sites, spectral karyotyping, high-resolution genome copy number, transcriptome, and transgene copy number analyses and in vivo tumorigenicity. Of 44 independent integration events, <5% were exonic and 85% of modified cells had integrated ≤2 transgene(s). Expression of 95.6% of genes was unaltered in modified cells. Only three small regions showed genome copy number changes that did not correlate with altered gene expression or integration sites. Spectral karyotyping revealed rare nonrecurrent occurrence of three different translocations. Integrase-modified cells were not tumorigenic in immunocompromised mice for at least 4 months. Stable integration of a human factor VIII (FVIII) construct conferred durable FVIII secretion in vitro. Xenoimplantation of FVIII-secreting CLECs in immunocompetent hemophilic mice achieved significant phenotypic correction. Pre-evaluated clonal populations of phiC31 integrase–modified CLECs could be useful as bioimplants for monogenic diseases such as hemophilia. PMID:20424600

  9. Integrated test plan for crosswell compressional and shear wave seismic tomography for site characterization at the VOC Arid Site

    SciTech Connect

    Elbring, G.J.; Narbutovskih, S.M.

    1994-02-01

    This integrated test plan describes the demonstration of the crosswell acoustic tomography technique as part of the Volatile Organic Compounds-Arid Integrated Demonstration (VOC-Arid ID). The purpose of this demonstration is to image the subsurface seismic velocity structure and to relate the resulting velocity model to lithology and saturation. In fiscal year (FY) 1994 an initial fielding will test three different downhole sources at two different sites at the Hanford US Department of Energy facility to identify which sources will provide the energy required to propagate between existing steel-cased wells at these two sites. Once this has been established, a second fielding will perform a full compressional and shear wave tomographic survey at the most favorable site. Data reduction, analysis, and interpretation of this full data set will be completed by the end of this fiscal year. Data collection for a second survey will be completed by the end of the fiscal year, and data reduction for this data set will be completed in FY 1995. The specific need is detailed subsurface characterization with minimum intrusion. This technique also has applications for long term vadose zone monitoring for both Resource Conservation and Recovery Act (RCRA) waste storage facilities and for remediation monitoring. Images produced are continuous between boreholes. This is a significant improvement over the single point data derived solely from core information. Saturation changes, either naturally occurring (e.g., perched water tables) or remediation induced (e.g., water table mounding from injection wells or during inwell air sparging) could be imaged. These crosswell data allow optimal borehole placement for groundwater remediation, associated monitoring wells and possibly evaluation of the effective influence of a particular remediation technique.

  10. Site-specific integration of adeno-associated virus involves partial duplication of the target locus

    PubMed Central

    Henckaerts, Els; Dutheil, Nathalie; Zeltner, Nadja; Kattman, Steven; Kohlbrenner, Erik; Ward, Peter; Clément, Nathalie; Rebollo, Patricia; Kennedy, Marion; Keller, Gordon M.; Linden, R. Michael

    2009-01-01

    A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences. PMID:19372372

  11. Transgene expression after rep-mediated site-specific integration into chromosome 19.

    PubMed

    Philpott, Nicola J; Gomos, Janette; Falck-Pedersen, Erik

    2004-01-01

    We have used a plasmid-based transfection model of the adeno-associated virus (AAV) Rep-mediated site-specific integration (RMSSI) pathway to characterize the stability and expression of a site-specifically integrated transgene (either green fluorescent protein [GFP] or chloramphenicol acetyltransferase [CAT]). Three plasmids containing the AAV p5 integration efficiency element (p5IEE) have been used to study integration and transgene expression in HeLa cells: (1) pRepGFP(itr+) contains both AAV ITRs, rep, and p5IEE and can be used as either a plasmid or rAAV vehicle for integration; (2) pRepGFP(itr-) contains the AAV rep gene and the p5IEE; (3) pAd-p5CAT contains only the 138-bp p5IEE of AAV. The data presented demonstrate that in the absence of drug selection, all three constructs undergo site-specific integration (efficiencies of between 10 and 40% of transduced cell lines). At 6 weeks posttransfection most cell lines that underwent RMSSI also expressed the appropriate transgene product. By 18 weeks posttransfection cell lines that were established with rep in cis to the transgene showed a decline in transgene expression as well as a loss of transgene DNA. In many cell lines, there appears to be transgene-containing DNA that does not contribute to gene expression. Data support a model of gene expression and transgene instability through a Rep-mediated pathway. In contrast to rep-containing cell lines, clonal cell lines containing p5IEECAT (with Rep provided in trans) maintained both the integrated transgene and transgene expression throughout the entire experimental time course (18 weeks). PMID:14965377

  12. Polybrene: Observations on cochlear hair cell necrosis and minimal lentiviral transduction of cochlear hair cells.

    PubMed

    Han, Miaomiao; Yu, Dongzhen; Song, Qiang; Wang, Jiping; Dong, Pin; He, Jingchun

    2015-07-23

    Polybrene is widely used to enhance viral transduction; however, little is known about the utility thereof, in enhancing lentiviral transduction of cochlear cells. In the present study, we examined the cytotoxic effects of polybrene, and the further effects thereof, on lentiviral transduction of cochlear cells, especially sensory hair cells. Cochlear basilar membranes of newborn rats were cultured and treated with 0.1-10 μg/mL polybrene for 24h to explore the potential development of ototoxicity. PI staining and TUNEL detection were used to evaluate necrosis or apoptosis of hair cell. Various doses of lentivirus-GFP were added to cochlear organotypic cultures with safe concentrations of polybrene, incubated for 24h, and cultured (in the absence of the virus and polybrene) for a further 48 h. Transduction efficiencies were evaluated. The results showed that polybrene at 0.1 μg/mL was safe to cochlear cells, and 0.5-10 μg/mL concentration induced hair cell necrosis in a dose-dependent manner. However, supporting cells were not damaged. Lentiviral vectors transduced into cochlear cells and 0.1 μg/mL polybrene enhanced transduction efficiency. However, hair cells were hardly transduced with lentiviral vectors either alone or in the presence of 0.1 μg/mL polybrene. The use of polybrene to aid lentiviral transduction of cochlear hair cells requires further attention. PMID:26071903

  13. The structure of adenovirus type 12 DNA integration sites in the hamster cell genome.

    PubMed Central

    Knoblauch, M; Schröer, J; Schmitz, B; Doerfler, W

    1996-01-01

    Foreign DNA can integrate into the genomes of mammalian cells, and this process plays major roles in viral oncogenesis and in the generation of transgenic organisms and will be important in evolving regimens for human somatic gene therapy. In the present study, the insertion sites of adenovirus type 12 (Ad12) DNA genomes have been analyzed in detail in the Ad12-transformed hamster cell line T637, its revertants, which have lost most of the >20 Ad12 genome equivalents integrated chromosomally in cell line T637, and in the Ad12-induced tumor T191. Some of these junction sites have been molecularly cloned, and the nucleotide sequences at the sites of transition between viral and cellular DNAs have been determined. The sites of linkage between the hamster cellular and the foreign (viral) DNA are characterized by the frequent occurrence of patch homologies between the recombination partners. The cellular junction sites investigated here are not transcriptionally active. One of the cellular DNA sequences abutting the right Ad12 DNA terminus in cell line T637 (os2) is represented only once in the hamster genome and has a strikingly low abundance of 5'-CG-3' dinucleotide sequences. One 5'-GCGC-3' sequence close to the Ad12 DNA integration site is heavily methylated in normal cells, Ad12-transformed cells, and Ad12-induced tumor cells. The second such sequence is more remote from the junction site, is partly methylated in BHK21 hamster cells, and shows differences in methylation in different Ad12-transformed cell lines. This site is unmethylated in liver DNA. The cellular DNA sequence at the site of Ad12 linkage in the tumor T191 exhibits homologies to highly repetitive sequences of the Alu family and to an origin of hamster DNA replication containing an Alu element. A number of junction sites between Ad12 DNA and hamster or mouse DNA in Ad12-transformed cell lines or Ad12-induced tumor cell lines, investigated here and previously, are characterized by stem-loop structures

  14. 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. PMID:23995555

  15. Implementation of an integrated CERCLA-NEPA environmental process at a FUSRAP site

    SciTech Connect

    Devgun, J.S.; Beskid, N.J.; Robertson, R.C.; Atkin, R.G.

    1989-01-01

    The Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) of 1980 and its amendment in 1986 through the Superfund Amendments and Reauthorization Act (SARA) have significantly affected the environmental compliance process. Remedial actions at contaminated sites now must satisfy the requirements of not only the National Environmental Policy Act (NEPA) but also CERCLA, as amended. For planning and conducting remedial actions under the Formerly Utilized Sites Remedial Action Program (FUSRAP), the US Department of Energy has developed and implemented an integrated process aimed at satisfying the requirements of both NEPA and CERCLA, as amended. The integrated approach involves a single, comprehensive environmental process that results in a single set of documentation. The process is streamlined, efficient, and cost effective and it minimizes confusion to the public. This paper discusses the need for, and the elements of, the integrated process as applied to FUSRAP sites. The implementation of the process is illustrated through a case study of FUSRAP sites in Tonawanda, New York. 5 figs.

  16. 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. PMID:27069793

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. Integrated environmental site characterization involving geochemistry, geophysics, and geology: A shortcut to remediation

    SciTech Connect

    Viellenave, J.; Slatten, M.; Church, G.; Anderson, M.

    1996-11-01

    Environmental site characterization processes have evolved from simple drill-and-sample routines into more sophisticated evaluations of increasingly complex problems involving a variety of contaminants. Strategic integration of several geoscience tools into a more holistic approach benefits the site owner/operator by developing a synoptic perspective of the site at the earliest possible time, allowing for more selective and focused use of the expensive and invasive technologies. The ultimate effect is a better site characterization, including attention to difficult PRP issues, lower liability, fewer risks of bypassing potentially hazardous contaminant accumulations, and a result that is more targeted to environmental and human health risks. An integrated site investigation system requires good geology and hydrology, but is properly augmented by use of modem and sophisticated geochemical and geophysical tools. Establishing characterization objectives is critical in deciding what geoscience tool(s) to deploy in any given situation. For each tool, critical criteria are identified that will enable the user to best decide which to use for what purposes.

  3. 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

  4. 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. PMID:25918715

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

    PubMed Central

    Luo, Yumei; 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. PMID:25918715

  6. Identifying Human Kinase-Specific Protein Phosphorylation Sites by Integrating Heterogeneous Information from Various Sources

    PubMed Central

    Li, Tingting; Du, Pufeng; Xu, Nanfang

    2010-01-01

    Phosphorylation is an important type of protein post-translational modification. Identification of possible phosphorylation sites of a protein is important for understanding its functions. Unbiased screening for phosphorylation sites by in vitro or in vivo experiments is time consuming and expensive; in silico prediction can provide functional candidates and help narrow down the experimental efforts. Most of the existing prediction algorithms take only the polypeptide sequence around the phosphorylation sites into consideration. However, protein phosphorylation is a very complex biological process in vivo. The polypeptide sequences around the potential sites are not sufficient to determine the phosphorylation status of those residues. In the current work, we integrated various data sources such as protein functional domains, protein subcellular location and protein-protein interactions, along with the polypeptide sequences to predict protein phosphorylation sites. The heterogeneous information significantly boosted the prediction accuracy for some kinase families. To demonstrate potential application of our method, we scanned a set of human proteins and predicted putative phosphorylation sites for Cyclin-dependent kinases, Casein kinase 2, Glycogen synthase kinase 3, Mitogen-activated protein kinases, protein kinase A, and protein kinase C families (avaiable at http://cmbi.bjmu.edu.cn/huphospho). The predicted phosphorylation sites can serve as candidates for further experimental validation. Our strategy may also be applicable for the in silico identification of other post-translational modification substrates. PMID:21085571

  7. 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.

  8. 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.

  9. 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.

  10. 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.

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

    PubMed

    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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. Cloning and sequencing of viral integration site in human fibroblasts immortalized by simian virus 40.

    PubMed

    Yano, O; Hirano, H; Karasaki, Y; Higashi, K; Nakamura, H; Akiya, S; Gotoh, S

    1991-02-01

    We have analyzed cellular DNA sequences at the viral genome integration site in a human fibroblast cell line VA13 immortalized by simian virus 40 (SV40). The computer analysis of the junctional cellular DNA sequences did not show any homology to the DNA sequences previously reported. This suggests that immortalization by SV40 was not induced by the destruction of any known oncogene or anti-oncogene at the integration site. We did not find the precise substantial sequence homology at the junctional site between the cellular DNA and SV40 DNA, indicating that the recombination mechanism involved does not require precise sequence homology and therefore, SV40 genome was probably not integrated by homologous recombination. Short direct and inverted repeats of 5 to 29 nucleotides were found in the junctional cellular and SV40 DNA. Cellular DNA abutting SV40 DNA was found by the Northern blot analysis to be expressed in diploid human fibroblasts and SV40-transformed cells. The nature of this RNA is now under study. PMID:1851675

  17. 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

  18. 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.

  19. The integration profile of EIAV-based vectors.

    PubMed

    Hacker, Caroline V; Vink, Conrad A; Wardell, Theresa W; Lee, Sheena; Treasure, Peter; Kingsman, Susan M; Mitrophanous, Kyriacos A; Miskin, James E

    2006-10-01

    Lentiviral vectors based on equine infectious anemia virus (EIAV) stably integrate into dividing and nondividing cells such as neurons, conferring long-term expression of their transgene. The integration profile of an EIAV vector was analyzed in dividing HEK293T cells, alongside an HIV-1 vector as a control, and compared to a random dataset generated in silico. A multivariate regression model was generated and the influence of the following parameters on integration site selection determined: (a) within/not within a gene, (b) GC content within 20 kb, (c) within 10 kb of a CpG island, (d) gene density within a 2-Mb window, and (e) chromosome number. The majority of the EIAV integration sites (68%; n = 458) and HIV-1 integration sites (72%; n = 162) were within a gene, and both vectors favored AT-rich regions. Sites within genes were examined using a second model to determine the influence of the gene-specific parameters, gene region, and transcriptional activity. Both EIAV and HIV-1 vectors preferentially integrated within active genes. Unlike the gammaretrovirus MLV, EIAV and HIV-1 vectors do not integrate preferentially into the promoter region or the 5' end of the transcription unit. PMID:16950499

  20. 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

  1. Lentiviral vectors for induction of self-differentiation and conditional ablation of dendritic cells.

    PubMed

    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

    2011-08-01

    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

  2. Lentiviral vectors with CMV or MHCII promoters administered in vivo: immune reactivity versus persistence of expression.

    PubMed

    Kimura, Takahiro; Koya, Richard C; Anselmi, Laura; Sternini, Catia; Wang, He-Jing; Comin-Anduix, Begonya; Prins, Robert M; Faure-Kumar, Emmanuelle; Rozengurt, Nora; Cui, Yan; Kasahara, Noriyuki; Stripecke, Renata

    2007-07-01

    Lentiviral vectors (LVs) are potential tools for genetic vaccination. To improve the safety of LV vaccines, we evaluated the selectivity, bio-distribution, persistence of expression, and immune potency of vesicular stomatitis virus G (VSV-G)-pseudotyped vectors transcriptionally targeted to antigen presenting cells (APCs) through a major histocompatibility complex class II (MHCII) promoter. Control vectors contained the ubiquitous cytomegalovirus (CMV) promoter. Bio-distribution studies after intravenous injections of LVs expressing green fluorescent protein (GFP) or luciferase were conducted by a combination of flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (RT-Q-PCR) and whole-body bioluminescence analyses. GFP-expressing vectors showed selective expression in MHCII(+) cells of spleen and LV-CMV-GFP administration produced noticeable spleen inflammation, whereas LV-MHCII-GFP did not. Long-term optical imaging analyses of C57BL/6 mice injected with LV-CMV-LUC showed diminishing luciferase expression in the liver and spleen over time. Vaccination/boost with LV-CMV expressing the melanoma antigen tyrosinase-related protein 2 (TRP2) yielded dose-dependent antigen-specific CD8(+) T-cell reactivity and high protection against B16 melanoma challenge. Unexpectedly, administration of LVs containing the MHCII promoter resulted in persistence of luciferase expression and viral integration in MHCII(+) splenocytes and virtually no CD8(+) T-cell responses against TRP2. These studies reveal that APC transduction by LVs could lead to immune reactivity (LV-CMV) or persistence of transgene expression (LV-MHCII), providing a relevant paradigm for vaccination and gene replacement approaches. PMID:17505480

  3. 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

  4. 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

  5. Understanding Transcription Factor Regulation by Integrating Gene Expression and DNase I Hypersensitive Sites

    PubMed Central

    Wang, Guohua; Wang, Fang; Huang, Qian; Li, Yu; Liu, Yunlong; Wang, Yadong

    2015-01-01

    Transcription factors are proteins that bind to DNA sequences to regulate gene transcription. The transcription factor binding sites are short DNA sequences (5–20 bp long) specifically bound by one or more transcription factors. The identification of transcription factor binding sites and prediction of their function continue to be challenging problems in computational biology. In this study, by integrating the DNase I hypersensitive sites with known position weight matrices in the TRANSFAC database, the transcription factor binding sites in gene regulatory region are identified. Based on the global gene expression patterns in cervical cancer HeLaS3 cell and HelaS3-ifnα4h cell (interferon treatment on HeLaS3 cell for 4 hours), we present a model-based computational approach to predict a set of transcription factors that potentially cause such differential gene expression. Significantly, 6 out 10 predicted functional factors, including IRF, IRF-2, IRF-9, IRF-1 and IRF-3, ICSBP, belong to interferon regulatory factor family and upregulate the gene expression levels responding to the interferon treatment. Another factor, ISGF-3, is also a transcriptional activator induced by interferon alpha. Using the different transcription factor binding sites selected criteria, the prediction result of our model is consistent. Our model demonstrated the potential to computationally identify the functional transcription factors in gene regulation. PMID:26425553

  6. 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

  7. PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

    PubMed

    Song, Jiangning; Tan, Hao; 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

  8. 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.

  9. Complement regulatory proteins are incorporated into lentiviral vectors and protect particles against complement inactivation.

    PubMed

    Schauber-Plewa, C; Simmons, A; Tuerk, M J; Pacheco, C D; Veres, G

    2005-02-01

    Lentiviral vectors pseudotyped with G glycoprotein from vesicular stomatitis virus (VSV-G) and baculovirus gp64 are inactivated by human complement. The extent of vector inactivation in serum from individual donors was examined and results showed wide donor-dependent variation in complement sensitivity for VSV-G-pseudotyped lentivectors. Amphotropic envelope (Ampho)-pseudotyped vectors were generally resistant to serum from all donors, while gp64-pseudotyped vectors were inactivated but showed less donor-to-donor variation than VSV-G. In animal sera, the vectors were mostly resistant to inactivation by rodent complement, whereas canine complement caused a moderate reduction in titer. In a novel advance for the lentiviral vector system, human complement-resistant-pseudotyped lentivector particles were produced through incorporation of complement regulatory proteins (CRPs). Decay accelerating factor (DAF)/CD55 provided the most effective protection using this method, while membrane cofactor protein (MCP)/CD46 showed donor-dependent protection and CD59 provided little or no protection against complement inactivation. Unlike previous approaches using CRPs to produce complement-resistant viral vectors, CRP-containing lentivectors particles were generated for this study without engineering the CRP molecules. Thus, through overexpression of native DAF/CD55 in the viral producer cell, an easy method was developed for generation of lentiviral vectors that are almost completely resistant to inactivation by human complement. Production of complement-resistant lentiviral particles is a critical step toward use of these vectors for in vivo gene therapy applications. PMID:15550926

  10. 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

  11. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.

    PubMed

    Mazarakis, N D; Azzouz, M; Rohll, J B; Ellard, F M; Wilkes, F J; Olsen, A L; Carter, E E; Barber, R D; Baban, D F; Kingsman, S M; Kingsman, A J; O'Malley, K; Mitrophanous, K A

    2001-09-15

    In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease. PMID:11590128

  12. 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").

  13. 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.

  14. 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.

  15. 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.

  16. Radionuclide disequilibria studies for investigating the integrity of potential nuclear waste disposal sites: subseabed studies.

    SciTech Connect

    Laul, J.C.; Thomas, C.W.; Petersen, M.R.; Perkins, R.W.

    1981-09-01

    This study of subseabed sediments indicates that natural radionuclides can be employed to define past long-term migration rates and thereby evaluate the integrity of potential disposal sites in ocean sediments. The study revealed the following conclusions: (1) the sedimentation rate of both the long and short cores collected in the North Pacific is 2.5 mm/1000 yr or 2.5 m/m.yr in the upper 3 meters; (2) the sedimentation rate has been rather constant over the last one million years; and (3) slow diffusive processes dominate within the sediment. Reworking of the sediment by physical processes or organisms is not observed.

  17. 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.

  18. 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

  19. 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.

  20. In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

    PubMed

    Engelhorn, M; Boccard, F; Murtin, C; Prentki, P; Geiselmann, J

    1995-08-11

    The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell. PMID:7659518

  1. In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

    PubMed

    Engelhorn, M; Boccard, F; Murtin, C; Prentki, P; Geiselmann, J

    1995-09-11

    The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell. PMID:7567442

  2. 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

  3. 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

  4. Integrating care for high-risk patients in England using the virtual ward model: lessons in the process of care integration from three case sites

    PubMed Central

    Lewis, Geraint; Vaithianathan, Rhema; Wright, Lorraine; Brice, Mary R; Lovell, Paul; Rankin, Seth; Bardsley, Martin

    2013-01-01

    Background Patients at high risk of emergency hospitalisation are particularly likely to experience fragmentation in care. The virtual ward model attempts to integrate health and social care by offering multidisciplinary case management to people at high predicted risk of unplanned hospitalisation. Objective To describe the care practice in three virtual ward sites in England and to explore how well each site had achieved meaningful integration. Method Case studies conducted in Croydon, Devon and Wandsworth during 2011–2012, consisting of semi-structured interviews, workshops, and site visits. Results Different versions of the virtual wards intervention had been implemented in each site. In Croydon, multidisciplinary care had reverted back to one-to-one case management. Conclusions To integrate successfully, virtual ward projects should safeguard the multidisciplinary nature of the intervention, ensure the active involvement of General Practitioners, and establish feedback processes to monitor performance such as the number of professions represented at each team meeting. PMID:24250284

  5. 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

  6. Correction of murine β-thalassemia after minimal lentiviral gene transfer and homeostatic in vivo erythroid expansion

    PubMed Central

    Negre, Olivier; Fusil, Floriane; Colomb, Charlotte; Roth, Shoshannah; Gillet-Legrand, Beatrix; Henri, Annie; Beuzard, Yves; Bushman, Frederic; Leboulch, Philippe

    2011-01-01

    A challenge for gene therapy of genetic diseases is to maintain corrected cell populations in subjects undergoing transplantation in cases in which the corrected cells do not have intrinsic selective advantage over nontransduced cells. For inherited hematopoietic disorders, limitations include inefficient transduction of stem cell pools, the requirement for toxic myelosuppression, and a lack of optimal methods for cell selection after transduction. Here, we have designed a lentiviral vector that encodes human β-globin and a truncated erythropoietin receptor, both under erythroid-specific transcriptional control. This truncated receptor confers enhanced sensitivity to erythropoietin and a benign course in human carriers. Transplantation of marrow transduced with the vector into syngenic thalassemic mice, which have elevated plasma erythropoietin levels, resulted in long-term correction of the disease even at low ratios of transduced/untransduced cells. Amplification of the red over the white blood cell lineages was self-controlled and averaged ∼ 100-fold instead of ∼ 5-fold for β-globin expression alone. There was no detectable amplification of white blood cells or alteration of hematopoietic homeostasis. Notwithstanding legitimate safety concerns in the context of randomly integrating vectors, this approach may prove especially valuable in combination with targeted integration or in situ homologous recombination/repair and may lower the required level of pretransplantation myelosuppression. PMID:21436071

  7. Characterization of Equine Infectious Anemia Virus Integration in the Horse Genome

    PubMed Central

    Liu, Qiang; Wang, Xue-Feng; Ma, Jian; He, Xi-Jun; Wang, Xiao-Jun; Zhou, Jian-Hua

    2015-01-01

    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. PMID:26102582

  8. 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/. PMID:23599922

  9. Research plan for integrated ecosystem and pollutant monitoring at remote wilderness study sites

    SciTech Connect

    Bruns, D.A.; Wiersma, G.B.

    1988-03-01

    This research plan outlines an approach to the measurement of pollutants and ecosystem parameters at remote, high-elevation, wilderness study sites. A multimedia, systems approach to environmental monitoring is emphasized. The primary purpose of the research is to apply and field test a technical report entitled ''Guidelines for measuring the physical, chemical, and biological condition of wilderness ecosystems.'' This document intended to provide Federal Land Managers with information to establish environmental monitoring programs in wilderness areas. To date, this monitoring document has yet to be evaluated under rigorous field conditions at a remote, high-elevation Rocky Mountain site. For the purpose of field testing approaches to monitoring of pollutants and ecosystems in remote, wilderness areas, evaluation criteria were developed. These include useability, cost-effectiveness, data variability, alternative approaches, ecosystems conceptual approach, and quality assurance. Both the Forest Service and INEL environmental monitoring techniques will be evaluated with these criteria. Another objective of this research plan is to obtain an integrated data base on pollutants and ecosystem structure and function at a remote study site. The methods tested in this project will be used to acquire these data from a systems approach. This includes multimedia monitoring of air and water quality, soils, and forest, stream, and lake ecosystems. 71 refs., 1 fig., 9 tabs.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26569308

  11. Characterization of the lactococcal temperate phage TP901-1 and its site-specific integration.

    PubMed Central

    Christiansen, B; Johnsen, M G; Stenby, E; Vogensen, F K; Hammer, K

    1994-01-01

    The temperate lactococcal phage TP901-1, induced by UV light from Lactococcus lactis subsp. cremoris 901-1, was characterized. The restriction map was found to be circular, and the packaging of TP901-1 DNA was concluded to occur by a headful mechanism. The pac region was localized on the 38.4-kb phage genome. TP901-1 belongs to the class of P335 phages (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989). Evidence is presented that the phages TP936-1 (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989) and C3-T1 (A. W. Jarvis, V. R. Parker, and M. B. Bianchin, Can. J. Microbiol. 38:398-404, 1992) are very closely related to or are identical to TP901-1. The lytically propagated TP901-1 phages were able to lysogenize both indicator strains Lactococcus cremoris 3107 and Wg2. Lysogenization resulted in site-specific integration of the phage genome into the bacterial chromosome. Only one chromosomal attB site was found in 20 independent lysogens. The attP region of TP901-1 and the attL and attR regions were cloned and sequenced. The results showed a core region of only 5 bp, in which the recombination occurs, followed after a 1-bp mismatch by a 7-bp identical region, TCAAT(T/C)AAGGTAA. This result was further verified by sequencing of the attB region obtained by PCR. An integration vector was constructed with the 6.5-kb EcoRI fragment from TP901-1 containing attP. This vector also functions in the plasmid-free strains, MG1363 and LM0230 with only one specific attB site, strongly indicating a more general use of the TP901-1-based integration vector in lactococci. Images PMID:8106318

  12. Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

    PubMed Central

    McClements, W L; Enquist, L W; Oskarsson, M; Sullivan, M; Vande Woude, G F

    1980-01-01

    Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus. Images PMID:6255187

  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. 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

  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. 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.

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

    PubMed

    Ou, Li; Przybilla, Michael J; Koniar, Brenda L; Whitley, Chester B

    2016-09-01

    Mucopolysaccharidosis type I (MPS I) is a lysosomal disease caused by α-l-iduronidase (IDUA) deficiency and accumulation of glycosaminoglycans (GAG). Lentiviral vector encoding correct IDUA cDNA could be used for treating MPS I. To optimize the lentiviral vector design, 9 constructs were designed by combinations of various promoters, enhancers, and codon optimization. After in vitro transfection into 293FT cells, 5 constructs achieved the highest IDUA activities (5613 to 7358 nmol/h/mg protein). These 5 candidate vectors were then tested by injection (1 × 10(7) TU/g) into neonatal MPS I mice. After 30 days, one vector, CCEoIDW, achieved the highest IDUA levels: 2.6% of wildtype levels in the brain, 9.9% in the heart, 200% in the liver and 257% in the spleen. CCEoIDW achieved the most significant GAG reduction: down 49% in the brain, 98% in the heart, 100% in the liver and 95% in the spleen. Further, CCEoIDW had the lowest transgene frequency, especially in the gonads (0.03 ± 0.01 copies/100 cells), reducing the risk of insertional mutagenesis and germ-line transmission. Therefore, CCEoIDW is selected as the optimal lentiviral vector for treating MPS I disease and will be applied in large animal preclinical studies. Further, taken both in vitro and in vivo comparisons together, codon optimization, use of EF-1α promoter and woodchuck hepatitis virus posttranscriptional response element (WPRE) could enhance transgene expression. These results provided a better understanding of factors contributing efficient transgene expression in lentiviral gene therapies. PMID:27556013

  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. PMID:25048992

  20. 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.

  1. HIV Provirus Stably Reproduces Parental Latent and Induced Transcription Phenotypes Regardless of the Chromosomal Integration Site

    PubMed Central

    Hashemi, Farhad B.; Barreto, Kris; Bernhard, Wendy; Hashemi, Pargol; Lomness, Adam

    2016-01-01

    ABSTRACT Understanding the mechanisms of HIV proviral latency is essential for development of a means to eradicate infection and achieve a cure. We have previously described an in vitro latency model that reliably identifies HIV expression phenotypes of infected cells using a dual-fluorescence reporter virus. Our results have demonstrated that ∼50% of infected cells establish latency immediately upon integration of provirus, a phenomenon termed early latency, which appears to occur by mechanisms that are distinct from epigenetic silencing observed with HIV provirus that establishes productive infections. In this study, we have used a mini-dual HIV reporter virus (mdHIV) to compare the long-term stability of provirus produced as early latent or productive infections using Jurkat-Tat T cell clones. Cloned lines bearing mdHIV provirus integrated at different chromosomal locations display unique differences in responsiveness to signaling agonists and chromatin-modifying compounds, and they also produce characteristic expression patterns from the 5′ long terminal repeat (LTR) dsRed and internal EIF1α-enhanced green fluorescent protein (EIF1α-eGFP) reporters. Furthermore, reporter expression profiles of single cell sorted subcultures faithfully reproduce expression profiles identical to that of their original parental population, following prolonged growth in culture, without shifting toward expression patterns resembling that of cell subclones at the time of sorting. Comparison of population dispersion coefficient (CV) and mean fluorescence intensity (MFI) of the subcloned lines showed that both untreated and phorbol myristate acetate (PMA)-ionomycin-stimulated cultures produce expression patterns identical to those of their parental lines. These results indicate that HIV provirus expression characteristics are strongly influenced by the epigenetic landscape at the site of chromosomal integration. IMPORTANCE There is currently considerable interest in development

  2. Large-scale discovery of insertion hotspots and preferential integration sites of human transposed elements

    PubMed Central

    Levy, Asaf; Schwartz, Schraga; Ast, Gil

    2010-01-01

    Throughout evolution, eukaryotic genomes have been invaded by transposable elements (TEs). Little is known about the factors leading to genomic proliferation of TEs, their preferred integration sites and the molecular mechanisms underlying their insertion. We analyzed hundreds of thousands nested TEs in the human genome, i.e. insertions of TEs into existing ones. We first discovered that most TEs insert within specific ‘hotspots’ along the targeted TE. In particular, retrotransposed Alu elements contain a non-canonical single nucleotide hotspot for insertion of other Alu sequences. We next devised a method for identification of integration sequence motifs of inserted TEs that are conserved within the targeted TEs. This method revealed novel sequences motifs characterizing insertions of various important TE families: Alu, hAT, ERV1 and MaLR. Finally, we performed a global assessment to determine the extent to which young TEs tend to nest within older transposed elements and identified a 4-fold higher tendency of TEs to insert into existing TEs than to insert within non-TE intergenic regions. Our analysis demonstrates that TEs are highly biased to insert within certain TEs, in specific orientations and within specific targeted TE positions. TE nesting events also reveal new characteristics of the molecular mechanisms underlying transposition. PMID:20008508

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Moving target indicating radar applications in an integrated site security suite

    SciTech Connect

    Appenzeller, R.C. )

    1991-01-01

    The integration of a small, lightweight, low power consumption radar into a site security sensor suite can provide several key advantages in the ability to detect vehicles and personnel over large ground areas. This paper presents rationale for the inclusion of a man-portable Moving Target Indicator (MTI) radar in several security scenarios and outlines the technical specifics of a candidate radar. The Department of Energy (DOE) is currently investigating the effectiveness of a combination of optical sensors in concert with a scanning narrow beam radar at the Nevada Test Site in Mercury, Nevada. Demonstration results from these previous test activities are included herein. Of particular interest is the complimentary nature of this sensor suite where the large field of view achievable with radar allows the optical sensors to be used as pinpoint target classification devices. The inclusion of a radar minimizes operator fatigue caused by watching cameras scanning in azimuth and elevation. Advances in the areas of nuisance alarm rejection and improved range detection against single personnel targets were made in 1990 and this capability is included in the current production version.

  8. 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.

  9. The InterPro database, an integrated documentation resource for protein families, domains and functional sites.

    PubMed

    Apweiler, R; Attwood, T K; Bairoch, A; Bateman, A; Birney, E; Biswas, M; Bucher, P; Cerutti, L; Corpet, F; Croning, M D; Durbin, R; Falquet, L; Fleischmann, W; Gouzy, J; Hermjakob, H; Hulo, N; Jonassen, I; Kahn, D; Kanapin, A; Karavidopoulou, Y; Lopez, R; Marx, B; Mulder, N J; Oinn, T M; Pagni, M; Servant, F; Sigrist, C J; Zdobnov, E M

    2001-01-01

    Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk. PMID:11125043

  10. InterPro: an integrated documentation resource for protein families, domains and functional sites.

    PubMed

    Mulder, Nicola J; Apweiler, Rolf; Attwood, Terri K; Bairoch, Amos; Bateman, Alex; Binns, David; Biswas, Margaret; Bradley, Paul; Bork, Peer; Bucher, Phillip; Copley, Richard; Courcelle, Emmanuel; Durbin, Richard; Falquet, Laurent; Fleischmann, Wolfgang; Gouzy, Jerome; Griffith-Jones, Sam; Haft, Daniel; Hermjakob, Henning; Hulo, Nicolas; Kahn, Daniel; Kanapin, Alexander; Krestyaninova, Maria; Lopez, Rodrigo; Letunic, Ivica; Orchard, Sandra; Pagni, Marco; Peyruc, David; Ponting, Chris P; Servant, Florence; Sigrist, Christian J A

    2002-09-01

    The exponential increase in the submission of nucleotide sequences to the nucleotide sequence database by genome sequencing centres has resulted in a need for rapid, automatic methods for classification of the resulting protein sequences. There are several signature and sequence cluster-based methods for protein classification, each resource having distinct areas of optimum application owing to the differences in the underlying analysis methods. In recognition of this, InterPro was developed as an integrated documentation resource for protein families, domains and functional sites, to rationalise the complementary efforts of the individual protein signature database projects. The member databases - PRINTS, PROSITE, Pfam, ProDom, SMART and TIGRFAMs - form the InterPro core. Related signatures from each member database are unified into single InterPro entries. Each InterPro entry includes a unique accession number, functional descriptions and literature references, and links are made back to the relevant member database(s). Release 4.0 of InterPro (November 2001) contains 4,691 entries, representing 3,532 families, 1,068 domains, 74 repeats and 15 sites of post-translational modification (PTMs) encoded by different regular expressions, profiles, fingerprints and hidden Markov models (HMMs). Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (2,141,621 InterPro hits from 586,124 SWISS-PROT and TrEMBL protein sequences). The database is freely accessible for text- and sequence-based searches. PMID:12230031

  11. The InterPro database, an integrated documentation resource for protein families, domains and functional sites

    PubMed Central

    Apweiler, R.; Attwood, T. K.; Bairoch, A.; Bateman, A.; Birney, E.; Biswas, M.; Bucher, P.; Cerutti, L.; Corpet, F.; Croning, M. D. R.; Durbin, R.; Falquet, L.; Fleischmann, W.; Gouzy, J.; Hermjakob, H.; Hulo, N.; Jonassen, I.; Kahn, D.; Kanapin, A.; Karavidopoulou, Y.; Lopez, R.; Marx, B.; Mulder, N. J.; Oinn, T. M.; Pagni, M.; Servant, F.; Sigrist, C. J. A.; Zdobnov, E. M.

    2001-01-01

    Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1 000 000 hits from 462 500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk. PMID:11125043

  12. 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

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

    PubMed

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

    2016-01-01

    Most transgenic technologies rely on the oocyte as a substrate for genetic modification. Transgenics animals are usually generated by the injection of the gene constructs (including lentiviruses encoding gene constructs or modified embryonic stem cells) into the pronucleus of a fertilized egg followed by the transfer of the injected embryos into the uterus of a foster mother. Male germ cells also have potential as templates for transgenic development. We have previously shown that mature sperm can be utilized as template for lentiviral transduction and as such used to generate transgenic mice efficiently with germ line capabilities. We provide here a detailed protocol that is relatively simple, to establish transgenic mice using lentivirally transduced spermatozoa. This protocol employs a well-established lentiviral gene delivery system (usual for somatic cells) delivering a variety of transgenes to be directly used with sperm, and the subsequent use of these modified sperm in in vitro fertilization studies and embryo transfer into foster female mice, for the establishment of transgenic mice. PMID:27317176

  14. 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. PMID:27099176

  15. 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. PMID:20163250

  16. 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

  17. 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. PMID:27317179

  18. 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

    2015-01-01

    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

  19. A bicistronic lentiviral vector-based method for differential transsynaptic tracing of neural circuits.

    PubMed

    Ohashi, Yohei; Tsubota, Tadashi; Sato, Ayana; Koyano, Kenji W; Tamura, Keita; Miyashita, Yasushi

    2011-01-01

    We developed a bicistronic HIV1-derived lentiviral vector system co-expressing green fluorescent protein (AcGFP1) and wheat germ agglutinin (WGA) mediated by picornaviral 2A peptide. This system was first applied to the analysis of the rat cerebellar efferent pathways. When the lentiviral vector was injected into a specific lobule, the local Purkinje cell population (first-order neurons) was efficiently infected and co-expressed both AcGFP1 and WGA protein. In the second-order neurons in the cerebellar and vestibular nuclei, WGA but not AcGFP1 protein was differentially detected, demonstrating that the presence of AcGFP1 protein enables discrimination of first-order neurons from second-order neurons. Furthermore, WGA protein was detected in the contralateral ventrolateral thalamic nucleus (third-order nucleus). This system also successfully labeled rat cortical pathways from the primary somatosensory cortex and monkey cerebellar efferent pathways. Thus, this bicistronic lentiviral vector system is a useful tool for differential transsynaptic tracing of neural pathways originating from local brain regions. PMID:20816792

  20. 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

  1. A Simple and Effective Method to Generate Lentiviral Vectors for Ex Vivo Gene Delivery to Mature Human Peripheral Blood Lymphocytes

    PubMed Central

    Yang, Shicheng; Karne, Neel K.; Goff, Stephanie L.; Black, Mary A.; Xu, Hui; Bischof, Daniela; Cornetta, Kenneth; Rosenberg, Steven A.; Morgan, Richard A.

    2012-01-01

    Abstract 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. PMID:22515320

  2. 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

  3. 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. PMID:22515320

  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. PMID:23299800

  5. 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

  6. Preferential integration of a transfected marker gene into spontaneously expressed fragile sites of a breast cancer cell line.

    PubMed

    Matzner, Isabel; Savelyeva, Larissa; Schwab, Manfred

    2003-01-28

    Common fragile sites are non-randomly distributed unstable chromosomal regions thought to be hot spots for recombination. They appear as gaps, breaks and triradial figures when cells are cultured under conditions that inhibit replication or repair of DNA. The removal of replication-inhibitory challenges is followed by repair activation to restore the DNA damage at the fragile site. The breast cancer cell line MDA-MB-436 has a spontaneous and non-random expression pattern of fragile sites that appear to be related to the complex pattern of chromosomal rearrangements. The high frequency of which fragile sites are spontaneously activated should make MDA-MB-436 cells a powerful tool to study in greater detail the DNA sequences of a multiplicity of fragile sites. Here, we have explored if the DNA at spontaneously activated fragile sites in MDA-MB-436 cells can be genetically tagged by the repair-mediated insertion of an exogenously supplied drug resistance gene. The cells were transfected with pSV2Neo, stably transfected clones were selected with neomycin, and the sites of pSV2Neo integration were determined by fluorescent in situ hybridization. Eighty-eight of 100 isolated clones had a non-random distribution of a total of 112 pSV2Neo integrations. Of these, 95 integrations (85%) coincide with the position at which non-random gaps and breaks appear in the MDA-MB-436 cells. Forty-nine (44%) of the 112 integrations appeared to be at position of known fragile sites, 46 (41%) were at the non-random chromosomal sites not previously described as "true" fragile sites. It is possible, however, that these non-random instabilities signal of genomic regions equivalent to fragile sites, that either have not previously been detected due to low level expression or that are activated in a tissue- or cell-type-specific manner. Collectively, our results show a preferential integration of exogenous DNA into fragile sites and other non-random regions of high genomic instability in MDA

  7. 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

  8. Comparative analysis of HIV-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuronal gene transfer

    PubMed Central

    Federici, Thais; Kutner, Robert; Zhang, Xian-Yang; Kuroda, Hitoshi; Tordo, Noël; Boulis, Nicholas M; Reiser, Jakob

    2009-01-01

    Background The delivery of therapeutic genes to the central nervous system (CNS) using viral vectors represents an appealing strategy for the treatment of nerve injury and disorders of the CNS. Important factors determining CNS targeting include tropism of the viral vectors and retrograde transport of the vector particles. Retrograde transport of equine anemia virus (EIAV)-based lentiviral vectors pseudotyped with the glycoprotein derived from the Rabies virus RabERA strain from peripheral muscle to spinal motor neurons (MNs) was previously reported. Despite therapeutic effects achieved in mouse models of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the efficiency of this approach needs to be improved for clinical translation. To date there has not been a quantitative assessment of pseudotyped HIV-1-based lentiviral vectors to transduce MNs. Here, we describe quantitative tests to analyze the retrograde transport capacity of HIV-1 vectors pseudotyped with the G glycoprotein derived from Rabies and Rabies-related viruses (Lyssaviruses). Methods With a view toward optimizing the retrograde transport properties of HIV-1-based lentiviral vectors, we compared the glycoproteins from different enveloped viruses belonging to the Rhabdoviridae family, genus Lyssavirus, and evaluated their ability to transduce specific cell populations and promote retrograde axonal transport. We first tested the transduction performance of these pseudotypes in vitro in SH-SY5Y neuroblastoma cells, NSC-34 neuroblastoma-spinal cord hybrid cells, and primary mixed spinal cord and pure astrocyte cultures. We then analyzed the uptake and retrograde transport of these pseudotyped vectors in vitro, using Campenot chambers. Finally, intraneural injections were performed to evaluate the in vivo retrograde axonal transport of these pseudotypes. Results Both the in vitro and in vivo studies demonstrated that lentiviral vectors pseudotyped with the glycoprotein derived from the

  9. The Skogaryd Research Site - Integration of terrestrial and freshwater greenhouse gas sources and sinks

    NASA Astrophysics Data System (ADS)

    Klemedtsson, L.

    2012-04-01

    atmosphere. The budgets have ignored dissolved carbon (C) and nitrogen (N) transport in water to streams and lakes and the subsequent exchange between the atmosphere and surface waters. Aquatic habitats can be significant net sources of CO2 and methane (CH4) and potential hot spots for N2O release, all important for natural greenhouse gas (GHG) emissions. Inland waters need to be included in the C and GHG balances for terrestrial landscapes. This project aims to quantify GHG balances at the landscape scale in forested regions that include land-atmosphere, land-water, and water-atmosphere exchange of CO2, CH4 and N2O. Different terrestrial and aquatic ecosystems will be linked holistically, using site specific techniques at different scales, from aircraft (km2) to chambers (m2) to develop integrated models that can be used to quantify net GHG flux for management strategies. The LAGGE project involves six Swedish universities and site is open for more cooperations.

  10. 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

  11. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4(+) T cells.

    PubMed

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F; Metzner, Karin J

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4(+) T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4(+) T cells were over-represented in gene hotspots identified in CD4(+) T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4(+) T cells. PMID:27067385

  12. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4+ T cells

    PubMed Central

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F.; Metzner, Karin J.

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4+ T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4+ T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4+ T cells were over-represented in gene hotspots identified in CD4+ T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4+ T cells. PMID:27067385

  13. Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo.

    PubMed

    Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L; Stavans, Joel

    2014-05-20

    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

  14. Seismic aftershock monitoring for on-site inspection purposes. Experience from Integrated Field Exercise 2008.

    NASA Astrophysics Data System (ADS)

    Labak, P.; Arndt, R.; Villagran, M.

    2009-04-01

    One of the sub-goals of the Integrated Field Experiment in 2008 (IFE08) in Kazakhstan was testing the prototype elements of the Seismic aftershock monitoring system (SAMS) for on-site inspection purposes. The task of the SAMS is to collect the facts, which should help to clarify nature of the triggering event. Therefore the SAMS has to be capable to detect and identify events as small as magnitude -2 in the inspection area size up to 1000 km2. Equipment for 30 mini-arrays and 10 3-component stations represented the field equipment of the SAMS. Each mini-array consisted of a central 3-component seismometer and 3 vertical seismometers at the distance about 100 m from the central seismometer. The mini-arrays covered approximately 80% of surrogate inspection area (IA) on the territory of former Semipalatinsk test site. Most of the stations were installed during the first four days of field operations by the seismic sub-team, which consisted of 10 seismologists. SAMS data center comprised 2 IBM Blade centers and 8 working places for data archiving, detection list production and event analysis. A prototype of SAMS software was tested. Average daily amount of collected raw data was 15-30 GB and increased according to the amount of stations entering operation. Routine manual data screening and data analyses were performed by 2-6 subteam members. Automatic screening was used for selected time intervals. Screening was performed using the Sonoview program in frequency domain and using the Geotool and Hypolines programs for screening in time domain. The screening results were merged into the master event list. The master event list served as a basis of detailed analysis of unclear events and events identified to be potentially in the IA. Detailed analysis of events to be potentially in the IA was performed by the Hypoline and Geotool programs. In addition, the Hyposimplex and Hypocenter programs were also used for localization of events. The results of analysis were integrated

  15. 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

  16. Response of a simian immunodeficiency virus (SIVmac251) to raltegravir: a basis for a new treatment for simian AIDS and an animal model for studying lentiviral persistence during antiretroviral therapy

    PubMed Central

    2010-01-01

    Background In this study we successfully created a new approach to ART in SIVmac251 infected nonhuman primates. This drug regimen is entirely based on drugs affecting the pre-integration stages of replication and consists of only two nucleotidic/nucleosidic reverse transcriptase inhibitors (Nt/NRTIs) and raltegravir, a promising new drug belonging to the integrase strand transfer inhibitor (INSTI) class. Results In acutely infected human lymphoid CD4+ T-cell lines MT-4 and CEMx174, SIVmac251 replication was efficiently inhibited by raltegravir, which showed an EC90 in the low nanomolar range. This result was confirmed in primary macaque PBMCs and enriched CD4+ T cell fractions. In vivo monotherapy with raltegravir for only ten days resulted in reproducible decreases in viral load in two different groups of animals. When emtricitabine (FTC) and tenofovir (PMPA) were added to treatment, undetectable viral load was reached in two weeks, and a parallel increase in CD4 counts was observed. In contrast, the levels of proviral DNA did not change significantly during the treatment period, thus showing persistence of this lentiviral reservoir during therapy. Conclusions In line with the high conservation of the three main amino acids Y143, Q148 and N155 (responsible for raltegravir binding) and molecular docking simulations showing similar binding modes of raltegravir at the SIVmac251 and HIV-1 IN active sites, raltegravir is capable of inhibiting SIVmac251 replication both in tissue culture and in vivo. This finding may help to develop effective ART regimens for the simian AIDS model entirely based on drugs adopted for treatment in humans. This ART-treated AIDS nonhuman primate model could be employed to find possible strategies for virus eradication from the body. PMID:20233398

  17. Selection of Novel Vesicular Stomatitis Virus Glycoprotein Variants from a Peptide Insertion Library for Enhanced Purification of Retroviral and Lentiviral Vectors

    PubMed Central

    Yu, Julie H.; Schaffer, David V.

    2006-01-01

    The introduction of new features or functions that are not present in an original protein is a significant challenge in protein engineering. For example, modifications to vesicular stomatitis virus glycoprotein (VSV-G), which is commonly used to pseudotype retroviral and lentiviral vectors for gene delivery, have been hindered by a lack of structural knowledge of the protein. We have developed a transposon-based approach that randomly incorporates designed polypeptides throughout a protein to generate saturated insertion libraries and a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel designed functionality. This method was applied to VSV-G in order to construct a comprehensive library of mutants whose combined members have a His6 tag inserted at likely every site in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high-titer viral vectors and could be purified by Ni-nitrilotriacetic acid affinity chromatography. Column purification of vectors reduced protein and DNA impurities more than 5,000-fold and 14,000-fold, respectively, from the viral supernatant. This substantially improved purity elicited a weaker immune response in the brain, without altering the infectivity or tropism from wild-type VSV-G-pseudotyped vectors. This work applies a powerful new tool for protein engineering to construct novel viral envelope variants that can greatly improve the safety and use of retroviral and lentiviral vectors for clinical gene therapy. Furthermore, this approach of library generation and selection can readily be extended to other challenges in protein engineering. PMID:16537595

  18. 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

  19. 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

  20. Dietary vitamin A regulates wingless-related MMTV integration site signaling to alter the hair cycle

    PubMed Central

    Suo, Liye; Sundberg, John P; Everts, Helen B.

    2016-01-01

    Alopecia areata 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 alopecia areata in the graft-induced C3H/HeJ mouse model of alopecia areata. In this study we also found that dietary vitamin A, in a dose dependent manner, activated the hair follicle stem cells 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 WNT signaling) and levels of WNT7A (wingless-related MMTV integration site 7A) 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 stem cells. PMID:25361771

  1. Myeloid ecotropic viral integration site 1 (MEIS) 1 involvement in embryonic implantation

    PubMed Central

    Xu, Bei; Geerts, Dirk; Qian, Kun; Zhang, Hanwang; Zhu, Guijin

    2008-01-01

    BACKGROUND The HOXA10 homeobox gene controls embryonic uterine development and adult endometrial receptivity. The three-amino-acid loop extension (TALE) family homeobox genes like myeloid ecotropic viral integration site 1 (MEIS) provide enhanced target gene activation and specificity in HOX-regulated cellular processes by acting as HOX cofactors. METHODS AND RESULTS Analysis of an Affymetrix data set in the public domain showed high expression of MEIS1 in human endometrium. MEIS1 expression was confirmed during the human menstrual cycle by RT–PCR and in situ hybridization and was increased during the secretory compared with proliferative phase of the cycle (P = 0.0001), the time of implantation. To assess the importance of maternal Meis1 expression in a mouse model, the uteri of Day 2 pregnant mice were injected with Meis1 over-expression or small interfering RNA (siRNA) constructs. Blocking Meis1 expression by siRNA before implantation significantly reduced average implantation rates (P = 0.00001). Increased or decreased Meis1 expression significantly increased or decreased the expression of integrin β3, a transcriptional target of HOXA10 and an important factor in early embryo-endometrium interactions (P = 0.006). Manipulating Meis1 expression before implantation also dramatically affected the number of pinopodes, uterine endometrial epithelial projections that develop at the time of endometrial receptivity. CONCLUSIONS The results suggest that in mouse, meis1 contributes to regulating endometrial development during the menstrual cycle and establishing the conditions necessary for implantation. PMID:18408019

  2. Assessing Hydrologic Impacts of Land Configuration Changes Using an Integrated Hydrologic Model at the Rocky Flats Environmental Technology Site, Colorado

    NASA Astrophysics Data System (ADS)

    Prucha, R. H.; Dayton, C. S.; Hawley, C. M.

    2002-12-01

    The Rocky Flats Environmental Technology Site (RFETS) in Golden, Colorado, a former Department of Energy nuclear weapons manufacturing facility, is currently undergoing closure. The natural semi-arid interaction between surface and subsurface flow at RFETS is complex and complicated by the industrial modifications to the flow system. Using a substantial site data set, a distributed parameter, fully-integrated hydrologic model was developed to assess the hydrologic impact of different hypothetical site closure configurations on the current flow system and to better understand the integrated hydrologic behavior of the system. An integrated model with this level of detail has not been previously developed in a semi-arid area, and a unique, but comprehensive, approach was required to calibrate and validate the model. Several hypothetical scenarios were developed to simulate hydrologic effects of modifying different aspects of the site. For example, some of the simulated modifications included regrading the current land surface, changing the existing surface channel network, removing subsurface trenches and gravity drain flow systems, installing a slurry wall and geotechnical cover, changing the current vegetative cover, and converting existing buildings and pavement to permeable soil areas. The integrated flow model was developed using a rigorous physically-based code so that realistic design parameters can simulate these changes. This code also permitted evaluation of changes to complex integrated hydrologic system responses that included channelized and overland flow, pond levels, unsaturated zone storage, groundwater heads and flow directions, and integrated water balances for key areas. Results generally show that channel flow offsite decreases substantially for different scenarios, while groundwater heads generally increase within the reconfigured industrial area most of which is then discharged as evapotranspiration. These changes have significant implications to

  3. Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

    PubMed

    Brown, D P; Idler, K B; Katz, L

    1990-04-01

    The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. PMID:2180909

  4. Analysis of factor VIII mediated suppression of lentiviral vector titres.

    PubMed

    Radcliffe, P A; Sion, C J M; Wilkes, F J; Custard, E J; Beard, G L; Kingsman, S M; Mitrophanous, K A

    2008-02-01

    Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) are near normal, RNA genome levels are reduced and measurement of integrated copies revealed the virus is severely defective in its ability to transduce target cells. This is due to the absence of sufficient vesicular stomatitis virus glycoprotein (VSV-G) envelope on viral particles derived from cells expressing FVIII. By using an internal tissue-specific promoter, that has low activity in the producer cells, to drive expression of FVIII we have overcome this inhibitory effect allowing us to generate titres approaching those obtained with vector genomes encoding reporter genes. Furthermore, we report that codon optimization of the full-length FVIII gene increased vector titres approximately 10-fold in addition to substantially improving expression per integrated vector copy. PMID:18046428

  5. 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

  6. Low Impact Development Design—Integrating Suitability Analysis and Site Planning for Reduction of Post-Development Stormwater Quantity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 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...

  8. 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

  9. Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

    PubMed Central

    Mao, Yingying; Yan, Renhe; Li, Andrew; Zhang, Yanling; Li, Jinlong; Du, Hongyan; Chen, Baihong; Wei, Wenjin; Zhang, Yi; Sumners, Colin; Zheng, Haifa; Li, Hongwei

    2015-01-01

    Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors. Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution. Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction

  10. 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

  11. Combining microseismic and geomechanical observations to interpret storage integrity at the In Salah CCS site

    NASA Astrophysics Data System (ADS)

    Goertz-Allmann, Bettina P.; Kühn, Daniela; Oye, Volker; Bohloli, Bahman; Aker, Eyvind

    2014-07-01

    We present results from microseismic monitoring and geomechanical analysis obtained at the industrial-scale CO2 sequestration site at the In Salah gas development project in Algeria. More than 5000 microseismic events have been detected at a pilot monitoring well using a master event cross-correlation method. The microseismic activity occurs in four distinct clusters and thereof three clearly correlate with injection rates and wellhead pressures. These event clusters are consistent with a location within the reservoir interval. However, due to insufficient network geometry there are large uncertainties on event location. We estimate a fracture pressure of 155 bar (at the wellhead) from the comparison of injection pressure and injection rate and conclude that reservoir fracture pressure of the injection horizon has most likely been exceeded occasionally, accompanied by increased microseismic activity. Our analysis of 3-D ray tracing for direct and converted phases suggests that one of the event clusters is located at a shallower depth than the reservoir injection interval. However, this event cluster is most likely unrelated to changes in the injection activity at a single well, as the event times do not correlate with the wellhead pressures. Furthermore, this event cluster shows b-values close to one, indicating re-activated natural or tectonic seismicity on pre-existing weakness zones rather than injection induced seismicity. Analysis of event azimuths and significant shear wave splitting of up to 5 per cent provide further valuable insight into fluid migration and fracture orientation at the reservoir level. Although only one geophone was available during the critical injection period, the microseismic monitoring of CO2 injection at In Salah is capable of addressing some of the most relevant questions about fluid migration and reservoir integrity. An improved monitoring array with larger aperture and higher sensitivity is highly recommended, as it could greatly

  12. 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

  13. Type II pneumocyte-restricted green fluorescent protein expression after lentiviral transduction of lung epithelial cells.

    PubMed

    Wunderlich, Stephanie; Gruh, Ina; Winkler, Monica E; Beier, Jennifer; Radtke, Kerstin; Schmiedl, Andreas; Groos, Stephanie; Haverich, Axel; Martin, Ulrich

    2008-01-01

    Type II alveolar epithelial (AT2) cell-specific reporter expression has been highly useful in the study of embryology and alveolar regeneration in transgenic mice. Technologies enabling efficient gene transfer and cell type-restricted transgene expression in AT2 cells would allow for correction of AT2 cell-based diseases such as genetic surfactant deficiencies. Moreover, such approaches are urgently required to investigate differentiation of AT2 cells from adult and embryonic stem cells of other species than mouse. Using a human surfactant protein C (SP-C) promoter fragment, we have constructed lentiviral vectors enabling AT2-restricted transgene expression and identification of stem cell-derived AT2 cells. Lung epithelial cell lines M3E3/C3, H441, RLE-6TN, A549, MLE-12, and MLE-15 were characterized at the molecular and ultrastructural levels to identify cell lines useful to assess the cell type specificity of our vector constructs. After transduction, no green fluorescent protein (GFP) expression was observed in nontarget cells including bronchial H441 cells, pulmonary A549 cells, fibroblasts, smooth muscle cells, and endothelial cells. In contrast, and in correlation with endogenous SP-C expression, lentiviral transduction resulted in stable GFP expression in MLE-12 and MLE-15 AT2 cells. In conclusion, we have constructed a lentiviral vector mediating SP-C promoter-dependent GFP expression. Transgene expression strictly corresponds with an AT2 phenotype of the transduced cells. In particular, the generated vector should facilitate local alveolar gene therapy and investigation of alveolar regeneration and stem cell differentiation. PMID:18052721

  14. Primate lentiviral Nef proteins deregulate T-cell development by multiple mechanisms

    PubMed Central

    2013-01-01

    Background A nef gene is present in all primate lentiviral genomes and is important for high viral loads and progression to AIDS in human or experimental macaque hosts of HIV or SIV, respectively. In these hosts, infection of the thymus results in a decreased output of naive T cells that may contribute to the development of immunodeficiency. We have previously shown that HIV-1 subtype B Nef proteins can block human T-cell development. However, the underlying mechanism(s) and the conservation of this Nef function between different groups of HIV and SIV remained to be determined. Results We investigated whether reduction of thymic output is a conserved function of highly divergent lentiviral Nef proteins including those from both types of human immunodeficiency viruses (HIV-1 and HIV-2), their direct simian counterparts (SIVcpz, SIVgor and SIVsmm, respectively), and some additional SIV strains. We found that expression of most of these nef alleles in thymocyte progenitors impaired T-cell development and reduced thymic output. For HIV-1 Nef, binding to active p21 protein (Cdc42/Rac)-activated kinase (PAK2) was a major determinant of this function. In contrast, selective disruption of PAK2 binding did not eliminate the effect on T-cell development of SIVmac239 Nef, as was shown by expressing mutants in a newly discovered PAK2 activating structural motif (PASM) constituted by residues I117, H121, T218 and Y221, as well as previously described mutants. Rather, down-modulation of cell surface CD3 was sufficient for reduced thymic output by SIVmac Nef, while other functions of SIV Nefs contributed. Conclusions Our results indicate that primate lentiviral Nef proteins impair development of thymocyte precursors into T cells in multiple ways. The interaction of HIV-1 Nef with active PAK2 by HIV-1 seem to be most detrimental, and downregulation of CD3 by HIV-2 and most SIV Nef proteins sufficient for reduced thymic output. Since the reduction of thymic output by Nef is a

  15. Lentiviral-based approach for the validation of cancer therapeutic targets in vivo.

    PubMed

    Ambrogio, Chiara; Stern, Patrick; Scuoppo, Claudio; Kranz, Harald; Barbacid, Mariano; Santamaría, David

    2014-10-01

    Despite the pressing need for novel cancer treatments, our improved understanding of tumor biology is not being successfully translated into better therapies. Here we present a lentiviral vector that enables in vivo validation of cancer therapeutic targets when combined with existing cancer animal models that faithfully reproduce the natural history of human disease. Unlike the conventional genetic approaches with targeted alleles, the outlined experimental strategy could be used to assess the preclinical efficacy of a growing number of putative therapeutic hits in a rapid and cost-effective manner. PMID:25312087

  16. Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

    SciTech Connect

    Zhou Qi; Schneider, Irene C.; Gallet, Manuela; Kneissl, Sabrina; Buchholz, Christian J.

    2011-05-10

    The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.

  17. Azimuthal Resistivity Investigation of an Unconfined Aquifer at the Hanford Integrated Field Research Challenge Site

    NASA Astrophysics Data System (ADS)

    Greenwood, W. J.; Ward, A. L.; Versteeg, R. J.; Johnson, T. C.; Draper, K.

    2009-12-01

    Developing a robust large-scale groundwater contaminate transport model requires quantifying the effect of heterogeneity and anisotropy on solute transport. Here we investigated the feasibility of using surface azimuthal resistivity methods to characterize near-surface anisotropy and heterogeneity in order to improve the conceptual model for uranium transport through unconsolidated sediment at the Integrated Field Research Challenge Site (IFRC) which borders the Columbia River. A generalized azimuthal resistivity array was constructed with seven telescoping radii and 15° rotations between each electrode. Azimuthal array data were acquired by multiplexing with the MPT-DAS1 system connected to 172 surface electrodes. Array geometries included the square array, arrow array, offset wenner and equatorial dipole-dipole. Effective depths of exploration ranged between 5 and 57 m. Results from the upper 5m of exploration depth exhibit an isotropic resistivity which is consistent with the excavation and homogonous fill depth of the waste ponds at the IFRC. Exploration depths beyond 5 m are influenced by the Hanford and Ringold Formations. These formations exhibit a strong anisotropic resistivity which increases with depth. Assuming that the response is entirely controlled by hydrologic anisotropy, these azimuthal resistivity data suggest a preferential path with a mean azimuth between 150° and 170°. This azimuthal resistivity trend coincides with an incision feature in the Ringold formation measured in a suite of core logs and is consistent with the trajectory of a tracer plume from an injection test conducted in March 2009. Surface azimuthal resistivity methods may also have application in characterizing localized anisotropy and heterogeneity within shallow alluvial deposits at Hanford allowing for the optimal placement of tracer injections and borehole electrodes.

  18. 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

  19. Integration of surface and upper air measurements for determining evapotranspiration at a high mountain site

    NASA Astrophysics Data System (ADS)

    Connell, Bernadette Helen

    An integrated approach using surface and upper air measurements was used to estimate evapotranspiration in a high mountain area. The first part of this work focused on identifying potential errors introduced into the results due to the characteristics of the sonde instrument and the dynamic environment it was used in. The second part of this work focused on estimating the evapotranspiration. Sources of error were reviewed for radiosonde measurements in the Atmospheric Boundary Layer and focusing on two radiosonde models manufactured by Atmospheric Instrumentation Research, Inc. Temperature and humidity lag errors and wind errors were analyzed. Errors in measurement of azimuth and elevation angles and pressure over short time intervals and at higher altitudes introduced wind vector errors greater than 5 m s-1. Mean temperature and humidity lag errors were small, but individual large lag errors occurred with dramatic changes in the environment, such as near the surface or at the top of the boundary layer. Dual sonde flights showed mean instrument error comparable to lag error. Vertical profiles of potential temperature and specific humidity measured by radiosondes within the surface boundary layer were used to estimate Bowen Ratio at a high elevation alpine site for 2 cases representing moist/windy and dry/calm conditions. The Bowen Ratio was combined with surface global radiation measurements to estimate evaporation (ET) through an energy balance approach. Weather modified vegetation characteristics and their spatial distribution reflect the large local differences in the latent (ET) and sensible heat fluxes measured a 2 surface stations close by. The sonde estimates correlated well (R2 = 0.90) with a weighted average of the surface measured ET in both an undisturbed high pressure ridge system characterized by a dry deep convective boundary layer and during the passage of a weak frontal system characterized by a shallower mechanically mixed moist boundary layer.

  20. INTEGRATIVE ASSESSMENT OF BENTHIC EFFECTS FROM REMEDIAL ACTIVITIES AT SUPERFUND SITES

    EPA Science Inventory

    At contaminated sediment sites, the Superfund program must decide whether to leave the site alone or select a remedial option. These decisions are based in part on the relative risks to the environment and health posed by each alternative. Whatever decision is made at a site, the...

  1. Anti-Apoptotic Effects of Lentiviral Vector Transduction Promote Increased Rituximab Tolerance in Cancerous B-Cells

    PubMed Central

    Ranjbar, Benyamin; Krogh, Louise Bechmann; Laursen, Maria Bach; Primo, Maria Nascimento; Marques, Sara Correia; Dybkær, Karen; Mikkelsen, Jacob Giehm

    2016-01-01

    Diffuse large B-cell lymphoma (DLBCL) is characterized by great genetic and clinical heterogeneity which complicates prognostic prediction and influences treatment efficacy. The most common regimen, R-CHOP, consists of a combination of anthracycline- and immuno-based drugs including Rituximab. It remains elusive how and to which extent genetic variability impacts the response and potential tolerance to R-CHOP. Hence, an improved understanding of mechanisms leading to drug tolerance in B-cells is crucial, and modelling by genetic intervention directly in B-cells is fundamental in such investigations. Lentivirus-based gene vectors are widely used gene vehicles, which in B-cells are an attractive alternative to potentially toxic transfection-based methodologies. Here, we investigate the use of VSV-G-pseudotyped lentiviral vectors in B-cells for exploring the impact of microRNAs on tolerance to Rituximab. Notably, we find that robust lentiviral transduction of cancerous B-cell lines markedly and specifically enhances the resistance of transduced germinal center B-cells (GCBs) to Rituximab. Although Rituximab works partially through complement-mediated cell lysis, increased tolerance is not achieved through effects of lentiviral transduction on cell death mediated by complement. Rather, reduced levels of PARP1 and persistent high levels of CD43 in Rituximab-treated GCBs demonstrate anti-apoptotic effects of lentiviral transduction that may interfere with the outcome and interpretation of Rituximab tolerance studies. Our findings stress that caution should be exercised exploiting lentiviral vectors in studies of tolerance to therapeutics in DLBCL. Importantly, however, we demonstrate the feasibility of using the lentiviral gene delivery platform in studies addressing the impact of specific microRNAs on Rituximab responsiveness. PMID:27045839

  2. Transgenic sheep generated by lentiviral vectors: safety and integration analysis of surrogates and their offspring

    PubMed Central

    Cornetta, Kenneth; Tessanne, Kimberly; Long, Charles; Yao, Jing; Satterfield, Carey; Westhusin, Mark

    2012-01-01

    The safety of HIV-1 based vectors was evaluated during the production of transgenic sheep. Vectors were introduced into the perivitelline space of in vivo derived one-cell sheep embryos by microinjection then transferred into the oviducts of recipient females. At 60–70 days of gestation, a portion of the recipients were euthanized and tissues collected from both surrogates and fetuses. Other ewes were allowed to carry lambs to term. Inadvertent transfer of vector from offspring to surrogates was evaluated in 330 blood and tissue samples collected from 57 ewes that served as embryo recipients. Excluding uterine contents, none of the samples tested positive for vector, indicating that that the vector did not cross the fetal maternal interface and infect surrogate ewes. Evaluating ewes, fetuses and lambs for replication competent lentivirus (RCL); 84 serum samples analyzed for HIV-1 capsid by ELISA and over 600 blood and tissue samples analyzed by quantitative PCR for the VSV-G envelopes revealed no evidence of RCL. Results of these experiments provide further evidence as to the safety of HIV-1 based vectors in animal and human applications. PMID:23180364

  3. 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. PMID:25082736

  4. 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

  5. Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children.

    PubMed Central

    Yaginuma, K; Kobayashi, H; Kobayashi, M; Morishima, T; Matsuyama, K; Koike, K

    1987-01-01

    Attention was directed to hepatitis B virus (HBV) integration in tissues obtained from an hepatocellular carcinoma (HCC) of an 11-year-old boy and from the liver of his 6-year-old brother, who had chronic active hepatitis. Multiple HBV DNA integration sites were demonstrated in both tissues. Cell population(s) in the HCC and liver from the patient with chronic active hepatitis were assumed to be heterogeneous with regard to HBV integration. The integrated forms in the two tissues showed similar genetic organization without gross rearrangement. The location of one of the virus-chromosomal junctions was restricted to the 5'-end region of the minus-strand DNA of HBV. The experimental results support our previous model for the mechanism of HBV integration, in which minus-strand replicative intermediates integrate into chromosomal DNA. The integrated HBV DNAs were conserved in the same region of the viral genome, spanning from the C gene through the S gene to the X gene, which contains intrinsic promoter-enhancer sequences. Images PMID:3033312

  6. The piggyBac transposon displays local and distant reintegration preferences and can cause mutations at noncanonical integration sites.

    PubMed

    Li, Meng Amy; Pettitt, Stephen J; Eckert, Sabine; Ning, Zemin; Rice, Stephen; Cadiñanos, Juan; Yusa, Kosuke; Conte, Nathalie; Bradley, Allan

    2013-04-01

    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

  7. 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

  8. Integral Equations in the Study of Polar and Ionic Interaction Site Fluids

    NASA Astrophysics Data System (ADS)

    Howard, Jesse J.; Pettitt, B. Montgomery

    2011-10-01

    We consider some of the current integral equation approaches and application to model polar liquid mixtures. We show the use of multidimensional integral equations and in particular progress on the theory and applications of three dimensional integral equations. The IEs we consider may be derived from equilibrium statistical mechanical expressions incorporating a classical Hamiltonian description of the system. We give example including salt solutions, inhomogeneous solutions and systems including proteins and nucleic acids.

  9. Integral equations in the study of polar and ionic interaction site fluids

    PubMed Central

    Howard, Jesse J.

    2011-01-01

    In this review article we consider some of the current integral equation approaches and application to model polar liquid mixtures. We consider the use of multidimensional integral equations and in particular progress on the theory and applications of three dimensional integral equations. The IEs we consider may be derived from equilibrium statistical mechanical expressions incorporating a classical Hamiltonian description of the system. We give example including salt solutions, inhomogeneous solutions and systems including proteins and nucleic acids. PMID:22383857

  10. 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. PMID:15876215

  11. Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy

    PubMed Central

    Negro-Demontel, María Luciana; Saccardo, Paolo; Giacomini, Cecilia; Yáñez-Muñoz, Rafael Joaquín; Ferrer-Miralles, Neus; Vazquez, Esther; Villaverde, Antonio; Peluffo, Hugo

    2014-01-01

    Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available. Gene therapy applications have emerged as important alternatives for the treatment of diverse nervous system injuries. New strategies are evolving with the notion that each particular pathological condition may require a specific vector. Moreover, the lack of detailed comparative studies between different vectors under similar conditions hampers the selection of an ideal vector for a given pathological condition. The potential use of lentiviral vectors versus several modular protein-based nanovectors was compared using a controlled cortical impact model of TBI under the same gene therapy conditions. We show that variables such as protein/DNA ratio, incubation volume, and presence of serum or chloroquine in the transfection medium impact on both nanovector formation and transfection efficiency in vitro. While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days. No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1β or using neurological sticky tape test. In fact, both vector types induced functional improvement per se. PMID:26015985

  12. A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors.

    PubMed

    Miskin, J; Chipchase, D; Rohll, J; Beard, G; Wardell, T; Angell, D; Roehl, H; Jolly, D; Kingsman, S; Mitrophanous, K

    2006-02-01

    Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination. PMID:16208418

  13. Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages

    PubMed Central

    Pan, Hui; Mostoslavsky, Gustavo; Eruslanov, Evgeny; Kotton, Darrell N.

    2008-01-01

    In-depth studies of innate immunity require efficient genetic manipulation of macrophages, which is especially difficult in primary macrophages. We have developed a lentiviral system for inducible gene expression both in macrophage cell lines and in primary macrophages. A transgenic mouse strain C3H.TgN(SRA-rtTA) that expresses reverse tetracycline transactivator (rtTA) under the control of macrophage-specific promoter, a modified human scavenger receptor A (SR-A) promoter was generated. For gene delivery, we constructed a dual-promoter lentiviral vector, in which expression of a “gene-of-interest” is driven by a doxycycline-inducible promoter and the expression of a selectable surface marker is driven by an independent constitutive promoter UBC. This vector is used for transduction of bone marrow-derived macrophage precursors. The transduced cells can be enriched to 95–99% purity using marker-specific monoclonal antibodies, expanded and differentiated into mature macrophages or myeloid dendritic cells. We also successfully used this approach for inducible protein expression in hard to transfect macrophage cell lines. Because many proteins, which are expressed by activated or infected macrophages, possess cytotoxic, anti-proliferative or pro-apoptotic activities, generation of stable macrophage cell lines that constitutively express those proteins is impossible. Our method will be especially useful to study immunity-related macrophage proteins in their physiological context during macrophage activation or infection. PMID:17967462

  14. Rapid generation of dendritic cell specific transgenic mice by lentiviral vectors.

    PubMed

    Zhang, Jinyu; Zou, Liyun; Liu, Qin; Li, Jingyi; Zhou, Jingran; Wang, Yong; Li, Na; Liu, Ting; Wei, Hong; Wu, Min; Wan, Ying; Wu, Yuzhang

    2009-12-01

    Dendritic cell (DC) specific transgenic mice are a most important model for investigating dendritic cell functions in vivo. Recently, lentivirus mediated gene transfer has become a powerful and convenient method for generation of transgenic mice. We cloned a 1.2 kb CD11c promoter and constructed a lentiviral vector, which efficiently drove DC-specific expression in vitro. After microinjection of purified virus into the perivitelline space of single-cell embryo, more than 80% newborn mice were transgenic and 7 F0 founders were rapidly generated in 2 months. GFP was strictly expressed in CD11c+ cells in spleens, thymus and lymph nodes of the transgenic mice. Importantly, the physiological characteristics and functions of DCs in the transgenic mice were not altered by the specific expression. These results indicate that this vector could be used to rapidly prepare DC-specific transgenic mice. Thus, this lentiviral vector system may provide a convenient and useful tool to study the properties of DCs in vivo. PMID:19468852

  15. [Construction and identification of recombinant lentiviral vector of hNoc4L gene].

    PubMed

    Wang, Tingting; Wang, Shujuan; Yan, Jinghua

    2010-11-01

    Formation and nuclear export of pre-ribosomes requires many nucleolar complexes, hNoc4L which contains a conserved Noc doman is a homolog of nucleolar complex associated 4 (S. cerevisiae), but its function is completely unclear. Here, we successfully got the recombinant lentiviral vector p113.7-EF1-hNoc4L-Flag by replacing the U6 promoter in p113.7 with EF1alpha promoter, and then inserted hNoc4L to down-stream of the EF1alpha prompter. We determined the transduction efficiency in different mammalian cell lines based on lentiviral packaging system. Subsequently, we analyzed the immunogenicity of the recombinant lentivirus and stable expression of hNoc4L in RAW264.7 cells. The results showed that the recombinant lentivirus characterized a high transduction efficiency, long-term expression and low immunogenicity. Therefore, we pave the way for further identification of the biological activity of hNoc4L protein during ribosome biogenesis in mammalian. PMID:21284218

  16. Correction of Methylmalonic Aciduria In Vivo Using a Codon-Optimized Lentiviral Vector

    PubMed Central

    Wong, Edward S.Y.; McIntyre, Chantelle; Peters, Heidi L.; Ranieri, Enzo; Anson, Donald S.

    2014-01-01

    Abstract Methylmalonic aciduria is a rare disorder of organic acid metabolism with limited therapeutic options, resulting in high morbidity and mortality. Positive results from combined liver/kidney transplantation suggest, however, that metabolic sink therapy may be efficacious. Gene therapy offers a more accessible approach for the treatment of methylmalonic aciduria than organ transplantation. Accordingly, we have evaluated a lentiviral vector–mediated gene transfer approach in an in vivo mouse model of methylmalonic aciduria. A mouse model of methylmalonic aciduria (Mut−/−MUTh2) was injected intravenously at 8 weeks of age with a lentiviral vector that expressed a codon-optimized human methylmalonyl coenzyme A mutase transgene, HIV-1SDmEF1αmurSigHutMCM. Untreated Mut−/−MUTh2 and normal mice were used as controls. HIV-1SDmEF1αmurSigHutMCM-treated mice achieved near-normal weight for age, and Western blot analysis demonstrated significant methylmalonyl coenzyme A enzyme expression in their livers. Normalization of liver methylmalonyl coenzyme A enzyme activity in the treated group was associated with a reduction in plasma and urine methylmalonic acid levels, and a reduction in the hepatic methylmalonic acid concentration. Administration of the HIV-1SDmEF1αmurSigHutMCM vector provided significant, although incomplete, biochemical correction of methylmalonic aciduria in a mouse model, suggesting that gene therapy is a potential treatment for this disorder. PMID:24568291

  17. Expression Profiles of Vpx/Vpr Proteins Are Co-related with the Primate Lentiviral Lineage

    PubMed Central

    Sakai, Yosuke; Miyake, Ariko; Doi, Naoya; Sasada, Hikari; Miyazaki, Yasuyuki; Adachi, Akio; Nomaguchi, Masako

    2016-01-01

    Viruses of human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency virus (SIV) lineages carry a unique accessory protein called Vpx. Vpx is essential or critical for viral replication in natural target cells such as macrophages and T lymphocytes. We have previously shown that a poly-proline motif (PPM) located at the C-terminal region of Vpx is required for its efficient expression in two strains of HIV-2 and SIVmac, and that the Vpx expression levels of the two clones are significantly different. Notably, the PPM sequence is conserved and confined to Vpx and Vpr proteins derived from certain lineages of HIV-2/SIVs. In this study, Vpx/Vpr proteins from diverse primate lentiviral lineages were experimentally and phylogenetically analyzed to obtain the general expression picture in cells. While both the level and PPM-dependency of Vpx/Vpr expression in transfected cells varied among viral strains, each viral group, based on Vpx/Vpr amino acid sequences, was found to exhibit a characteristic expression profile. Moreover, phylogenetic tree analyses on Gag and Vpx/Vpr proteins gave essentially the same results. Taken together, our study described here suggests that each primate lentiviral lineage may have developed a unique expression pattern of Vpx/Vpr proteins for adaptation to its hostile cellular and species environments in the process of viral evolution. PMID:27536295

  18. Highly Efficient Large-Scale Lentiviral Vector Concentration by Tandem Tangential Flow Filtration

    PubMed Central

    Cooper, Aaron R.; Patel, Sanjeet; Senadheera, Shantha; Plath, Kathrin; Kohn, Donald B.; Hollis, Roger P.

    2014-01-01

    Large-scale lentiviral vector (LV) concentration can be inefficient and time consuming, often involving multiple rounds of filtration and centrifugation. This report describes a simpler method using two tangential flow filtration (TFF) steps to concentrate liter-scale volumes of LV supernatant, achieving in excess of 2000-fold concentration in less than 3 hours with very high recovery (>97%). Large volumes of LV supernatant can be produced easily through the use of multi-layer flasks, each having 1720 cm2 surface area and producing ~560 mL of supernatant per flask. Combining the use of such flasks and TFF greatly simplifies large-scale production of LV. As a demonstration, the method is used to produce a very high titer LV (>1010 TU/mL) and transduce primary human CD34+ hematopoietic stem/progenitor cells at high final vector concentrations with no overt toxicity. A complex LV (STEMCCA) for induced pluripotent stem cell generation is also concentrated from low initial titer and used to transduce and reprogram primary human fibroblasts with no overt toxicity. Additionally, a generalized and simple multiplexed real- time PCR assay is described for lentiviral vector titer and copy number determination. PMID:21784103

  19. Lentiviral gene therapy using cellular promoters cures type 1 Gaucher disease in mice.

    PubMed

    Dahl, Maria; Doyle, Alexander; Olsson, Karin; Månsson, Jan-Eric; Marques, André R A; Mirzaian, Mina; Aerts, Johannes M; Ehinger, Mats; Rothe, Michael; Modlich, Ute; Schambach, Axel; Karlsson, Stefan

    2015-05-01

    Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease. PMID:25655314

  20. Expression Profiles of Vpx/Vpr Proteins Are Co-related with the Primate Lentiviral Lineage.

    PubMed

    Sakai, Yosuke; Miyake, Ariko; Doi, Naoya; Sasada, Hikari; Miyazaki, Yasuyuki; Adachi, Akio; Nomaguchi, Masako

    2016-01-01

    Viruses of human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency virus (SIV) lineages carry a unique accessory protein called Vpx. Vpx is essential or critical for viral replication in natural target cells such as macrophages and T lymphocytes. We have previously shown that a poly-proline motif (PPM) located at the C-terminal region of Vpx is required for its efficient expression in two strains of HIV-2 and SIVmac, and that the Vpx expression levels of the two clones are significantly different. Notably, the PPM sequence is conserved and confined to Vpx and Vpr proteins derived from certain lineages of HIV-2/SIVs. In this study, Vpx/Vpr proteins from diverse primate lentiviral lineages were experimentally and phylogenetically analyzed to obtain the general expression picture in cells. While both the level and PPM-dependency of Vpx/Vpr expression in transfected cells varied among viral strains, each viral group, based on Vpx/Vpr amino acid sequences, was found to exhibit a characteristic expression profile. Moreover, phylogenetic tree analyses on Gag and Vpx/Vpr proteins gave essentially the same results. Taken together, our study described here suggests that each primate lentiviral lineage may have developed a unique expression pattern of Vpx/Vpr proteins for adaptation to its hostile cellular and species environments in the process of viral evolution. PMID:27536295

  1. Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice

    PubMed Central

    Dahl, Maria; Doyle, Alexander; Olsson, Karin; Månsson, Jan-Eric; Marques, André R A; Mirzaian, Mina; Aerts, Johannes M; Ehinger, Mats; Rothe, Michael; Modlich, Ute; Schambach, Axel; Karlsson, Stefan

    2015-01-01

    Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease. PMID:25655314

  2. Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients.

    PubMed

    Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Lozano, M Luz; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

    2009-06-01

    Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs). PMID:19277017

  3. Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients

    PubMed Central

    Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Luz Lozano, M; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

    2009-01-01

    Previous clinical trials based on the genetic correction of purified CD34+ cells with γ-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34+ cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34+ cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34− mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs). PMID:19277017

  4. Lack of evidence of conserved lentiviral sequences in pigs with post weaning multisystemic wasting syndrome.

    PubMed Central

    Bratanich, A; Lairmore, M; Heneine, W; Konoby, C; Harding, J; West, K; Vasquez, G; Allan, G; Ellis, J

    1999-01-01

    In order to investigate the role of retroviruses in the recently described porcine postweaning multisystemic wasting syndrome (PMWS) serum and leukocytes were screened for reverse transcriptase (RT) activity, and tissues were examined for the presence of conserved lentiviral sequences using degenerate primers in a polymerase chain reaction (PCR). Serum and stimulated leukocytes from the blood and lymph nodes from pigs with PMWS, as well as from control pigs had RT activity that was detected by the sensitive Amp-RT assay. A 257-bp fragment was amplified from DNA from the blood and bone marrow of pigs with PMWS. This fragment was identical in size to conserved lentiviral sequences that were amplified from plasmids containing DNA from several lentiviruses. Cloning and sequencing of the fragment from affected pigs, however, did not reveal homology with the recognized lentiviruses. Together the results of these analyses suggest that the RT activity present in tissues from control and affected pigs is the result of endogenous retrovirus expression, and that a lentivirus is not a primary pathogen in PMWS. Images Figure 1. Figure 2. PMID:10480463

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

    PubMed

    Cheng, Pei-Hsun; Chang, Yu-Fan; Mao, Su-Han; Lin, Hsiu-Lien; Chen, Chuan-Mu; Yang, Shang-Hsun

    2016-10-01

    Transgenic animals are important in vivo models for biological research. However, low transgenic rates are commonly reported in the literature. Lentiviral transgenesis is a promising method that has greater efficiency with regard to generating transgenic animals, although the transgenic rate of this approach is highly dependent on different transgenes and concentrated lentiviruses. In this study, we modified a method to concentrate lentiviruses using a table centrifuge, commonly available in most laboratories, and carried out analysis of the transgenic efficiency in mice. Based on 26 individual constructs and 627 live pups, we found that the overall transgenic rate was more than 30%, which is higher than obtained with pronuclear microinjection. In addition, we did not find any significant differences in transgenic efficiency when the size of inserts was less than 5000 bp. These results not only show that our modified method can successfully generate transgenic mice but also suggest that this approach could be generally applied to different constructs when the size of inserts is less than 5000 bp. It is anticipated that the results of this study can help encourage the wider laboratory use of lentiviral transgenesis in mice. PMID:27264740

  6. Pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from Chandipura and Piry viruses.

    PubMed

    Hu, Shuang; Mohan Kumar, Dipu; Sax, Chelsea; Schuler, Clayton; Akkina, Ramesh

    2016-01-15

    While the envelope glycoprotein of vesicular stomatitis virus (VSV-G) is widely used for pseudotyping of lentiviral vectors, sub-optimal gene transfer into certain cell types and its sensitivity to inactivation by human complement hinders its broader applications. To find alternative candidates, here we evaluated two serologically distinct novel viral envelopes derived from Chandipura (CNV-G) and Piry (PRV-G) vesiculoviruses. Both permitted generation of high titer psuedotyped lentiviral vectors with a capacity for high efficiency gene transfer into various cell types from different species. In human lymphoid and hematopoietic stem cells, their transduction efficiency was significantly lower than that of VSV-G. However, both novel envelopes were found to be more resistant to inactivation by human serum complement compared to VSV-G. Thus CNV-G and PRV-G envelopes can be harnessed for multiple uses in the future based on the cell type that needs to be gene transduced and possibly for in vivo gene transfer. PMID:26650691

  7. The Eastern Lau Basin Integrated Studies Site (ISS): Recent Progress and Future Plans

    NASA Astrophysics Data System (ADS)

    Wiens, D.; Martinez, F.; Langmuir, C.; Tivey, M.; Childress, J.; Fisher, C.; Wheat, G.; Perfit, M.; Blackman, D.; Kim, S.

    2004-12-01

    Rapid progress is being made in understanding the Eastern Lau Spreading Center (ELSC) Integrated Studies Site, which is a new focus area in the Ridge 2000 Program. The ELSC, located in the western Pacific near Tonga, is a 390 km-long first-order ridge segment that displays a broad range of effects of the back-arc environment. Its southern end, at only 40 km from the Tonga arc volcanic front, is propagating southward into a back-arc rift. Its northern end is 100 km from the volcanic front and terminates at a large, nontransform offset. The ELSC undergoes substantial and systematic changes in primary parameters affecting crustal accretion including spreading rate and mantle source composition. As a consequence it displays large changes along its length in lava chemistry, axial depth and morphology, melt lens characteristics, and crustal thickness and structure. A focus of the work at the ELSC is to understand how changes in these forcing functions affect crustal accretion, hydrothermal venting, and faunal composition and abundance. A geophysical/hydrothermal study of the entire ELSC during an initial R2K cruise in April-May 2004 (PI: Martinez) disclosed a surprisingly high level of hydrothermal activity along the ELSC. This survey involved shipboard multibeam, two deep-towed sonars (DSL120A and IMI30), and concurrent MAPR/Chemical Scanner, CTD/rosette tow-yos, and vertical casts. Hydrothermal activity as indicated by water column plumes increases toward the north, even though magmatic robustness decreases. An ancillary study (PI: Thurnherr) also deployed autonomous floats during this cruise to investigate deep circulation patterns that affect hydrothermal plumes and faunal dispersal. A second cruise in September 2004 (PI: Langmuir) focused on locating vent sources using ABE, petrological sampling, and determining water column properties. Three additional cruises are scheduled during 2005. The first (PI: Tivey) will provide an initial characterization of vent fields

  8. 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

    ... to protect the security and integrity of urine collections? 40.43 Section 40.43 Transportation Office... PROGRAMS Collection Sites, Forms, Equipment and Supplies Used in DOT Urine Collections § 40.43 What steps must operators of collection sites take to protect the security and integrity of urine collections?...

  9. 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

    ... to protect the security and integrity of urine collections? 40.43 Section 40.43 Transportation Office... PROGRAMS Collection Sites, Forms, Equipment and Supplies Used in DOT Urine Collections § 40.43 What steps must operators of collection sites take to protect the security and integrity of urine collections?...

  10. 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. PMID:22503155

  11. 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.

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

    PubMed

    Grandchamp, Nicolas; Altémir, Dorothée; Philippe, Stéphanie; Ursulet, Suzanna; Pilet, Héloïse; Serre, Marie-Claude; Lenain, Aude; Serguera, Che; Mallet, Jacques; Sarkis, Chamsy

    2014-01-01

    Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable. PMID:24956106

  13. Gene therapy for hemophilia B with liver-specific element mediated by Rep-RBE site-specific integration system.

    PubMed

    Xu, Zhengxin; Ye, Juan; Zhang, Amin; Xie, Linjun; Shen, Qi; Xue, Jinglun; Chen, Jinzhong

    2015-02-01

    Adeno-associated virus (AAV) is a nonpathogenic virus capable of targeting human chromosome 19 for integration at AAVS1 site, and a 16 bp Rep binding element (RBE) sequence of AAV was sufficient for mediating this specific integration in the presence of AAV regulation proteins (Rep). Previously, we cotransduced 2 plasmids, pRBE-CMV-hFIX and pRC, into the AAVS1 transgenic mice by hydrodynamic injection, and a long-term expression of human coagulation Factor IX (hFIX) was observed. The corresponding AAVS1 locus site-specific integrations were verified by nested polymerase chain reaction. In this study, we established a novel hFIX expression plasmid, pRBE-HCR-hAAT-hFIX, driven by a liver-specific promoter by replacing the CMV promoter of pRBE-CMV-hFIX with a humanized promoter consisting of HCR-hAAT. The expression of hFIX in vitro was almost the same in transient transfection of pRBE-CMV-hFIX or pRBE-HCR-hAAT-hFIX. AAVS1-specific integrations were identified both in mice transfected with pRC/pRBE-CMV-hFIX cocktail and pRC/pRBE-HCR-hAAT-hFIX cocktail. However, the expression of hFIX of pRBE-HCR-hAAT-hFIX mice was higher and persisted longer. It achieved more than 1% of normal plasma hFIX concentration and maintained for 240 days. The result suggested that RBE-HCR-hAAT element could improve the expression of hFIX and present potential usage of Rep-RBE site-specific integration in gene therapy for hemophilia B. PMID:25295466

  14. Specific Targeting of Human Interleukin (IL)-13 Receptor α2-Positive Cells with Lentiviral Vectors Displaying IL-13

    PubMed Central

    Ou, Wu; Marino, Michael P.; Suzuki, Akiko; Joshi, Bharat; Husain, Syed R.; Maisner, Andrea; Galanis, Evanthia; Puri, Raj K.

    2012-01-01

    Abstract The ability to selectively and efficiently target transgene delivery to specific cell types in vitro and in vivo remains one of the formidable challenges in gene therapy. Lentiviral vectors have several advantages that make them attractive as gene delivery vehicles and their tropism can be altered through pseudotyping, allowing transgene delivery to specific populations of cells. The human interleukin-13 receptor α2 (IL-13Rα2) is uniquely overexpressed in many different human tumors, making it an attractive target for cancer therapy. In this study, we examined whether IL-13Rα2-positive tumor cells can be specifically targeted with lentiviral vector pseudotypes containing a truncated fusion (F) protein derived from measles virus (MV) and a tail-truncated and receptor-blind MV hemagglutinin (H) protein bearing IL-13 at the C terminus. The retargeted lentiviral vector efficiently transduced cells that express high levels of IL-13Rα2, but not cells expressing low levels of IL-13Rα2 in vitro. In vivo, it specifically targeted IL-13Rα2-positive glioma cell xenografts in immunodeficient mice in the context of subcutaneous and intracranial glioma models. Similar lentiviral vectors may be developed for targeting other tumors expressing specific cell surface receptors. PMID:22612657

  15. Inhibition of Intracellular Antiviral Defense Mechanisms Augments Lentiviral Transduction of Human Natural Killer Cells: Implications for Gene Therapy

    PubMed Central

    Sutlu, Tolga; Nyström, Sanna; Gilljam, Mari; Stellan, Birgitta; Applequist, Steven E.

    2012-01-01

    Abstract Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approach for cancer treatment. Yet, optimization of highly efficient and clinically applicable gene transfer protocols for NK cells still presents a challenge. In this study, we aimed at identifying conditions under which optimum lentiviral gene transfer to NK cells can be achieved. Our results demonstrate that stimulation of NK cells with interleukin (IL)-2 and IL-21 supports efficient transduction using a VSV-G pseudotyped lentiviral vector. Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. We were able to boost the efficiency of lentiviral genetic modification on average 3.8-fold using BX795, an inhibitor of the TBK1/IKKɛ complex acting downstream of RIG-I, MDA-5, and TLR3. We have also observed that the use of BX795 enhances lentiviral transduction efficiency in a number of human and mouse cell lines, indicating a broadly applicable, practical, and safe approach that has the potential of being applicable to various gene therapy protocols. PMID:22779406

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

    SciTech Connect

    Graf, Marcus; Ludwig, Christine; Kehlenbeck, Sylvia; Jungert, Kerstin; Wagner, Ralf . E-mail: ralf.wagner@klinik.uni-regensburg.de

    2006-09-01

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

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

    PubMed Central

    Askou, Anne Louise; Aagaard, Lars; Kostic, Corinne; Arsenijevic, Yvan; Hollensen, Anne Kruse; Bek, Toke; Jensen, Thomas Gryesten; Mikkelsen, Jacob Giehm; Corydon, Thomas Juhl

    2015-01-01

    Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration. PMID:26052532

  18. 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.

  19. 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. PMID:26921546

  20. 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)

  1. Role of base excision repair in maintaining the genetic and epigenetic integrity of CpG sites

    PubMed Central

    Bellacosa, Alfonso; Drohat, Alexander C.

    2016-01-01

    Cytosine methylation at CpG dinucleotides is a central component of epigenetic regulation in vertebrates, and the base excision repair (BER) pathway is important for maintaining both the genetic stability and the methylation status of CpG sites. This perspective focuses on two enzymes that are of particular importance for the genetic and epigenetic integrity of CpG sites, Methyl Binding Domain 4 (MBD4) and Thymine DNA Glycosylase (TDG). We discuss their capacity for countering C to T mutations at CpG sites, by initiating base excision repair of G·T mismatches generated by deamination of 5-methylcytosine (5mC). We also consider their role in active DNA demethylation, including pathways that are initiated by oxidation and/or deamination of 5mC. PMID:26021671

  2. 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).

  3. Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase.

    PubMed

    De Paepe, Annelies; De Buck, Sylvie; Nolf, Jonah; Van Lerberge, Els; Depicker, Ann

    2013-07-01

    Random T-DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T-DNA copies, the risk of mutating the host genome and the difficulty of stacking well-defined traits. Therefore, recombination systems have been proposed to integrate the T-DNA at a pre-selected site in the host genome. Here, we demonstrate the capacity of the ϕC31 integrase (INT) for efficient targeted T-DNA integration. Moreover, we show that the iterative site-specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre-selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T-DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site-specific integration (SSI) of this T-DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium-mediated integration, allowing the serial integration of transgenic DNA sequences in plants. PMID:23574114

  4. 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.

  5. 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.

  6. Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors

    PubMed Central

    2011-01-01

    Background Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages. Results Human blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1α promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocyte-derived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494. Conclusions This report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages. PMID:21281514

  7. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

    PubMed Central

    Weitzman, M D; Kyöstiö, S R; Kotin, R M; Owens, R A

    1994-01-01

    AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration. Images PMID:8016070

  8. 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.

  9. 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. PMID:27191866

  10. Integrated technologies to evaluate the impact of air emissions at a petroleum recovery Superfund site

    SciTech Connect

    Zarus, G.M.; Wagaman, S.A.; Trespalacios, M.J.; Turpin, R.D.; Forrester, T.; Wilder, L.; Meyer, J.; Hansen, M.

    1995-12-31

    The US Environmental Protection Agency`s Environmental Response Team (US EPA/ERT) was requested by US EPA Region 6 to verify or refute the presence of volatile organic air emissions at a petroleum recovery Superfund (PRS) site. Several environmental protection and public health organizations were involved with evaluating the air emissions at this PRS site, including: Region 6 of the US EPA, the Agency for Toxic Substances and Disease Registry (ATSDR), and the US EPA/ERT. Each group brought a unique expertise which helped in identifying sources, determining their emission rates, evaluating their impact, and monitoring during remedial activities. This report describes the technologies used at the PRS site and some of the results obtained. The compounds of concern included: benzene, bis(2-chloroethyl)ether, 1,2-dichloroethene, methylene chloride, styrene, toluene, 1,1,2-trichloroethane, and vinyl chloride.

  11. Integration of site-specific health information: Agency for Toxic Substances and Disease Registry health assessments

    SciTech Connect

    Lesperance, A.M.; Siegel, M.R.

    1990-12-01

    The Agency for Toxic Substances and Disease Registry is required to conduct a health assessment of any site that is listed on or proposed for the US Environmental Protection Agency's National Priorities List. Sixteen US Department of Energy (DOE) sites currently fall into this category. Health assessments contain a qualitative description of impacts to public health and the environment from hazardous waste sites, as well as recommendations for actions to mitigate or eliminate risk. Because these recommendations may have major impacts on compliance activities at DOE facilities, the health assessments are an important source of information for the monitoring activities of DOE's Office of Environmental Compliance (OEC). This report provides an overview of the activities involved in preparing the health assessment, its role in environmental management, and its key elements.

  12. Scientific Opportunities for Monitoring at Environmental Remediation Sites (SOMERS): Integrated Systems-Based Approaches to Monitoring

    SciTech Connect

    Bunn, Amoret L.; Wellman, Dawn M.; Deeb, Rula A.; Hawley, Elizabeth L.; Truex, Michael J.; Peterson, Mark; Freshley, Mark D.; Pierce, Eric M.; McCord, John; Young, Michael H.; Gilmore, Tyler J.; Miller, Rick; Miracle, Ann L.; Kaback, Dawn; Eddy-Dilek, Carol; Rossabi, Joe; Lee, Michelle H.; Bush, Richard P.; Beam , Paul; Chamberlain, G. M.; Marble, Justin; Whitehurst, Latrincy; Gerdes, Kurt D.; Collazo, Yvette

    2012-05-15

    Through an inter-disciplinary effort, DOE is addressing a need to advance monitoring approaches from sole reliance on cost- and labor-intensive point-source monitoring to integrated systems-based approaches such as flux-based approaches and the use of early indicator parameters. Key objectives include identifying current scientific, technical and implementation opportunities and challenges, prioritizing science and technology strategies to meet current needs within the DOE complex for the most challenging environments, and developing an integrated and risk-informed monitoring framework.

  13. Stable gene transfer to the nervous system using a non-primate lentiviral vector.

    PubMed

    Mitrophanous, K; Yoon, S; Rohll, J; Patil, D; Wilkes, F; Kim, V; Kingsman, S; Kingsman, A; Mazarakis, N

    1999-11-01

    We have constructed a non-primate lentiviral vector system based on the equine infectious anaemia virus (EIAV). This system is able to transduce both dividing and non-dividing cells, including primary cultured hippocampal neurons and neurons and glia in the adult rat central nervous system (CNS), at efficiencies comparable with HIV-based vectors. We demonstrate that the only EIAV proteins required for this activity are gag/pol and that the only accessory protein required for vector production is rev. In addition, we show that the pol encoded dUTPase activity that is found in all non-primate lentiviruses is not required. The vectors can be pseudotyped with a range of envelopes including rabies G and MLV 4070A and can be concentrated to high titres. The ability of EIAV to infect mitotically inactive cells makes this vector an attractive alternative to the immunodeficiency viruses for gene therapy. PMID:10602376

  14. SK-HEP cells and lentiviral vector for production of human recombinant factor VIII.

    PubMed

    da Rosa, Nathalia Gonsales; Swiech, Kamilla; Picanço-Castro, Virgínia; Russo-Carbolante, Elisa Maria de Sousa; Soares Neto, Mario Abreu; de Castilho-Fernandes, Andrielle; Faça, Vitor Marcel; Fontes, Aparecida Maria; Covas, Dimas Tadeu

    2012-08-01

    Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs. PMID:22488441

  15. Airway gene transfer in a non-human primate: lentiviral gene expression in marmoset lungs.

    PubMed

    Farrow, N; Miller, D; Cmielewski, P; Donnelley, M; Bright, R; Parsons, D W

    2013-01-01

    Genetic therapies for cystic fibrosis (CF) must be assessed for safety and efficacy, so testing in a non-human primate (NHP) model is invaluable. In this pilot study we determined if the conducting airways of marmosets (n = 2) could be transduced using an airway pre-treatment followed by an intratracheal bolus dose of a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector (LacZ reporter). LacZ gene expression (X-gal) was assessed after 7 days and found primarily in conducting airway epithelia as well as in alveolar regions. The LacZ gene was not detected in liver or spleen via qPCR. Vector p24 protein bio-distribution into blood was transient. Dosing was well tolerated. This preliminary study confirmed the transducibility of CF-relevant airway cell types. The marmoset is a promising NHP model for testing and translating genetic treatments for CF airway disease towards clinical trials. PMID:23412644

  16. Integration of Rooftop Photovoltaic Systems in St. Paul Ford Site's Redevelopment Plans

    SciTech Connect

    Olis, D.; Mosey, G.

    2015-03-01

    The purpose of this analysis is to estimate how much electricity the redeveloped Ford Motor Company assembly plant site in St. Paul, Minnesota, might consume under different development scenarios and how much rooftop photovoltaic (PV) generation might be possible at the site. Because the current development scenarios are high-level, preliminary sketches that describe mixes of residential, retail, commercial, and industrial spaces, electricity consumption and available rooftop area for PV under each scenario can only be grossly estimated. These results are only indicative and should be used for estimating purposes only and to help inform development goals and requirements moving forward.

  17. 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.

  18. The role of HTLV-1 clonality, proviral structure, and genomic integration site in adult T-cell leukemia/lymphoma

    PubMed Central

    Cook, Lucy B.; Melamed, Anat; Niederer, Heather; Valganon, Mikel; Laydon, Daniel; Foroni, Letizia; Taylor, Graham P.; Matsuoka, Masao; Bangham, Charles R. M.

    2014-01-01

    Adult T-cell leukemia/lymphoma (ATL) occurs in ∼5% of human T-lymphotropic virus type 1 (HTLV-1)–infected individuals and is conventionally thought to be a monoclonal disease in which a single HTLV-1+ T-cell clone progressively outcompetes others and undergoes malignant transformation. Here, using a sensitive high-throughput method, we quantified clonality in 197 ATL cases, identified genomic characteristics of the proviral integration sites in malignant and nonmalignant clones, and investigated the proviral features (genomic structure and 5′ long terminal repeat methylation) that determine its capacity to express the HTLV-1 oncoprotein Tax. Of the dominant, presumed malignant clones, 91% contained a single provirus. The genomic characteristics of the integration sites in the ATL clones resembled those of the frequent low-abundance clones (present in both ATL cases and carriers) and not those of the intermediate-abundance clones observed in 24% of ATL cases, suggesting that oligoclonal proliferation per se does not cause malignant transformation. Gene ontology analysis revealed an association in 6% of cases between ATL and integration near host genes in 3 functional categories, including genes previously implicated in hematologic malignancies. In all cases of HTLV-1 infection, regardless of ATL, there was evidence of preferential survival of the provirus in vivo in acrocentric chromosomes (13, 14, 15, 21, and 22). PMID:24735963

  19. Integrated mild gasification processing at the Homer City Electric Power Generating Station site. Final report, July 1989--June 1993

    SciTech Connect

    Battista, J.J.; Zawadzki, E.A.

    1993-07-01

    A new process for the production of commercial grade coke, char, and carbon products has been evaluated by Penelec/NYSEG. The process, developed by Coal Technology Corporation, CTC, utilizes a unique screw reactor to produce a devolatilized char from a wide variety of coals for the production of commercial grade coke for use in blast furnaces, foundries, and other processes requiring high quality coke. This process is called the CTC Mild Gasification Process (MGP). The process economics are significantly enhanced by integrating the new technology into an existing power generating complex. Cost savings are realized by the coke producer, the coke user, and the electric utility company. Site specific economic studies involving the Homer City Generating Station site in Western Pennsylvania, confirmed that an integrated MGP at the Homer City site, using coal fines produced at the Homer City Coal Preparation Plant, would reduce capital and operating costs significantly and would enable the HC Owners to eliminate thermal dryers, obtain low cost fuel in the form of combustible gases and liquids, and obtain lower cost replacement coal on the spot market. A previous report, identified as the Interim Report on the Project, details the technical and economic studies.

  20. Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison.

    PubMed

    Wright, Edward; Temperton, Nigel J; Marston, Denise A; McElhinney, Lorraine M; Fooks, Anthony R; Weiss, Robin A

    2008-09-01

    Cross-neutralization between rabies virus (RABV) and two European bat lyssaviruses (EBLV-1 and -2) was analysed using lentiviral pseudotypes as antigen vectors. Glycoprotein (G-protein) cDNA from RABV challenge virus standard-11 (CVS-11) and EBLV-1 and -2 were cloned and co-expressed with human immunodeficiency virus (HIV) or murine leukemia virus (MLV) gag-pol and packageable green fluorescent protein (GFP) or luciferase reporter genes in human cells. The harvested lentiviral (HIV) vector infected over 40% of baby hamster kidney (BHK) target cells, providing high-titre pseudotype stocks. Tests on blinded antibody-positive (n=15) and -negative (n=45) sera, predetermined by the fluorescent antibody virus neutralization (FAVN) test approved by the World Health Organization (WHO) and Office International des Epizooties (OIE), revealed that the CVS-11 pseudotype assay had 100% concordance with FAVN and strongly correlated with neutralization titres (r2=0.89). Cross-neutralization tests using sera from RABV-vaccinated humans and animals on pseudotypes with CVS-11, EBLV-1 and EBLV-2 envelopes showed that the relative neutralization titres correlated broadly with the degree of G-protein diversity. Pseudotypes have three major advantages over live-virus neutralization tests: (i) they can be handled in low-biohazard-level laboratories; (ii) the use of reporter genes such as GFP or beta-galactosidase will allow the assay to be undertaken at low cost in laboratories worldwide; (iii) each assay requires <10 microl serum. This robust microassay will improve our understanding of the protective humoral immunity that current rabies vaccines confer against emerging lyssaviruses, and will be applicable to surveillance studies, thus helping to control the spread of rabies. PMID:18753230

  1. Integrating Vocational & Academic Education. A Handbook Featuring Four Demonstration Sites Including Students from Special Populations.

    ERIC Educational Resources Information Center

    Tindall, Lloyd W.; And Others

    This handbook describes the processes and techniques used to develop, implement, and evaluate four integrated vocational and academic learning programs in Wisconsin that included students from special populations. The handbook contains seven chapters. Chapter 1 presents an overview of the project, including the request for proposal process and…

  2. Implementation of an Integrated, Portable Transformer Condition Monitoring Instrument in the Classroom and On-Site

    ERIC Educational Resources Information Center

    Chatterjee, B.; Dey, D.; Chakravorti, S.

    2010-01-01

    The development of integrated, portable, transformer condition monitoring (TCM) equipment for classroom demonstrations as well as for student exercises conducted in the field is discussed. Demonstrations include experimentation with real-world transformers to illustrate concepts such as polarization and depolarization current through oil-paper…

  3. SITE EMERGING TECHNOLOGY SUMMARY: INTEGRATION OF PHOTOCATALYTIC OXIDATION WITH AIR STRIPPING OF CONTAMINATED AQUIFERS

    EPA Science Inventory

    In a recently completed test program, bench-scale laboratory studies at Arizona State University (ASU) in Tempe, AZ, and pilot-scale studies in a simulated field test situation at Zentox Corp in Ocala, FL, were performed to evaluate the integration of gas-solid ultraviolet (UV) p...

  4. Enhanced Potency of a Broadly Neutralizing HIV-1 Antibody In Vitro Improves Protection against Lentiviral Infection In Vivo

    PubMed Central

    Rudicell, Rebecca S.; Kwon, Young Do; Ko, Sung-Youl; Pegu, Amarendra; Louder, Mark K.; Georgiev, Ivelin S.; Wu, Xueling; Zhu, Jiang; Boyington, Jeffrey C.; Chen, Xuejun; Shi, Wei; Yang, Zhi-yong; Doria-Rose, Nicole A.; McKee, Krisha; O'Dell, Sijy; Schmidt, Stephen D.; Chuang, Gwo-Yu; Druz, Aliaksandr; Soto, Cinque; Yang, Yongping; Zhang, Baoshan; Zhou, Tongqing; Todd, John-Paul; Lloyd, Krissey E.; Eudailey, Joshua; Roberts, Kyle E.; Donald, Bruce R.; Bailer, Robert T.; Ledgerwood, Julie; Mullikin, James C.; Shapiro, Lawrence; Koup, Richard A.; Graham, Barney S.; Nason, Martha C.; Connors, Mark; Haynes, Barton F.; Rao, Srinivas S.; Roederer, Mario; Kwong, Peter D.

    2014-01-01

    ABSTRACT Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans. IMPORTANCE In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope

  5. Long-term integrating samplers for indoor air and sub slab soil gas at VI sites

    EPA Science Inventory

    Vapor intrusion (VI) site assessments are plagued by substantial spatial and temporal variability that makes exposure and risk assessment difficult. Most risk-based decision making for volatile organic compound (VOC) exposure in the indoor environment is based on health benchmark...

  6. Ambulatory Care in Medical Residency: Integration of Private Practitioner's Office with Traditional Sites.

    ERIC Educational Resources Information Center

    Bane, Susan; Criscione, Teri

    1983-01-01

    An Albany Medical College program utilizes private internists' offices as sites for resident ambulatory education. The private practitioner is perceived as an excellent teacher and role model who provides a positive effect on the long-term career goals of residents. (MLW)

  7. UAS Integration in the NAS Project Test Site Kick-off Meeting

    NASA Technical Reports Server (NTRS)

    Kopardekar, Parimal; Witzberger, Kevin; Hackenberg, Davis L.; Murphy, Jim

    2015-01-01

    This briefing was presented during the Test Site Kick Off Meeting to discuss the contract awards for Task 1 and Task 2. This briefing covered a high level overview for contract deliverables, Task 1 - UAS Traffic Management and Task 2, Live Virtual Constructive Distributed Environment.

  8. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    PubMed Central

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  9. EVALUATING NOVEL SENSORS INTEGRATED WITH A MEMBRANE INTERFACE PROBE FOR IN SITU CHARACTERIZATION OF CONTAMINATED SITES

    EPA Science Inventory

    This project will demonstrate use of several existing sensor technologies and validate a new innovative suite of sensor technologies interfaced with an in-situ probe, for real-time characterization and delineation of VOCs at contaminated sites. Field tests will be performed at v...

  10. Reference site selection for wetland condition assessments: Integrating best professional judgement and objective selection criteria

    EPA Science Inventory

    The National Wetlands Condition Assessment (NWCA), one of a series of water assessments being conducted by states, tribes, the U.S. Environmental Protection Agency (EPA), and other partners, surveyed over 900 wetland sites across the lower 48 states during Summer 2011. The NWCA ...

  11. An Integrated Decision Model for Evaluating Educational Web Sites from the Fuzzy Subjective and Objective Perspectives

    ERIC Educational Resources Information Center

    Huang, Tony Cheng-Kui; Huang, Chih-Hong

    2010-01-01

    With advances in information and network technologies, lots of data have been digitized to reveal information for users by the construction of Web sites. Unfortunately, they are both overloading and overlapping in Internet so that users cannot distinguish their quality. To address this issue in education, Hwang, Huang, and Tseng proposed a group…

  12. Integrated Cropland and Grassland Flux Tower Observation Sites over Grazinglands for Quantifying Surface-Atmosphere Exchange

    NASA Astrophysics Data System (ADS)

    Mahan, H. R.; Wagle, P.; Bajgain, R.; Zhou, Y.; Basara, J. B.; Xiao, X.; Duckles, J. M.; Steiner, J. L.; Starks, P. J.; Northup, B. K.

    2014-12-01

    Quantifying methane (CH4), carbon dioxide (CO2), and water vapor fluxes between land surface and boundary layer using the eddy covariance method have many applicable uses across several disciplines. Three eddy flux towers have been established over no-till winter wheat (Triticum aestivum L.), and native and improved pastures at the USDA ARS Grazinglands Research Laboratory, El Reno, OK. An additional tower will be established in fall 2014 over till winter wheat. Each flux site is equipped with an eddy covariance system, PhenoCam, COSMOS, and in-situ observations of soil and atmospheric state variables. The objective of this research is to measure, compare, and model the land-atmosphere exchange of CO2, water vapor, and CH4 in different land cover types and management practices (till vs no-till, grazing vs no-grazing, native vs improved pasture). Models that focus on net ecosystem CO2 exchange (NEE), gross primary production (GPP), evapotranspiration (ET), and CH4 fluxes can be improved by the cross verification of these measurements. Another application will be to link the in-situ measurements with satellite remote sensing in order to scale-up flux measurements from small spatial scales to local and regional scales. Preliminary data analysis from the native grassland site revealed that CH4 concentration was negligible (~ 0), and it increased significantly when cattle were introduced into the site. In summer 2014, daily ET magnitude was about 4-5 mm day-1 and the NEE magnitude was 4-5 g C day-1 at the native grassland site. Further analysis of data for all the sites for longer temporal periods will enhance understanding of biotic and abiotic factors that govern carbon, water, and energy exchanges between the land surface and atmosphere under different land cover and management systems. The research findings will help predict the responses of these ecosystems to management practices and global environmental change in the future.

  13. Integrated wastewater management planning for DOE`s Rocky Flats Environmental Technology Site

    SciTech Connect

    Hopkins, J.; Barthel, J.; Wheeler, M.; Conroy, K.

    1996-02-01

    Rocky Mountain Remediation Services, L.L.C. (RMRS), jointly formed by Morrison Knudsen Corporation and BNFL Inc., provides international experience in the nuclear, environmental, waste management, decontamination and decommissioning (D&D) , and project management industry. The company is currently the environmental restoration, waste management, and D&D subcontractor for Kaiser-Hill Company at the Rocky Flats Environmental Technology Site (RFETS). RMRS offers unique solutions and state-of-the-art technology to assist in resolving the issues that face industries today. RMRS has been working on methods to improve cost savings recognized at RFETS, through application of unique technologies and process engineering. RMRS prepared and is implementing a strategy that focused on identifying an approach to improve cost savings in current wastewater treatment systems and to define a low-cost, safe and versatile wastewater treatment system for the future. Development of this strategy, was targeted by Department of Energy (DOE) Headquarters, DOE Rocky Flats Field Office and Kaiser-Hill as a ``Project Breakthrough`` where old concepts were thrown out the door and the project goals and objectives were developed from the groundup. The objectives of the strategy developed in a project break through session with DOE included lower lifecycle costs, shutdown of one of two buildings at RFETS, Building 374 or Building 774, reduced government capital investment, and support of site closure program goals, identified as the site`s Accelerated Site Action Plan (ASAP). The recommended option allows for removal of water treatment functions from Building 374, the existing process wastewater treatment facility. This option affords the lowest capital cost, lowest unit operating cost, lowest technical management risk, greatest support of ASAP phasing and provides the greatest flexibility for design with unforeseen future needs.

  14. HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

    Cancer.gov

    Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

  15. HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

    Cancer.gov

    Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

  16. The role of short-range Cys171-Cys178 disulfide bond in maintaining cutinase active site integrity: A molecular dynamics simulation

    SciTech Connect

    Matak, Mehdi Youssefi; Moghaddam, Majid Erfani

    2009-12-11

    Understanding structural determinants in enzyme active site integrity can provide a good knowledge to design efficient novel catalytic machineries. Fusarium solani pisi cutinase with classic triad Ser-His-Asp is a promising enzyme to scrutinize these structural determinants. We performed two MD simulations: one, with the native structure, and the other with the broken Cys171-Cys178 disulfide bond. This disulfide bond stabilizes a turn in active site on which catalytic Asp175 is located. Functionally important H-bonds and atomic fluctuations in catalytic pocket have been changed. We proposed that this disulfide bond within active site can be considered as an important determinant of cutinase active site structural integrity.

  17. Novel integration sites at the distal 3' end of the c-myb locus in retrovirus-induced promonocytic leukemias.

    PubMed

    Nazarov, V; Wolff, L

    1995-06-01

    In BALB/c nu/nu and sublethally irradiated DBA/2 mice, promonocytic leukemia was induced by intravenous inoculation of Friend murine leukemia virus (F-MuLV) strain C57 in conjunction with intraperitoneal injection of pristane. These tumors appear to be identical morphologically to previously reported ones induced by other MuLVs, such as Moloney, amphotropic 4070A, and F-MuLV FB29, which most commonly have provirus integrations in the 5' end of the c-myb locus. Interestingly, 2 of the 16 F-MuLV-induced tumors had viruses integrated in the distal 3' end of c-myb. To determine the precise locations of these integrations, it was necessary to clone sequences encoding the 3' c-myb exons and to prepare a physical map of this region. Exons 10 to 15 were positioned on the map, and it was found that the proviruses in the aforementioned tumors were located within narrow region in the beginning of the large (greater than 11 kb) intron 14. The predicted protein product encoded by the affected alleles is truncated by 38 amino acids. This represents a novel virus integration site which is most likely associated with oncogenic activation of the c-myb gene during leukemogenesis. PMID:7745739

  18. Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

    PubMed

    Liu, Xianglei; Lin, Jun; Hu, Haifeng; Zhou, Bin; Zhu, Baoquan

    2016-01-01

    As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production. PMID:26672454

  19. Integrated chemical management system: A tool for managing chemical information at the Rocky Flats Environmental Technology Site

    SciTech Connect

    Costain, D.

    1995-07-01

    The Integrated Chemical Management System is a computer-based chemical information at the Rocky Flats Environmental Technology Site. Chemical containers are identified by bar code labels and information on the type, quantity and location of chemicals are tracked on individual data bases in separate buildings. Chemical inventories from multiple buildings are uploaded to a central sitewide chemical data base where reports are available from Product, Waste, and Chemical Use modules. Hazardous chemical information is provided by a separate Material Safety Data Sheet module and excess chemicals are traded between chemical owners and users with the aid of the Chemical Exchange Module.

  20. A new integrated approach to field GC/MS for on-site analysis

    SciTech Connect

    Eckenrode, B.

    1997-12-31

    There is a large amount of redundancy in commercially available standalone peripheral sampling devices especially with regard to the power supplies, controllers, and embedded intelligence. The integration of sampling control as well as the sampling/desorbing devices reduces the need for many cumbersome {open_quotes}external{close_quotes} sample handling methods. The integration of sampling and analysis strategies also provides a simpler portability profile for the overall system. In the design of portable analytical instrumentation, developers inevitably confront tradeoffs between overall system performance and the issues of size, weight, power requirements, and cost. It is clear that to a limited degree most field chemists will compromise size, weight, power requirements, and cost before they compromise performance. As we develop smaller and lighter systems, it is our intent to provide an instrumentation platform that is adaptable and upgradable, allowing the customer a choice of system compromises.

  1. Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes

    PubMed Central

    Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B.

    2016-01-01

    Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5′→3′, 3′ →5′ or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically. Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm PMID:27515825

  2. Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes.

    PubMed

    Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B

    2016-01-01

    Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5'→3', 3' →5' or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically.Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm. PMID:27515825

  3. Development of an integrated strategy for the disposal of solid low level waste at BNFL`s Drigg site

    SciTech Connect

    Higson, S.G.

    1989-11-01

    During the past 12 months, the first phase of a major upgrading of disposal operations at Drigg has been completed. This has involved the introduction of waste containerization and orderly emplacement in open concrete vaults. A further phase over the next few years will involve the introduction of compaction of all suitable waste. While the current upgrade has clearly resulted in a major improvement in the visual impact and management control of the site, the desire to implement such an improvement on a timescale consistent with the short term need for new facilities at Drigg has not allowed sufficient time for a detailed assessment of the full implications of the proposed system. This paper describes the development of the strategy for upgrading the Drigg site, highlights improvements that have been implemented as the project has progressed and outlines major outstanding concerns, particularly in relation to long term site management, that may eventually lead to a requirement for further optimization of the overall strategy. Progress under the Drigg Technical Development Programme is reviewed with specific emphasis on the preliminary results of engineering studies aimed at defining an integrated strategy that will meet the requirements of both acceptable visual impact and long term site stability and safety.

  4. Preoperational test report, cross-site transfer system integrated test (POTR-007)

    SciTech Connect

    Pacquet, E.A.

    1998-04-02

    This report documents the results obtained during the performance of Preoperational Test POTP-007, from December 12, 1997 to March 27, 1998. The main objectives were to demonstrate the operation of the following Cross-Site Transfer System components: Booster pumps P-3125A and P-3125B interlocks and controls, both local and remote; Booster pump P-3125A and P-3125B and associated variable speed drives VSD-1 and VSD-2 performance in both manual and automatic modes; and Water filling, circulation, venting and draining of the transfer headers (supernate and slurry line). As described in reference 1, the following components of the Cross-Site Transfer System that would normally be used during an actual waste transfer, are not used in this specific test: Water Flush System; Valving and instrumentation associated with the 241-SY-A valve pit jumpers; and Valving and instrumentation associated with the 244-A lift station.

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

    PubMed Central

    2011-01-01

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

  6. Complete Genome Sequence of Streptomyces parvulus 2297, Integrating Site-Specifically with Actinophage R4

    PubMed Central

    Miura, Takamasa; Harada, Chizuko; Guo, Yong; Narisawa, Kazuhiko; Ohta, Hiroyuki; Takahashi, Hideo; Shirai, Makoto

    2016-01-01

    Streptomyces parvulus 2297, which is a host for site-specific recombination according to actinophage R4, is derived from the type strain ATCC 12434. Species of S. parvulus are known as producers of polypeptide antibiotic actinomycins and have been considered for industrial applications. We herein report for the first time the complete genome sequence of S. parvulus 2297. PMID:27563047

  7. Complete Genome Sequence of Streptomyces parvulus 2297, Integrating Site-Specifically with Actinophage R4.

    PubMed

    Nishizawa, Tomoyasu; Miura, Takamasa; Harada, Chizuko; Guo, Yong; Narisawa, Kazuhiko; Ohta, Hiroyuki; Takahashi, Hideo; Shirai, Makoto

    2016-01-01

    Streptomyces parvulus 2297, which is a host for site-specific recombination according to actinophage R4, is derived from the type strain ATCC 12434. Species of S. parvulus are known as producers of polypeptide antibiotic actinomycins and have been considered for industrial applications. We herein report for the first time the complete genome sequence of S. parvulus 2297. PMID:27563047

  8. Integration of plant-based canopy sensors for site-specific nitrogen management

    NASA Astrophysics Data System (ADS)

    Shiratsuchi, Luciano Shozo

    The soil's nitrogen (N) supply can vary drastically in the field, spatially as well as temporally making any soil prediction difficult even with very detailed mapping. Consequently, a plant-based approach wherein the measured canopy can indicate the N needs in a reactive and spatially-variable way can be a better approach than mapping, because integrate the soil N supply and translate the crop need on-the-go. The first experiment evaluated the performance of various spectral indices for sensing N status of corn, where spectral variability might be confounded by water-induced variations in crop reflectance. We found that water and previous crops effects on vegetation indices (VI) must be considered, and also that some VIs are less susceptible to water with good ability for N differentiation. In the second experiment, the objective was to develop an approach that relies on local soil conditions as well as on active canopy sensor measurements for real-time adjustment of N application rate. We found that local variations in plant N availability must be considered to determine the optimal N rate on-the-go, and that the localized reference incorporated the spatial variability of the N-rich plot. Next, we determined the correlation between active canopy sensors assessments of N availability and ultrasonic sensor measurements of canopy height at several growth stages for corn. We found strong correlations between both sensors and that they had similar abilities to distinguish N-mediated differences in canopy development. The integrated use of both sensors improved the N estimation compared to the isolated use of either sensor. Based on these strong correlations, we developed an N recommendation algorithm based on ultrasonic plant height measurements to be used for on-the-go variable rate N application. Lastly, we evaluated the crop water status using infrared thermometry integrated with optical and ultrasonic sensors, we concluded that the integration of sensors was

  9. Integrated risk and recovery monitoring of ecosystem restorations on contaminated sites

    USGS Publications Warehouse

    Hooper, Michael J.; Glomb, Stephen J.; Harper, David; Hoelzle, Timothy B.; McIntosh, Lisa M.; Mulligan, David R.

    2016-01-01

    Ecological restorations of contaminated sites balance the human and ecological risks of residual contamination with the benefits of ecological recovery and the return of lost ecological function and ecosystem services. Risk and recovery are interrelated dynamic conditions, changing as remediation and restoration activities progress through implementation into long-term management and ecosystem maturation. Monitoring restoration progress provides data critical to minimizing residual contaminant risk and uncertainty, while measuring ecological advancement toward recovery goals. Effective monitoring plans are designed concurrently with restoration plan development and implementation and are focused on assessing the effectiveness of activities performed in support of restoration goals for the site. Physical, chemical, and biotic measures characterize progress toward desired structural and functional ecosystem components of the goals. Structural metrics, linked to ecosystem functions and services, inform restoration practitioners of work plan modifications or more substantial adaptive management actions necessary to maintain desired recovery. Monitoring frequency, duration, and scale depend on specific attributes and goals of the restoration project. Often tied to restoration milestones, critical assessment of monitoring metrics ensures attainment of risk minimization and ecosystem recovery. Finally, interpretation and communication of monitoring findings inform and engage regulators, other stakeholders, the scientific community, and the public. Because restoration activities will likely cease before full ecosystem recovery, monitoring endpoints should demonstrate risk reduction and a successional trajectory toward the condition established in the restoration goals. A detailed assessment of the completed project's achievements, as well as unrealized objectives, attained through project monitoring, will determine if contaminant risk has been minimized, if injured

  10. 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

    Ecological restorations of contaminated sites balance the human and ecological risks of residual contamination with the benefits of ecological recovery and the return of lost ecological function and ecosystem services. Risk and recovery are interrelated dynamic conditions, changing as remediation and restoration activities progress through implementation into long-term management and ecosystem maturation. Monitoring restoration progress provides data critical to minimizing residual contaminant risk and uncertainty, while measuring ecological advancement toward recovery goals. Effective monitoring plans are designed concurrently with restoration plan development and implementation and are focused on assessing the effectiveness of activities performed in support of restoration goals for the site. Physical, chemical, and biotic measures characterize progress toward desired structural and functional ecosystem components of the goals. Structural metrics, linked to ecosystem functions and services, inform restoration practitioners of work plan modifications or more substantial adaptive management actions necessary to maintain desired recovery. Monitoring frequency, duration, and scale depend on specific attributes and goals of the restoration project. Often tied to restoration milestones, critical assessment of monitoring metrics ensures attainment of risk minimization and ecosystem recovery. Finally, interpretation and communication of monitoring findings inform and engage regulators, other stakeholders, the scientific community, and the public. Because restoration activities will likely cease before full ecosystem recovery, monitoring endpoints should demonstrate risk reduction and a successional trajectory toward the condition established in the restoration goals. A detailed assessment of the completed project's achievements, as well as unrealized objectives, attained through project monitoring, will determine if contaminant risk has been minimized, if injured

  11. Apodized structures for the integration of defect sites into photonic lattices

    SciTech Connect

    Boguslawski, Martin Kelberer, Andreas; Rose, Patrick; Denz, Cornelia

    2014-09-15

    We introduce a versatile concept to optically induce photonic structures of local refractive index modulations as well as photonic lattices holding single defect sites. For a given structure, we develop a set of nondiffracting beams obtained by fractionalizing the corresponding spatial spectrum. By combining this set in a multiplexing procedure, we achieve an incoherent combination of all individual structures of the set resulting in a locally addressable refractive index manipulation. We exemplarily present experimental results for apodized, meaning locally confined index changes in a photorefractive crystal resembling a sixfold and a circular symmetric structure. By an additional multiplexing step, we furthermore create periodic photonic lattices featuring embedded defects.

  12. Integration of geological, geochemical, and geophysical spatial data of the Cement oil field, Oklahoma, test site

    USGS Publications Warehouse

    Termain, Patricia A.; Donovan, Terrence J.; Chavez, Pat S.

    1980-01-01

    Measurement pertaining to geology, geochemistry, and geophysics of the Cement oil field, Oklahoma, test site were collected employing both airborne sensors and ground-based data collection. The measurements include: (1) airborne gamma-ray spectrometry (supplying bismuth 214, thalium 208, and potassium 40 gamma-ray intensities); (2) aeromagnetic survey data; (3) multi-frequency airborne resistivity survey data (supplying apparent electrical resistivity of near surface materials); (4) gravity data; (5) geological and topographic maps; and (6) image data from Landsat MSS and U-2 photography.

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

    SciTech Connect

    McCormack, R.L.

    1995-08-01

    This document describes the plans of the Hanford Site for the safe interim storage of fissile materials. Currently, spent nuclear fuels reside in storage basins that have leaked in the past and are projected to leak in the future. Other problems in the basins include; sludge from decomposition, degraded cladding of fuel elements, and construction defects which make the basins seismically unsafe. This management plan describes the time and cost that it will take to implement a safe interim storage plan for the fissile materials.

  14. Integrated ecological hazard assessment of waste site soil extracts using FETAX and short-term fathead minnow teratogenesis assay

    SciTech Connect

    Fort, D.J.; Stover, E.L.; Bantle, J.A.

    1996-12-31

    Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a 96-h whole embryo-larval assay designed to detect environmental developmental toxicants for use in ecological hazard assessment. FETAX offers several advantages in integrated biological hazard assessment including, time- and cost-effectiveness, technical ease, and versatility. FETAX has undergone extensive intra- and more recently interlaboratory validation with known mammalian teratogens and non-teratogens. Ecological hazard evaluations of contaminated sediments, waste site soils, and complex surface and groundwaters have also been performed. An integrated hazard assessment study using FETAX, the conventional, Pimephales promelas 7-d teratogenecity test, and an abbreviated P. promelas teratogenecity test utilizing the general FETAX protocol was conducted with specific reference toxicants and aqueous extracts of contaminated hazardous waste site soils. Results from the studies indicated that FETAX can be used as a component of a battery of bioassays designed to assess potential ecological hazard. Furthermore, the generalized FETAX protocol may be useful with other species in evaluating developmental toxicity hazard.

  15. Integration of Multiple Field Methods in Characterizing a Field Site with Bayesian Inverse Modeling

    NASA Astrophysics Data System (ADS)

    Savoy, H.; Dietrich, P.; Osorio-Murillo, C. A.; Kalbacher, T.; Kolditz, O.; Ames, D. P.; Rubin, Y.

    2014-12-01

    A hydraulic property of a field can be expressed as a space random function (SRF), and the parameters of that SRF can be constrained by the Method of Anchored Distributions (MAD). MAD is a general Bayesian inverse modeling technique that quantifies the uncertainty of SRF parameters by integrating various direct local data along with indirect non-local data. An example is given with a high-resolution 3D aquifer analog with known hydraulic conductivity (K) and porosity (n) at every location. MAD is applied using different combinations of simulated measurements of K, n, and different scales of hydraulic head that represent different field methods. The ln(K) and n SRF parameters are characterized with each of the method combinations to assess the influence of the methods on the SRFs and their implications. The forward modeling equations are solved by the numerical modeling software OpenGeoSys (opengeosys.org) and MAD is applied with the software MAD# (mad.codeplex.com). The inverse modeling results are compared to the aquifer analog for success evaluation. The goal of the study is to show how integrating combinations of multi-scale and multi-type measurements from the field via MAD can be used to reduce the uncertainty in field-scale SRFs, as well as point values, of hydraulic properties.

  16. Integrated characterization of the geologic framework of a contaminated site in West Trenton, New Jersey

    USGS Publications Warehouse

    Ellefsen, Karl J.; Burton, William C.; Lacombe, Pierre J.

    2012-01-01

    Fractured sedimentary bedrock and groundwater at the former Naval Air Warfare Center in West Trenton, New Jersey (United States of America) are contaminated with chlorinated solvents. Predicting contaminant migration or removing the contaminants requires an understanding of the geology. Consequently, the geologic framework near the site was characterized with four different methods having different spatial scales: geologic field mapping, analyses of bedrock drill core, analyses of soil and regolith, and S-wave refraction surveys. A fault zone is in the southeast corner of the site and separates two distinct sedimentary formations; the fault zone dips (steeply) southeasterly, strikes northeasterly, and extends at least 550 m along its strike direction. Drill core from the fault zone is extensively brecciated and includes evidence of tectonic contraction. Approximately 300 m east of this fault zone is another fault zone, which offsets the contact between the two sedimentary formations. The S-wave refraction surveys identified both fault zones beneath soil and regolith and thereby provided constraints on their lateral extent and location.

  17. Integration of evolutionary and desolvation energy analysis identifies functional sites in a plant immunity protein

    PubMed Central

    Casasoli, Manuela; Federici, Luca; Spinelli, Francesco; Di Matteo, Adele; Vella, Nicoletta; Scaloni, Flavio; Fernandez-Recio, Juan; Cervone, Felice; De Lorenzo, Giulia

    2009-01-01

    Plant immune responses often depend on leucine-rich repeat receptors that recognize microbe-associated molecular patterns or pathogen-specific virulence proteins, either directly or indirectly. When the recognition is direct, a molecular arms race takes place where plant receptors continually and rapidly evolve in response to virulence factor evolution. A useful model system to study ligand-receptor coevolution dynamics at the protein level is represented by the interaction between pathogen-derived polygalacturonases (PGs) and plant polygalacturonase-inhibiting proteins (PGIPs). We have applied codon substitution models to PGIP sequences of different eudicotyledonous families to identify putative positively selected sites and then compared these sites with the propensity of protein surface residues to interact with protein partners, based on desolvation energy calculations. The 2 approaches remarkably correlated in pinpointing several residues in the concave face of the leucine-rich repeat domain. These residues were mutated into alanine and their effect on the recognition of several PGs was tested, leading to the identification of unique hotspots for the PGIP-PG interaction. The combined approach used in this work can be of general utility in cases where structural information about a pattern-recognition receptor or resistance-gene product is available. PMID:19372373

  18. A multi-site approach towards integrating environmental management in the wine production industry.

    PubMed

    Walsdorff, A; van Kraayenburg, M; Barnardt, C A

    2005-01-01

    Nine wineries and a water bottling plant situated in the Franschhoek Valley, South Africa, have decided to lead the way towards creating a safer and more sustainable environment in the Valley. As part of the Vignerons de Franschhoek ECO Association (VFEA), they adopted a multi-site approach and obtained, in December 2003, the first ISO 14001 multi-site certification in South Africa. This proactive approach allows them better overall environmental performances through mutual motivation, information sharing and comparison of practices by the different participating organisations of Franschhoek Valley. At the initiation of the project, the major concerns were associated with a lack of sound environmental management and training of employees. Of particular concern was also the lack of monitoring and measurement of environmental performance key indicators, especially in water consumption and wastewater disposal areas. Although the system is still in its juvenile stage, it already showed some effluent quality improvements through better wastewater management, better control over chemical stores as well as definite improvement in water management and increase in solid wastes recycling. The latter improvements will definitely entail long-term cost savings should the system be continually and properly maintained. It is the intention of the VFEA to create a more environmentally friendly and sustainable Valley by convincing others to be more environmentally responsive and expanding the ISO 14001 principles to other areas of the organisations including the farms, to other wineries and ultimately to the whole Franschhoek Valley. PMID:15771100

  19. Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation.

    PubMed

    Dhamne, Hemant; Chande, Ajit G; Mukhopadhyaya, Robin

    2014-01-01

    Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance. PMID:24325878

  20. Site-specific landslide assessment in Alpine area using a reliable integrated monitoring system

    NASA Astrophysics Data System (ADS)

    Romeo, Saverio; Di Matteo, Lucio; Kieffer, Daniel Scott

    2016-04-01

    Rockfalls are one of major cause of landslide fatalities around the world. The present work discusses the reliability of integrated monitoring of displacements in a rockfall within the Alpine region (Salzburg Land - Austria), taking into account also the effect of the ongoing climate change. Due to the unpredictability of the frequency and magnitude, that threatens human lives and infrastructure, frequently it is necessary to implement an efficient monitoring system. For this reason, during the last decades, integrated monitoring systems of unstable slopes were widely developed and used (e.g., extensometers, cameras, remote sensing, etc.). In this framework, Remote Sensing techniques, such as GBInSAR technique (Groung-Based Interferometric Synthetic Aperture Radar), have emerged as efficient and powerful tools for deformation monitoring. GBInSAR measurements can be useful to achieve an early warning system using surface deformation parameters as ground displacement or inverse velocity (for semi-empirical forecasting methods). In order to check the reliability of GBInSAR and to monitor the evolution of landslide, it is very important to integrate different techniques. Indeed, a multi-instrumental approach is essential to investigate movements both in surface and in depth and the use of different monitoring techniques allows to perform a cross analysis of the data and to minimize errors, to check the data quality and to improve the monitoring system. During 2013, an intense and complete monitoring campaign has been conducted on the Ingelsberg landslide. By analyzing both historical temperature series (HISTALP) recorded during the last century and those from local weather stations, temperature values (Autumn-Winter, Winter and Spring) are clearly increased in Bad Hofgastein area as well as in Alpine region. As consequence, in the last decades the rockfall events have been shifted from spring to summer due to warmer winters. It is interesting to point out that

  1. Insights Into Contamination Control For The Shuttle Payload Integration Facility (SPIF) At The Eastern Launch Site (ELS)

    NASA Astrophysics Data System (ADS)

    Pugel, Nancy J.

    1982-02-01

    The Shuttle Payload Integration Facility (SPIF) is a payload processing facility at the Eastern Launch Site (ELS), Cape Canaveral Air Force Station (CCAFS) which has been designed to accommodate assembly, test, and checkout operations for a number of Shuttle payloads, many of which are contamination sensitive. In general, the SPIF was designed to meet class 100,000, per Fed. Std. 209B (Reference 1), cleanliness requirements and operate as a class 100,000 clean area. However, due to the expansiveness of the building (which includes several airlocks, a transfer aisle, and integration cells), the variety of hardware to be processed through the facility (payloads, upper states, and support equipment), and the variety of operations to be performed for these hardware, many different contamination control methods must be carefully integrated and implemented to maintain an overall clean environment. In instances where the operation being performed is not generally compatible with the clean environment, uniaue and innovative contamination control methods must be developed with respect to ease of implementation as well as effectiveness of contamination control.

  2. B-cell reconstitution after lentiviral vector–mediated gene therapy in patients with Wiskott-Aldrich syndrome

    PubMed Central

    Castiello, Maria Carmina; Scaramuzza, Samantha; Pala, Francesca; Ferrua, Francesca; Uva, Paolo; Brigida, Immacolata; Sereni, Lucia; van der Burg, Mirjam; Ottaviano, Giorgio; Albert, Michael H.; Grazia Roncarolo, Maria; Naldini, Luigi; Aiuti, Alessandro; Villa, Anna; Bosticardo, Marita

    2015-01-01

    Background Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene–corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. Objective Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. Methods We evaluated B-cell counts, B-cell subset distribution, B cell–activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. Results After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19+CD21−CD35− and CD21low B cells and a reduction in B cell–activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients. Conclusions We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic

  3. Happier Together: Integrating a Wellness Application into a Social Network Site

    NASA Astrophysics Data System (ADS)

    Munson, Sean A.; Lauterbach, Debra; Newman, Mark W.; Resnick, Paul

    What are the benefits and drawbacks of integrating health and wellness interventions into existing online social network websites? In this paper, we report on a case study of deploying the Three Good Things positive psychology exercise as a Facebook application. Our experience shows that embedding a wellness intervention in an existing social website is a viable option. In particular, we find adherence rates on par with or better than many other Internet-based wellness interventions. We also gained insights about users' privacy and audience concerns that inform the design of social network-based wellness applications. Participants did not want all of their entries to be shared with all their Facebook friends, both because they did not want others to know some things and because they did not want to clutter others' newsfeeds. Users found it compelling, however, to interact with their friends around some "Good Things" they had posted.

  4. Application of an integrated real time investigation to expedite the acquisition of pre-design data at a Superfund site

    SciTech Connect

    Gelb, S.B.; Fitzpatrick, R.; Koller, D.

    1995-12-31

    At a Superfund Site in EPA Region 2, McLaren/Hart implemented its Integrated Real-Time Approach (IRTA), with EPA approval, to obtain pre-design data in support of the pump and treat remedy required by the ROD. The flexibility of the IRTA program allowed the lateral extent of a dissolved VOC plume in the shallow aquifer to be delineated, and resulted in the identification of previously unknown contaminated source areas. The IRTA approach employed a multi-disciplinary field team consisting of a hydrogeologists functioning as the Field Team Manager, an on-site chemist and sampling technicians. Rapid sample collection was achieved with a variety of equipment operated by McLaren/Hart`s Field Services Division. Samples were analyzed on-site by the McLaren/Hart Mobile laboratory. Analytical data were reviewed and evaluated by the field team as the information was generated, allowing informed selection of subsequent sampling locations. Groundwater samples were initially collected from areas known to be contaminated, and then proceeded outward until the lateral extent of the VOC plume had been defined. Soil samples were then collected from areas where the groundwater displayed the highest VOC concentrations. As such, the versatility of IRTA allowed for cost-effective groundwater plume delineation and source area identification.

  5. Lung endothelial HO-1 targeting in vivo using lentiviral miRNA regulates apoptosis and autophagy during oxidant injury

    PubMed Central

    Zhang, Yi; Jiang, Ge; Sauler, Maor; Lee, Patty J.

    2013-01-01

    The lung endothelium is a major target for inflammatory and oxidative stress. Heme oxygenase-1 (HO-1) induction is a crucial defense mechanism during oxidant challenges, such as hyperoxia. The role of lung endothelial HO-1during hyperoxia in vivo is not well defined. We engineered lentiviral vectors with microRNA (miRNA) sequences controlled by vascular endothelium cadherin (VE-cad) to study the specific role of lung endothelial HO-1. Wild-type (WT) murine lung endothelial cells (MLECs) or WT mice were treated with lentivirus and exposed to hyperoxia (95% oxygen). We detected HO-1 knockdown (∼55%) specifically in the lung endothelium. MLECs and lungs showed approximately a 2-fold increase in apoptosis and ROS generation after HO-1 silencing. We also demonstrate for the first time that silencing endothelial HO-1 has the same effect on lung injury and survival as silencing HO-1 in multiple lung cell types and that HO-1 regulates caspase 3 activation and autophagy in endothelium during hyperoxia. These studies demonstrate the utility of endothelial-targeted gene silencing in vivo using lentiviral miRNA constructs to assess gene function and that endothelial HO-1 is an important determinant of survival during hyperoxia.—Zhang, Y., Jiang, G., Sauler, M., Lee, P. J. Lung endothelial HO-1 targeting in vivo using lentiviral miRNA regulates apoptosis and autophagy during oxidant injury. PMID:23771928

  6. Cleavage of Hemagglutinin-Bearing Lentiviral Pseudotypes and Their Use in the Study of Influenza Virus Persistence

    PubMed Central

    Sawoo, Olivier; Dublineau, Amélie; Batéjat, Christophe; Zhou, Paul; Manuguerra, Jean-Claude; Leclercq, India

    2014-01-01

    Influenza A viruses (IAVs) are a major cause of infectious respiratory human diseases and their transmission is dependent upon the environment. However, the role of environmental factors on IAV survival outside the host still raises many questions. In this study, we used lentiviral pseudotypes to study the influence of the hemagglutinin protein in IAV survival. High-titered and cleaved influenza-based lentiviral pseudoparticles, through the use of a combination of two proteases (HAT and TMPRSS2) were produced. Pseudoparticles bearing hemagglutinin proteins derived from different H1N1, H3N2 and H5N1 IAV strains were subjected to various environmental parameters over time and tested for viability through single-cycle infectivity assays. We showed that pseudotypes with different HAs have different persistence profiles in water as previously shown with IAVs. Our results also showed that pseudotypes derived from H1N1 pandemic virus survived longer than those derived from seasonal H1N1 virus from 1999, at high temperature and salinity, as previously shown with their viral counterparts. Similarly, increasing temperature and salinity had a negative effect on the survival of the H3N2 and H5N1 pseudotypes. These results showed that pseudotypes with the same lentiviral core, but which differ in their surface glycoproteins, survived differently outside the host, suggesting a role for the HA in virus stability. PMID:25166303

  7. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

    PubMed

    Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

    2015-01-01

    Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. PMID:26290434

  8. Lentiviral vector-mediated over-expression of Sox9 protected chondrocytes from IL-1β induced degeneration and apoptosis

    PubMed Central

    Lu, Huading; Zeng, Chun; Chen, Mingwei; Lian, Liyi; Dai, Yuhu; Zhao, Huiqing

    2015-01-01

    To explore whether the over-expression of Sry-related HMG box (Sox9) in degenerative chondrocytes is able to improve cell regeneration and protects cells from inflammation induced apoptosis, we generated a Sox9 over-expressing vector delivery system in which the Sox9 gene was inserted into a lentiviral vector. After infecting mouse chondrocytes with the Sox9-encoding vector, we observed a high level of gene transduction efficiency and achieved a high level of Sox9 expression in the infected chondrocytes. To explore whether over-expression of Sox9 is able to induce cell regeneration and improve cell survival, we induced Sox9 over-expression by lentiviral vector infection 48 hours before IL-1β treatment. The cells were infected with the reporter gene GFP-encoded lentiviral vector as a negative control or left uninfected. 48-hours after IL-1β treatment, the chrondrocytes treated with IL-1β alone, underwent a degenerative process, with elevated expression of MMP-3, MMP-13, ADAMTS-5 and ALP, but the cell specific anabolic proteins collagen II and aggrecan were significantly suppressed. The cells infected with the GFP reporter vector had no increased regeneration after IL-1β treatment. The results indicated that Sox9 is an important chondrocyte transcription factor, promoting chondrocyte regeneration and cell survival, which were mediated through affecting multiple cell differentiation as well as anti-apoptotic signaling pathways. PMID:26617711

  9. Site planning and integration fiscal year 1999 multi-year work plan (MYWP) update for WBS 1.8.2.1

    SciTech Connect

    SCHULTZ, E.A.

    1998-10-01

    The primary mission of the Site Planning and Integration (SP and I) project is to assist Fluor Daniel Project Direction to ensure that all work performed under the Project Hanford Management Contract (PHMC) is adequately planned, executed, controlled, and that performance is measured and reported in an integrated fashion. Furthermore, SP and I is responsible for the development, implementation, and management of systems and processes that integrate technical, schedule, and cost baselines for PHMC work.

  10. Hydro-mechanical simulations of well abandonment at the Ketzin pilot site for CO2 storage verify wellbore system integrity

    NASA Astrophysics Data System (ADS)

    Unger, Victoria; Kempka, Thomas

    2015-04-01

    In geological underground utilisation, operating and abandoned wells have been identified as a main potential leakage pathways for reservoir fluids. In the scope of the well abandonment procedure currently carried out at the Ketzin pilot site for CO2 storage in Germany, a hydro-mechanical model was built to carry out a coupled analysis of the integrity in the entire wellbore system. The main aim of the present study was to assess the impacts of stress changes associated with CO2 injection as well as the cement backfill undertaken in the scope of well abandonment. A numerical model comprising cement sheaths, steel casings, tubing, multiple packers and wellbore annuli was implemented to enable a detailed representation of the entire wellbore system. The numerical model grid has a horizontal discretisation of 5 m x 5 m to focus on near wellbore effects, whereby element sizes increase with increasing distance from the wellbore. Vertical grid discretisation uses a tartan grid type over the entire model thickness of 1,500 m to ensure a sufficient discretisation of all wellbore system elements as well as of the reservoir unit. The total number of elements amounts to 210,672. Mechanical model parameters were taken from geological, drilling, logging and laboratory test data based on Ketzin pilot site-specific information as well as related literature (Kempka et al., 2014). The coupled calculations were performed using an elasto-plastic constitutive law, whereby an initial simulation run ensured a static mechanical equilibrium to represent the initial state before the start of CO2 injection. Thereto, gravitational load of the overburden rocks and pore pressure distribution following available well logs were integrated for initial model parameterisation including a normal faulting stress regime defined by a horizontal to vertical total stress ratio of 0.85. A correction accounting for the temperature and pressure dependent CO2 density was carried out in advance of each

  11. Integrating geochemical survey and magnetic prospection on an archaeological site in SW-Turkey

    NASA Astrophysics Data System (ADS)

    Dirix, Katrijn; Muchez, Philippe; Degryse, Patrick; Mušič, Branko; Poblome, Jeroen

    2013-04-01

    In this study, geochemical and magnetic survey data from an archaeological site are combined, aiming to explore the potential of both techniques to complement each other. The site under study comprises a suburban area of 6 ha, situated in the Roman to Byzantine city of Sagalassos (Taurus Mountains, SW-Turkey). For the geochemical survey, a total of 120 soil samples were collected in two grids, with cell sizes of respectively 100 x 100 m and 20 x 20 m. After Aqua Regia destruction, Al, As, Ba, Ca, Cu, Co, Cr, Fe, K, Mn, Mg, Na, Ni, Pb, P, Sr, Ti, V and Zn were measured by inductively coupled plasma optical emission spectrometry (ICP-OES), using a Varian 720-ES apparatus. The magnetic survey was performed using a Geometrics G-858 magnetometer in gradient mode, along 0.5 m spaced transects. Results indicate that large scale chemical prospection with cell sizes of 100 x 100 m is capable of detecting human influence on soils by anomalous values of Cu, K, P and Zn, although the data are too coarse to aid geophysical interpretation. In the more detailed grid with cell sizes of 20 x 20 m, geochemical enrichments of Co, Cr, Fe, Mg, Mn, Ni and V were detected on a location displaying strong magnetic anomalies. These anomalies were found to result from a local change towards a more mafic lithology. In an area where magnetic readings displayed high-frequency magnetic noise, soil samples contained distinct concentrations of P, Cu, K and Zn, suggesting that the soils at this location were strongly altered by human processes, which resulted in enhanced magnetic susceptibilities of the soils. An uphill zone, lacking the strongly contrasting magnetic anomalies found elsewhere, was shown to spatially correspond with enrichments of As, Al, Ba and Pb. The association of Al, Ba and Pb was identified as tracer signature for weathered limestone material in the territory of Sagalassos. Therefore, it is thought that the observed changes reflect the presence of a limestone hill in this zone

  12. Integrated metagenomics and field measurements of polygon features at the NGEE-Arctic Barrow site

    NASA Astrophysics Data System (ADS)

    Tas, N.; Wu, Y.; Smith, L. J.; Ulrich, C.; Kneafsey, T. J.; Torn, M. S.; Hubbard, S. S.; Wullschleger, S. D.; Jansson, J.

    2013-12-01

    Arctic soils contain an estimated 12-42% of terrestrial carbon, most of which is sequestered in permafrost. High latitudes have experienced the greatest regional warming in recent decades and observations suggest that permafrost degradation is now commonly observed in the region. With increasing global temperatures, permafrost soils are becoming a potential source of greenhouse gas (GHG) emissions. Because of widespread permafrost thaw much of the soil organic matter may be available for rapid mineralization by microorganisms in the soil. Yet little is known about the vulnerability of permafrost and the potential response of soil microorganisms to availability of new carbon sources. On the Alaskan North Slope the collapse and rise of soil due to formation of ice wedges and permafrost thaw create distinct features called polygons. As part of the U.S. Department of Energy (DOE) Next Generation Ecosystem Experiment (NGEE) in the Arctic, we aimed to determine the distribution of microbial populations across a range of polygon features and to correlate the microbial data to GHG flux data. To determine the microbial community distribution and metabolic potential, we collected seasonally thawed active layer soil samples along two polygon transects (Site 0 and AB), including high-centered, transitional and low-centered polygons. Illumina HiSeq technology was used to sequence 16SrRNA genes and metagenomes from these active layer soils. The sequence data was correlated to GHG flux measurements and to environmental data from the site, including geophysical and geochemical soil characteristics. Both the microbial communities and the flux measurements varied along the polygon transect. Each polygon had a distinct microbial community structure; however, these microbial communities shared many metabolic capabilities. For example, many genes involved in degradation of chitin could be found all three polygons. Functional genes involved in methanogenesis and CH4-flux measurements

  13. RD2-MolPack-Chim3, a Packaging Cell Line for Stable Production of Lentiviral Vectors for Anti-HIV Gene Therapy

    PubMed Central

    Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo

    2013-01-01

    Abstract Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology. PMID:23767932

  14. THE INTEGRATION OF ENGINEERED AND INSTITUTIONAL CONTROLS: A CASE STUDY APPROACH WITH LESSONS LEARNED FROM PREVIOUSLY CLOSED SITES

    SciTech Connect

    Kevin M. Kostelnik; James H. Clarke; Jerry L. Harbour

    2005-02-01

    Environmental remediation efforts that are underway at hundreds of contaminated sites in the United States will not be able to remediate large portions of those sites to conditions that would permit unrestricted access. Rather, large volumes of waste materials, contaminated soils and cleanup residuals will have to be isolated either in place or in new, often on-site, disposal cells with long term monitoring, maintenance and institutional control needs. The challenge continues to be to provide engineering systems and controls that can ensure the protection of public health and the environment over very long time horizons (hundreds to perhaps thousands of years) with minimal intervention. Effective long term management of legacy hazardous and nuclear waste requires an integrated approach that addresses both the engineered containment and control system itself and the institutional controls and other responsibilities that are needed. Decisions concerning system design, monitoring and maintenance, and the institutional controls that will be employed are best done through a "risk-nformed, performance-based" approach. Such an approach should incorporate an analysis of potential "failure" modes and consequences for all important system features, together with lessons learned from experience with systems already in place. The authors will present the preliminary results of a case study approach that included several sites where contamination isolation systems including institutional controls have been implemented. The results are being used together with failure trees and logic diagrams that have been developed for both the engineered barriers and the institutional controls. The use of these analytical tools to evaluate the potential for different levels of failure and associated consequences will be discussed. Of special interest is the robustness of different approaches to providing long-term protection through redundancy and defense in depth.

  15. Climate changes inferred from integrated multi-site pollen data in northern Taiwan

    NASA Astrophysics Data System (ADS)

    Wang, Liang-Chi; Wu, Jiunn-Tzong; Lee, Teh-Quei; Lee, Pei-Fen; Chen, Su-Hwa

    2011-04-01

    In this study, we synthesized the fossil pollen data from 10 lake sediments and 2 land cores to integrate the alteration of forest covered areas in northern Taiwan with changes in humidity and temperature over the last 2000 years. The abundance of arboreal pollen, fern spores and Tsuga pollen in the pollen and spore assemblages were used as indicators. Our results suggested that the climate in northern Taiwan was stably cool and dry during 2000-1000 cal. yr BP, but changed to wet and warm during 1000-500 cal. yr BP, which corresponded to the Medieval Warm Period (MWP). In addition, an increased density and dispersal of Tsuga pollen corresponding to 500-200 cal. yr BP was observed, which corresponded to the Little Ice Age (LIA). In recent years, a decline in Tsuga pollen density and increased fern spore density has been observed, which indicates an increase in temperature associated with elevated rainfall. Based on the synthesized data set, we conducted GIS mapping of such changes in the north of Taiwan over time. The results revealed that the temporal and spatial climate changes could be inferred from the palynological GIS mapping method, and that the fluctuations in temperature over time matched well with the global climate events, including MWP, LIA and recent warming.

  16. An integrated methodology on the suitability of offshore sites for wind farm development

    NASA Astrophysics Data System (ADS)

    Patlakas, Platon; Galanis, George; Péray, Marie; Filipot, Jean-François; Kalogeri, Christina; Spyrou, Christos; Diamantis, Dimitris; Kallos, Gerorge

    2016-04-01

    During, the last decades the potential and interest in wind energy investments has been constantly increasing in the European countries. As technology changes rapidly, more and more areas can be identified as suitable for energy applications. Offshore wind farms perfectly illustrate how new technologies allow to build bigger, more efficient and resistant in extreme conditions wind power plants. The current work proposes an integrated methodology to determine the suitability of an offshore marine area for the development of wind farm structures. More specifically, the region of interest is evaluated based both on the natural resources, connected to the local environmental characteristics, and potential constrains set by anthropogenic or other activities. State of the art atmospheric and wave models and a 10-year hindcast database are utilized in conjunction with local information for a number of potential constrains, leading to a 5-scale suitability index for the whole area. In this way, sub regions are characterized, at a high resolution mode, as poorly or highly suitable for wind farm development, providing a new tool for technical/research teams and decision makers. In addition, extreme wind and wave conditions and their 50-years return period are analyzed and used to define the safety level of the wind farms structural characteristics.

  17. A dual promoter lentiviral vector for the in vivo evaluation of gene therapeutic approaches to axon regeneration after spinal cord injury.

    PubMed

    Löw, K; Blesch, A; Herrmann, J; Tuszynski, M H

    2010-05-01

    The identification of axon growth-promoting genes, and overexpression of these genes in central nervous system (CNS) neurons projecting to the spinal cord, has emerged as one potential approach to enhancing CNS regeneration. Assessment of the regenerative potential of candidate genes usually requires axonal tracing of spinal projections, ideally limited to neurons that express the candidate gene. Alternatively, coexpression of a reporter gene such as enhanced green fluorescent protein (GFP) from an internal ribosomal entry site can be used to identify neurons expressing the candidate gene, but this strategy does not label corticospinal axons in the spinal cord. We therefore developed a dual promoter lentiviral vector in which a potentially therapeutic transgene is expressed from the cytomegalovirus-enhanced chicken beta-actin promoter and the fluorescent protein copGFP is expressed from the elongation factor-1alpha promoter. The vector was constructed to be compatible with the Gateway recombination system for efficient introduction of transgenes through entry shuttle vectors. We show both simultaneous expression of a candidate and reporter gene in corticospinal and red nucleus neurons, and efficient labeling of their axons after lesions in the cervical spinal cord. This expression system is therefore an accurate and efficient means of screening candidate genes in vivo for enhancement of axonal growth. PMID:20200564

  18. Lentiviral-mediated over-expression of hyaluronan synthase-1 (HAS-1) decreases the cellular inflammatory response and results in regenerative wound repair.

    PubMed

    Caskey, Robert C; Allukian, Myron; Lind, Robert C; Herdrich, Benjamin J; Xu, Junwang; Radu, Antoneta; Mitchell, Marc E; Liechty, Kenneth W

    2013-01-01

    Fetal wounds have been found to have increased levels of high-molecular-weight hyaluronan (HMW-HA) compared with those of adults. The primary enzyme responsible for producing HMW-HA is hyaluronic acid synthase-1 (HAS-1). We hypothesized that over-expression of HAS-1 in adult dermal wounds would decrease inflammation and promote regenerative healing. To test this hypothesis, the flanks of adult C57Bl/6 mice were treated with a lentiviral construct containing either HAS-1-GFP or GFP transgenes. After 48 h, a 4-mm excisional wound was made at the site of treatment. Wounds were harvested at days 3, 7, or 28 after wounding. Wound phenotype was assessed by histology to examine tissue architecture and immunohistochemistry for CD45. At 7 and 28 days, lenti-HAS-1-treated wounds demonstrated the restoration of the normal dermal elements and organized collagen fiber orientation. In contrast, the lenti-GFP-treated wounds lacked normal dermal architecture and demonstrated a disorganized collagen scar. At 3 and 7 days, wounds treated with lenti-HAS-1 exhibited a significant decrease in the number of inflammatory cells when compared with wounds treated with lenti-GFP. Thus, HAS-1 over-expression promotes dermal regeneration, in part by decreasing the inflammatory response and by recapitulation of fetal extracellular matrix HMW-HA content. PMID:23149717

  19. USE OF PERFLUOROCARBON TRACER (PFT) TECHNOLOGY FOR SUBSURFACE BARRIER INTEGRITY VERIFICATION AT THE WALDO TEST SITE.

    SciTech Connect

    SULLIVAN,T.

    1999-06-01

    Testing of perfluorocarbon gas tracers (PFT) on a subsurface barrier with known flaws was conducted at the Waldo Test Site operated by Science and Engineering Associates, Inc (SEA). The tests involved the use of five unique PFTs with a different tracer injected along the interior of each wall of the barrier. A fifth tracer was injected exterior to the barrier to examine the validity of diffusion controlled transport of the PFTs. The PFTs were injected for three days at a nominal flow rate of 15 cm{sup 3}/min and concentrations in the range of a few hundred ppm. Approximately 65 liters of air laced with tracer was injected for each tracer. The tracers were able to accurately detect the presence of the engineered flaws. Two flaws were detected on the north and east walls, and one flaw was detected on the south and west walls. In addition, one non-engineered flaw at the seam between the north and east walls was also detected. The use of multiple tracers provided independent confirmation of the flaws and permitted a distinction between tracers arriving at a monitoring port after being released from a nearby flaw and non-engineered flaws. The PFTs detected the smallest flaw, 0.5 inches in diameter. Visual inspection of the data showed excellent agreement with the known flaw locations and the relative size of the flaws was accurately estimated. Simultaneous with the PFT tests, SEA conducted tests with another gas tracer sulfur hexafluoride (SF{sub 6}).

  20. Performance Assessment of a Low-Level Radioactive Waste Disposal Site using GoldSim Integrated Systems Model

    NASA Astrophysics Data System (ADS)

    Merrell, G.; Singh, A.; Tauxe, J.; Perona, R.; Dornsife, W.; grisak, G. E.; Holt, R. M.

    2011-12-01

    Texas Commission on Environmental Quality has approved licenses for four landfills at the Waste Control Specialists (WCS) site located in Andrews County, West Texas. The site includes a hazardous waste landfill and three landfills for radioactive waste. An updated performance assessment is necessary prior to acceptance of waste at the landfills. The updated performance assessment a) provides for more realistic and flexible dose modeling capabilities, b) addresses all plausible release and accident scenarios as they relate to the performance objectives, c) includes impact of climate and hydrologic scenarios that may impact long-term performance of the landfill, d) addresses impact of cover naturalization and degradation on the landfill, and e) incorporates uncertainty and sensitivity analysis for critical parameters. For the updated performance assessment, WCS has developed an integrated systems level performance assessment model using the GoldSim platform. GoldSim serves as a model for integrating all of the major components of a performance assessment, which include the radionuclide source term, facility design, environmental transport pathways, exposure scenarios, and radiological doses. Unlike many computer models that are based on first principles, GoldSim is a systems level model that can be used to integrate and abstract more complex sub-models into one system. This can then be used to assess the results into a unified model of the disposal system and environment. In this particular application, the GoldSim model consists of a) hydrogeologic model that simulates flow and transport through the Dockum geologic unit that underlies all of the waste facilities, b) waste cells that represent the containment unit and simulate degradation of waste forms, radionuclide leaching, and partitioning into the liquid and vapor phase within the waste unit, c) a cover system model that simulates upward diffusive transport from the underground repository to the atmosphere. In

  1. Assessing recreation impacts to cliffs in Shenandoah National Park: Integrating visitor observation with trail and recreation site measurements

    USGS Publications Warehouse

    Wood, K.T.; Lawson, S.R.; Marion, J.L.

    2006-01-01

    The rock outcrops and cliffs of Shenandoah National Park provide habitat for several rare and endangered plant and animal species, including the federally endangered Shenandoah Salamander (Plethodon shenandoah; Ludwig et al., 1993). The location of the well-known park tour road, Skyline Drive, along the ridgeline provides exceptional access to many outcrops and cliffs throughout the park for a large number of the park?s 1.2 million annual visitors. Consequently, visitor use of cliff areas has led to natural resource impacts, including marked decreases in size and vigor of known rare plant populations. Despite the clear ecological value and potential threats to the natural resources at cliff areas, managers possess little information on visitor use of cliff sites and presently have no formal planning document to guide management. Thus, a park wide study of cliff sites was initiated during the 2005 visitor use season. As part of this research effort, our study used an integrative approach to study recreational use and visitor-caused resource impacts at one of the more heavily visited cliff sites in the park: Little Stony Man Cliffs (LSMC). In particular, this study integrated data from resource impact measurements and visitor use observation to help assess the effects of recreational use on the natural resources of LSMC. Procedures derived from campsite and trail impact studies were used to measure and characterize the amount of visitor-caused resource impacts on LSMC (Marion & Leung, 2001; Marion, 1995). Visitor use observations were conducted on top of LSMC to document and characterize the type and amount of recreational use the cliffs receive and the behaviors of recreationists that may contribute to cliff-top resource impacts. Resource impact measurement data show trampling disturbance present at LSMC, characterized by vegetation loss, exposed soil, and root exposure. Documentation of informal trails, soil erosion, tree damage, and tree stumps provide further

  2. Munitions integrity and corrosion features observed during the HUMMA deep-sea munitions disposal site investigations

    NASA Astrophysics Data System (ADS)

    Silva, Jeff A. K.; Chock, Taylor

    2016-06-01

    An evaluation of the current condition of sea-disposed military munitions observed during the 2009 Hawaii Undersea Military Munitions Assessment Project investigation is presented. The 69 km2 study area is located south of Pearl Harbor, Oahu, Hawaii, and is positioned within a former deep-sea disposal area designated as Hawaii-05 or HI-05 by the United States Department of Defense. HI-05 is known to contain both conventional and chemical munitions that were sea-disposed between 1920 and 1951. Digital images and video reconnaissance logs collected during six remotely operated vehicle and 16 human-occupied vehicle surveys were used to classify the integrity and state of corrosion of the 1842 discarded military munitions (DMM) objects encountered. Of these, 5% (or 90 individual DMM objects) were found to exhibit a mild-moderate degree of corrosion. The majority (66% or 1222 DMM objects) were observed to be significantly corroded, but visually intact on the seafloor. The remaining 29% of DMM encountered were found to be severely corroded and breached, with their contents exposed. Chemical munitions were not identified during the 2009 investigation. In general, identified munitions known to have been constructed with thicker casings were better preserved. Unusual corrosion features were also observed, including what are termed here as 'corrosion skirts' that resembled the flow and cementation of corrosion products at and away from the base of many munitions, and 'corrosion pedestal' features resembling a combination of cemented corrosion products and seafloor sediments that were observed to be supporting munitions above the surface of the seafloor. The origin of these corrosion features could not be determined due to the lack of physical samples collected. However, a microbial-mediated formation hypothesis is presented, based on visual analysis, which can serve as a testable model for future field programs.

  3. Dr.VIS v2.0: an updated database of human disease-related viral integration sites in the era of high-throughput deep sequencing.

    PubMed

    Yang, Xiaobo; Li, Ming; Liu, Qi; Zhang, Yabing; Qian, Junyan; Wan, Xueshuai; Wang, Anqiang; Zhang, Haohai; Zhu, Chengpei; Lu, Xin; Mao, Yilei; Sang, Xinting; Zhao, Haitao; Zhao, Yi; Zhang, Xiaoyan

    2015-01-01

    Dr.VIS is a database of human disease-related viral integration sites (VIS). The number of VIS has grown rapidly since Dr.VIS was first released in 2011, and there is growing recognition of the important role that viral integration plays in the development of malignancies. The updated database version, Dr.VIS v2.0 (http://www.bioinfo.org/drvis or bminfor.tongji.edu.cn/drvis_v2), represents 25 diseases, covers 3340 integration sites of eight oncogenic viruses in human chromosomes and provides more accurate information about VIS from high-throughput deep sequencing results obtained mainly after 2012. Data of VISes for three newly identified oncogenic viruses for 14 related diseases have been added to this 2015 update, which has a 5-fold increase of VISes compared to Dr.VIS v1.0. Dr.VIS v2.0 has 2244 precise integration sites, 867 integration regions and 551 junction sequences. A total of 2295 integration sites are located near 1730 involved genes. Of the VISes, 1153 are detected in the exons or introns of genes, with 294 located up to 5 kb and a further 112 located up to 10 kb away. As viral integration may alter chromosome stability and gene expression levels, characterizing VISes will contribute toward the discovery of novel oncogenes, tumor suppressor genes and tumor-associated pathways. PMID:25355513

  4. OPERATIONS REVIEW OF THE SAVANNAH RIVER SITE INTEGRATED SALT DISPOSITION PROCESS - 11327

    SciTech Connect

    Peters, T.; Poirier, M.; Fondeur, F.; Fink, S.; Brown, S.; Geeting, M.

    2011-02-07

    The Savannah River Site (SRS) is removing liquid radioactive waste from its Tank Farm. To treat waste streams that are low in Cs-137, Sr-90, and actinides, SRS developed the Actinide Removal Process and implemented the Modular Caustic Side Solvent Extraction (CSSX) Unit (MCU). The Actinide Removal Process contacts salt solution with monosodium titanate to sorb strontium and select actinides. After monosodium titanate contact, the resulting slurry is filtered to remove the monosodium titanate (and sorbed strontium and actinides) and entrained sludge. The filtrate is transferred to the MCU for further treatment to remove cesium. The solid particulates removed by the filter are concentrated to {approx} 5 wt %, washed to reduce the sodium concentration, and transferred to the Defense Waste Processing Facility for vitrification. The CSSX process extracts the cesium from the radioactive waste using a customized solvent to produce a Decontaminated Salt Solution (DSS), and strips and concentrates the cesium from the solvent with dilute nitric acid. The DSS is incorporated in grout while the strip acid solution is transferred to the Defense Waste Processing Facility for vitrification. The facilities began radiological processing in April 2008 and started processing of the third campaign ('MarcoBatch 3') of waste in June 2010. Campaigns to date have processed {approx}1.2 million gallons of dissolved saltcake. Savannah River National Laboratory (SRNL) personnel performed tests using actual radioactive samples for each waste batch prior to processing. Testing included monosodium titanate sorption of strontium and actinides followed by CSSX batch contact tests to verify expected cesium mass transfer. This paper describes the tests conducted and compares results from facility operations. The results include strontium, plutonium, and cesium removal, cesium concentration, and organic entrainment and recovery data. Additionally, the poster describes lessons learned during operation

  5. A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs

    PubMed Central

    Souque, Philippe; Frenkiel, Marie-Pascale; Paulous, Sylvie; Garcìa-Nicolàs, Obdulio; Summerfield, Artur; Charneau, Pierre; Desprès, Philippe

    2015-01-01

    Background Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a “one health” strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. Methodology/Principal Findings A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. Conclusions/Significance Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great

  6. The Effects of Surgical Hand Scrubbing Protocols on Skin Integrity and Surgical Site Infection Rates: A Systematic Review.

    PubMed

    Liu, Liang Qin; Mehigan, Sinead

    2016-05-01

    This systematic review aimed to critically appraise and synthesize updated evidence regarding the effect of surgical-scrub techniques on skin integrity and the incidence of surgical site infections. Databases searched include the Cumulative Index to Nursing and Allied Health Literature, MEDLINE, Embase, and Cochrane Central. Our review was limited to eight peer-reviewed, randomized controlled trials and two nonrandomized controlled trials published in English from 1990 to 2015. Comparison models included traditional hand scrubbing with chlorhexidine gluconate or povidone-iodine against alcohol-based hand rubbing, scrubbing with a brush versus without a brush, and detergent-based antiseptics alone versus antiseptics incorporating alcohol solutions. Evidence showed that hand rubbing techniques are as effective as traditional scrubbing and seem to be better tolerated. Hand rubbing appears to cause less skin damage than traditional scrub protocols, and scrub personnel tolerated brushless techniques better than scrubbing using a brush. PMID:27129749

  7. Lentiviral-mediated delivery of Bcl-2 or GDNF protects against excitotoxicity in the rat hippocampus.

    PubMed

    Wong, Liang-Fong; Ralph, G Scott; Walmsley, Lucy E; Bienemann, Alison S; Parham, Stephen; Kingsman, Susan M; Uney, James B; Mazarakis, Nicholas D

    2005-01-01

    Nutrient deprivation during ischemia leads to severe insult to neurons causing widespread excitotoxic damage in specific brain regions such as the hippocampus. One possible strategy for preventing neurodegeneration is to express therapeutic proteins in the brain to protect against excitotoxicity. We investigated the utility of equine infectious anemia virus (EIAV)-based vectors as genetic tools for delivery of therapeutic proteins in an in vivo excitotoxicity model. The efficacy of these vectors at preventing cellular loss in target brain areas following excitotoxic insult was also assessed. EIAV vectors generated to overexpress the human antiapoptotic Bcl-2 or growth factor glial-derived neurotrophic factor (GDNF) genes protected against glutamate-induced toxicity in cultured hippocampal neurons. In an in vivo excitotoxicity model, adult Wistar rats received a unilateral dose of the glutamate receptor agonist N-methyl-D-aspartate to the hippocampus that induced a large lesion in the CA1 region. Neuronal loss could not be protected by prior transduction of a control vector expressing beta-galactosidase. In contrast, EIAV-mediated expression of Bcl-2 and GDNF significantly reduced lesion size thus protecting the hippocampus from excitotoxic damage. These results demonstrate that EIAV vectors can be effectively used to deliver putative neuroprotective genes to target brain areas and prevent cellular loss in the event of a neurological insult. Therefore these lentiviral vectors provide potential therapeutic tools for use in cases of acute neurotrauma such as cerebral ischemia. PMID:15585409

  8. Design of a regulated lentiviral vector for hematopoietic stem cell gene therapy of globoid cell leukodystrophy.

    PubMed

    Ungari, Silvia; Montepeloso, Annita; Morena, Francesco; Cocchiarella, Fabienne; Recchia, Alessandra; Martino, Sabata; Gentner, Bernhard; Naldini, Luigi; Biffi, Alessandra

    2015-01-01

    Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translation. PMID:26509184

  9. Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells

    PubMed Central

    Goyvaerts, C; De Groeve, K; Dingemans, J; Van Lint, S; Robays, L; Heirman, C; Reiser, J; Zhang, X-Y; Thielemans, K; De Baetselier, P; Raes, G; Breckpot, K

    2012-01-01

    Lentiviral vectors (LVs) provide unique opportunities for the development of immunotherapeutic strategies, as they transduce a variety of cells in situ, including antigen-presenting cells (APCs). Engineering LVs to specifically transduce APCs is required to promote their translation towards the clinic. We report on the Nanobody (Nb) display technology to target LVs to dendritic cells (DCs) and macrophages. This innovative approach exploits the budding mechanism of LVs to incorporate an APC-specific Nb and a binding-defective, fusion-competent form of VSV.G in the viral envelope. In addition to production of high titer LVs, we demonstrated selective, Nb-dependent transduction of mouse DCs and macrophages both in vitro and in situ. Moreover, this strategy was translated to a human model in which selective transduction of in vitro generated or lymph node (LN)-derived DCs and macrophages, was demonstrated. In conclusion, the Nb display technology is an attractive approach to generate LVs targeted to specific cell types. PMID:22241177

  10. Design of a regulated lentiviral vector for hematopoietic stem cell gene therapy of globoid cell leukodystrophy

    PubMed Central

    Ungari, Silvia; Montepeloso, Annita; Morena, Francesco; Cocchiarella, Fabienne; Recchia, Alessandra; Martino, Sabata; Gentner, Bernhard; Naldini, Luigi; Biffi, Alessandra

    2015-01-01

    Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translation. PMID:26509184

  11. Dual-regulated lentiviral vector for gene therapy of X-linked chronic granulomatosis.

    PubMed

    Chiriaco, Maria; Farinelli, Giada; Capo, Valentina; Zonari, Erika; Scaramuzza, Samantha; Di Matteo, Gigliola; Sergi, Lucia Sergi; Migliavacca, Maddalena; Hernandez, Raisa Jofra; Bombelli, Ferdinando; Giorda, Ezio; Kajaste-Rudnitski, Anna; Trono, Didier; Grez, Manuel; Rossi, Paolo; Finocchi, Andrea; Naldini, Luigi; Gentner, Bernhard; Aiuti, Alessandro

    2014-08-01

    Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development. PMID:24869932

  12. Role of Transgene Regulation in Ex Vivo Lentiviral Correction of Artemis Deficiency

    PubMed Central

    Multhaup, Megan M.; Podetz-Pedersen, Kelly M.; Karlen, Andrea D.; Olson, Erik R.; Gunther, Roland; Somia, Nikunj V.; Blazar, Bruce R.; Cowan, Morton J.

    2015-01-01

    Abstract Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID. PMID:25738323

  13. Reversal of Diabetes Through Gene Therapy of Diabetic Rats by Hepatic Insulin Expression via Lentiviral Transduction

    PubMed Central

    Elsner, Matthias; Terbish, Taivankhuu; Jörns, Anne; Naujok, Ortwin; Wedekind, Dirk; Hedrich, Hans-Jürgen; Lenzen, Sigurd

    2012-01-01

    Due to shortage of donor tissue a cure for type 1 diabetes by pancreas organ or islet transplantation is an option only for very few patients. Gene therapy is an alternative approach to cure the disease. Insulin generation in non-endocrine cells through genetic engineering is a promising therapeutic concept to achieve insulin independence in patients with diabetes. In the present study furin-cleavable human insulin was expressed in the liver of autoimmune-diabetic IDDM rats (LEW.1AR1/Ztm-iddm) and streptozotocin-diabetic rats after portal vein injection of INS-lentivirus. Within 5–7 days after the virus injection of 7 × 109 INS-lentiviral particles the blood glucose concentrations were normalized in the treated animals. This glucose lowering effect remained stable for the 1 year observation period. Human C-peptide as a marker for hepatic release of human insulin was in the range of 50–100 pmol/ml serum. Immunofluorescence staining of liver tissue was positive for insulin showing no signs of transdifferentiation into pancreatic β-cells. This study shows that the diabetic state can be efficiently reversed by insulin release from non-endocrine cells through a somatic gene therapy approach. PMID:22354377

  14. Large granular lymphocytes are universally increased in human, macaque, and feline lentiviral infection.

    PubMed

    Sprague, Wendy S; Apetrei, Cristian; Avery, Anne C; Peskind, Robert L; Vandewoude, Sue

    2015-10-15

    Large granular lymphocytes (LGLs) have only been anecdotally reported in HIV infection. We previously reported an LGL lymphocytosis in FIV-infected cats associated with a rise in FIV proviral loads and a marked neutropenia that persisted during chronic infection. Extensive immunophenotyping of peripheral blood mononuclear cells in cats chronically infected with FIV were identified LGLs as CD8lo(+)FAS(+); this cell population expanded commensurate with viral load. CD8lo(+)FAS(+) cells expressed similar levels of interferon-γ compared to CD8lo(+)FAS(+) cells from FIV-naive control animals, yet CD3ɛ expression, which was increased on total CD8(+) T cells in FIV-infected cats, was decreased on CD8lo(+)FAS(+) cells. Down-modulation of CD3 expression was reversed after culturing PBMC for 3 days in culture with ConA/IL-2. We identified CD8lo(+)FAS(+) LGLs to be polyclonal T cells lacking CD56 expression. Blood smears from HIV-infected individuals and SIVmac239-infected rhesus macaques revealed increased LGLs compared to HIV/SIV negative counterparts. In humans, there was no correlation with viral load or treatment and in macaques the LGLs arose in acute SIV infection with increases in viremia. This is the first report describing and partially characterizing LGL lymphocytosis in association with lentiviral infections in three different species. PMID:26292765

  15. A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

    PubMed

    Jimenez-Moreno, Carmen Maria; Herrera-Gomez, Irene de Gracia; Lopez-Noriega, Livia; Lorenzo, Petra Isabel; Cobo-Vuilleumier, Nadia; Fuente-Martin, Esther; Mellado-Gil, Jose Manuel; Parnaud, Geraldine; Bosco, Domenico; Gauthier, Benoit Raymond; Martin-Montalvo, Alejandro

    2015-01-01

    Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional β-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes. PMID:26122098

  16. Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB

    PubMed Central

    Georgiadis, Christos; Syed, Farhatullah; Petrova, Anastasia; Abdul-Wahab, Alya; Lwin, Su M.; Farzaneh, Farzin; Chan, Lucas; Ghani, Sumera; Fleck, Roland A.; Glover, Leanne; McMillan, James R.; Chen, Mei; Thrasher, Adrian J.; McGrath, John A.; Di, Wei-Li; Qasim, Waseem

    2016-01-01

    Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgammanull recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man. PMID:26763448

  17. Was cDNA sequences modulate transgene expression of was promoter-driven lentiviral vectors.

    PubMed

    Toscano, Miguel G; Benabdellah, Karim; Muñoz, Pilar; Frecha, Cecilia; Cobo, Marién; Martín, Francisco

    2009-11-01

    Abstract The development of vectors that express a therapeutic transgene efficiently and specifically in hematopoietic cells (HCs) is an important goal for gene therapy of hematological disorders. We have previously shown that a 500-bp fragment from the proximal Was gene promoter in a lentiviral vector (LV) was sufficient to achieve more than 100-fold higher levels of Wiskott-Aldrich syndrome protein in HCs than in nonhematopoietic cells (non-HCs). We show now that this differential was reduced up to 10 times when the enhanced green fluorescent protein gene (eGFP) was expressed instead of Was in the same LV backbone. Insertion of Was cDNA sequences downstream of eGFP in these LVs had a negative effect on transgene expression. This effect varied in different cell types but, overall, Was cDNA sequences increased the hematopoietic specificity of Was promoter-driven LV. We have characterized the minimal fragment required to increase hematopoietic specificity and have demonstrated that the mechanism involves Was promoter regulation and RNA processing. In addition, we have shown that Was cDNA sequences interfere with the enhancer activity of the woodchuck posttranscriptional regulatory element. These results represent the first data showing the role of Was intragenic sequences in gene regulation. PMID:19630517

  18. Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB.

    PubMed

    Georgiadis, Christos; Syed, Farhatullah; Petrova, Anastasia; Abdul-Wahab, Alya; Lwin, Su M; Farzaneh, Farzin; Chan, Lucas; Ghani, Sumera; Fleck, Roland A; Glover, Leanne; McMillan, James R; Chen, Mei; Thrasher, Adrian J; McGrath, John A; Di, Wei-Li; Qasim, Waseem

    2016-01-01

    Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgamma(null) recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man. PMID:26763448

  19. Use of a Closed Culture System to Improve the Safety of Lentiviral Vector Production.

    PubMed

    Wu, Tao; Bour, Gaëtan; Durand, Sarah; Lindner, Véronique; Gossé, Francine; Zona, Laetitia; Certoux, Jean-Marie; Diana, Michele; Baumert, Thomas F; Marescaux, Jacques; Mutter, Didier; Pessaux, Patrick; Robinet, Eric

    2015-12-01

    We evaluated the possibility of introducing a combination of six oncogenes into primary porcine hepatocytes (PPH) using a lentiviral vector (LV)-mediated gene transfer in order to develop a porcine hepatocellular carcinoma model based on autologous transplantation of ex vivo-transformed hepatocytes. The six oncogenes were introduced into three plasmids, hence enabling the production of LVs encoding a luciferase reporter gene and hTERT+p53(DD), cyclinD1+CDK4(R24C), and c-myc(T58A)+HRas(G21V) genes, respectively. In order to improve the protection of the laboratory personnel manipulating such LVs, we used a compact cell culture cassette (CliniCell(®) device) as a closed cell culture system. We demonstrated that the CliniCell device allows to produce LVs, through plasmid transfection of 293T cells, and, after transfer to a second cassette, to transduce PPH with a similar efficacy as conventional open cell culture systems such as flasks or Petri dishes. Additionally, it is possible to cryopreserve at -80°C the transduced cells, directly in the CliniCell device used for the transduction. In conclusion, the use of a closed culture system for the safe handling of oncogene-encoding LVs lays the foundation for the development of porcine tumor models based on the autologous transplantation of ex vivo-transformed primary cells. PMID:26467420

  20. Role of transgene regulation in ex vivo lentiviral correction of artemis deficiency.

    PubMed

    Multhaup, Megan M; Podetz-Pedersen, Kelly M; Karlen, Andrea D; Olson, Erik R; Gunther, Roland; Somia, Nikunj V; Blazar, Bruce R; Cowan, Morton J; McIvor, R Scott

    2015-04-01

    Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID. PMID:25738323

  1. Stable and Efficient Gene Transfer into the Retina Using an HIV-Based Lentiviral Vector

    NASA Astrophysics Data System (ADS)

    Miyoshi, Hiroyuki; Takahashi, Masayo; Gage, Fred H.; Verma, Inder M.

    1997-09-01

    The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

  2. Characterization of common marmoset dysgerminoma-like tumor induced by the lentiviral expression of reprogramming factors

    PubMed Central

    Yamaguchi, Saori; Marumoto, Tomotoshi; Nii, Takenobu; Kawano, Hirotaka; Liao, Jiyuan; Nagai, Yoko; Okada, Michiyo; Takahashi, Atsushi; Inoue, Hiroyuki; Sasaki, Erika; Fujii, Hiroshi; Okano, Shinji; Ebise, Hayao; Sato, Tetsuya; Suyama, Mikita; Okano, Hideyuki; Miura, Yoshie; Tani, Kenzaburo

    2014-01-01

    Recent generation of induced pluripotent stem (iPSCs) has made a significant impact on the field of human regenerative medicine. Prior to the clinical application of iPSCs, testing of their safety and usefulness must be carried out using reliable animal models of various diseases. In order to generate iPSCs from common marmoset (CM; Callithrix jacchus), one of the most useful experimental animals, we have lentivirally transduced reprogramming factors, including POU5F1 (also known as OCT3/4), SOX2, KLF4, and c-MYC into CM fibroblasts. The cells formed round colonies expressing embryonic stem cell markers, however, they showed an abnormal karyotype denoted as 46, X, del(4q), +mar, and formed human dysgerminoma-like tumors in SCID mice, indicating that the transduction of reprogramming factors caused unexpected tumorigenesis of CM cells. Moreover, CM dysgerminoma-like tumors were highly sensitive to DNA-damaging agents, irradiation, and fibroblast growth factor receptor inhibitor, and their growth was dependent on c-MYC expression. These results indicate that DNA-damaging agents, irradiation, fibroblast growth factor receptor inhibitor, and c-MYC-targeted therapies might represent effective treatment strategies for unexpected tumors in patients receiving iPSC-based therapy. PMID:24521492

  3. Editing T cell specificity towards leukemia by zinc-finger nucleases and lentiviral gene transfer

    PubMed Central

    Lombardo, Angelo; Magnani, Zulma; Liu, Pei-Qi; Reik, Andreas; Chu, Victoria; Paschon, David E.; Zhang, Lei; Kuball, Jurgen; Camisa, Barbara; Bondanza, Attilio; Casorati, Giulia; Ponzoni, Maurilio; Ciceri, Fabio; Bordignon, Claudio; Greenberg, Philip D.; Holmes, Michael C.; Gregory, Philip D.; Naldini, Luigi; Bonini, Chiara

    2016-01-01

    The transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. We designed zinc-finger nucleases (ZFNs) promoting the disruption of endogenous TCR β and α chain genes. ZFN-treated lymphocytes lacked CD3/TCR surface expression and expanded with IL-7 and IL-15. Upon lentiviral transfer of a TCR for the WT1 tumor antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near-purity, and proved superior in specific antigen recognition to matched TCR-transferred cells. In contrast to TCR-transferred cells, TCR edited lymphocytes did not mediate off-target reactivity while maintaining anti-tumor activity in vivo, thus demonstrating that complete editing of T-cell specificity generate tumor-specific lymphocytes with improved biosafety profile. PMID:22466705

  4. An integrated experimental program to understanding leakage from geologic carbon sequestration sites across scales

    NASA Astrophysics Data System (ADS)

    Clarens, A. F.; Wang, S.; Liang, B.; Peters, C. A.; Fitts, J. P.; Deng, H.; Ellis, B. R.

    2012-12-01

    angle decreased during the CO2 phase transition from supercritical or liquid to gas. These results suggest complex and nonlinear effects from wetting during leakage events. At intermediate scales, column experiments are underway in a 1 m column and a 10 m column is under construction. At Princeton, experimental work on fractured carbonate-rich caprocks has shown that preferential dissolution of calcite leads to significant alterations of fracture geometry. The evolution of fracture hydrodynamic properties, however, is more complex due to heterogeneous mineral distribution. Single- and multi-phase Computational Fluid Dynamics (CFD) simulations have been conducted to quantify changes in permeability and relative permeability due to geochemical alterations. Also, CFD calculations generate the detailed maps of velocity fields needed to study the interactions between fluid dynamics and mineral heterogeneity, which ultimately determine reaction rates. At the bench-scale, experiments are underway to study CO2 properties and reactions in porous and fractured sedimentary rocks. Inferences are drawn with the benefit of a suite of imaging methods. These experimental and modeling efforts are being integrated to isolate relevant physical and geochemical processes to generate the fundamental understanding needed to interpret field-scale observations.

  5. Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation.

    PubMed

    Park, Gun Wook; Kim, Jin Young; Hwang, Heeyoun; Lee, Ju Yeon; Ahn, Young Hee; Lee, Hyun Kyoung; Ji, Eun Sun; Kim, Kwang Hoe; Jeong, Hoi Keun; Yun, Ki Na; Kim, Yong-Sam; Ko, Jeong-Heon; An, Hyun Joo; Kim, Jae Han; Paik, Young-Ki; Yoo, Jong Shin

    2016-01-01

    Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed Integrated GlycoProteome Analyzer (I-GPA) including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database that we constructed, we created novel scoring algorithms with decoy glycopeptides, where 95 N-glycopeptides from standard α1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. The efficiency of I-GPA was demonstrated by automatically identifying 619 site-specific N-glycopeptides with FDR ≤ 1%, and simultaneously quantifying 598 N-glycopeptides, from human plasma samples that are known to contain highly glycosylated proteins. Thus, I-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes, which can be applied to biomarker discovery and ongoing global human proteome project. PMID:26883985

  6. Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation

    PubMed Central

    Park, Gun Wook; Kim, Jin Young; Hwang, Heeyoun; Lee, Ju Yeon; Ahn, Young Hee; Lee, Hyun Kyoung; Ji, Eun Sun; Kim, Kwang Hoe; Jeong, Hoi Keun; Yun, Ki Na; Kim, Yong-Sam; Ko, Jeong-Heon; An, Hyun Joo; Kim, Jae Han; Paik, Young-Ki; Yoo, Jong Shin

    2016-01-01

    Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed Integrated GlycoProteome Analyzer (I-GPA) including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database that we constructed, we created novel scoring algorithms with decoy glycopeptides, where 95 N-glycopeptides from standard α1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. The efficiency of I-GPA was demonstrated by automatically identifying 619 site-specific N-glycopeptides with FDR ≤ 1%, and simultaneously quantifying 598 N-glycopeptides, from human plasma samples that are known to contain highly glycosylated proteins. Thus, I-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes, which can be applied to biomarker discovery and ongoing global human proteome project. PMID:26883985

  7. Toward an integrated regional and on-site Earthquake Early Warning System for Southern Italy: test and performances

    NASA Astrophysics Data System (ADS)

    Emolo, A.; Zollo, A.; Elia, L.; Martino, C.; Colombelli, S.; Festa, G.; Iannaccone, G.

    2011-12-01

    We present an approach to Earthquake Early Warning for Southern Italy that integrates regional and on-site systems. The regional approach is based on the PRESTo (PRobabilistic and Evolutionary early warning SysTem) software platform. PRESTo processes the 3C acceleration data streams from the stations of the Irpinia Seismic Network (ISNet) for P-waves arrival detection and, once an event has been detected, provides location and magnitude estimations, and peak ground motion predictions at target sites. The earthquake location is obtained through an evolutionary, real-time probabilistic approach based on an equal differential time formulation profiting of information from both triggered and not-yet-triggered stations. The magnitude is estimated by an empirical relation that correlates it with the peak displacement measured on the first 2-4s of signal, starting at the detected P-wave. The peak ground motion parameters at a site of interest can be finally estimated by specific ground motion prediction equations once that the location and magnitude are known. All estimates are provided as a probability density functions with an uncertainty that typically decreases with time. Alarm messages can thus reach vulnerable structures before the destructive waves. PRESTo has been under continuous real-time testing during the past two years using data streaming from ISNet and has produced a bulletin of about a hundred low-magnitude events. Meanwhile, PRESTo has been tested off-line playing back in the system both real and synthetic seismograms for moderate-to-large events. The tests performed have shown that a stable solution is generally reached within 10s from the origin time when a dense seismic network is deployed in the source area. The on-site system is based on the real-time measurement, at near-source stations located at increasing distances from the earthquake epicenter, of the peak displacement and P-wave dominant period, measured on a 3s window after the first P arrival

  8. Integrative Multi-omic Analysis of Human Platelet eQTLs Reveals Alternative Start Site in Mitofusin 2.

    PubMed

    Simon, Lukas M; Chen, Edward S; Edelstein, Leonard C; Kong, Xianguo; Bhatlekar, Seema; Rigoutsos, Isidore; Bray, Paul F; Shaw, Chad A

    2016-05-01

    Platelets play a central role in ischemic cardiovascular events. Cardiovascular disease (CVD) is a major cause of death worldwide. Numerous genome-wide association studies (GWASs) have identified loci associated with CVD risk. However, our understanding of how these variants contribute to disease is limited. Using data from the platelet RNA and expression 1 (PRAX1) study, we analyzed cis expression quantitative trait loci (eQTLs) in platelets from 154 normal human subjects. We confirmed these results in silico by performing allele-specific expression (ASE) analysis, which demonstrated that the allelic directionality of eQTLs and ASE patterns correlate significantly. Comparison of platelet eQTLs with data from the Genotype-Tissue Expression (GTEx) project revealed that a number of platelet eQTLs are platelet specific and that platelet eQTL peaks localize to the gene body at a higher rate than eQTLs from other tissues. Upon integration with data from previously published GWASs, we found that the trait-associated variant rs1474868 coincides with the eQTL peak for mitofusin 2 (MFN2). Additional experimental and computational analyses revealed that this eQTL is linked to an unannotated alternate MFN2 start site preferentially expressed in platelets. Integration of phenotype data from the PRAX1 study showed that MFN2 expression levels were significantly associated with platelet count. This study links the variant rs1474868 to a platelet-specific regulatory role for MFN2 and demonstrates the utility of integrating multi-omic data with eQTL analysis in disease-relevant tissues for interpreting GWAS results. PMID:27132591

  9. Integrating nonlinear site effects in broadband ground motion models: Observations and simulations in the Los Angeles basin

    NASA Astrophysics Data System (ADS)

    Assimaki, D.; Steidl, J.; Li, W.

    2006-12-01

    Site-specific ground response analyses to strong seismic motion are currently required for the design of new components of the civil infrastructure on deep and/or soft sedimentary deposits, which prevail in many urban areas of moderate to high seismic risk in the US such as the Los Angeles basin. In these cases, it is necessary to arrive at realistic predictions of the nonlinear soil behavior, which require elaborate analyses to be employed that are associated, however, with considerable cost and effort. Currently, uncertainties exist regarding the methodology to be employed, the level of sophistication required that determines the number of input parameters, and the development of these parameters for implementation. These difficulties, further aggravated by the lack of well-documented validation studies, have thus far prohibited the integration of nonlinear analysis procedures in broadband ground motion simulations. We here present the parametric investigation of recorded and simulated ground motion time-histories for selected sites in the Los Angeles basin, conducted to examine the variability in ground response estimation as a function of the employed nonlinear soil model complexity, and the extent of geotechnical data availability in the near-surface. For this purpose, weak motion waveform inversion is first employed at selected sites in the basin, in absence of the necessary detailed low-strain dynamic soil property distribution in the near surface. Successively, the corresponding one-dimensional (1D) crustal velocity profiles are depicted at these locations based on the SCEC Community Velocity Model, and synthetic ground motion simulations are conducted for the rupture of a wide range of strike-slip fault geometries by means of the hybrid low/high-frequency method with correlated random source parameters. The lack of multiple strong motion seismic data at these sites necessitates the use of simulated ground motion time-histories for the generation of a

  10. Generation and usage of aequorin lentiviral vectors for Ca(2+) measurement in sub-cellular compartments of hard-to-transfect cells.

    PubMed

    Lim, Dmitry; Bertoli, Alessandro; Sorgato, M Catia; Moccia, Francesco

    2016-05-01

    Targeted aequorin-based Ca(2+) probes represent an unprecedented tool for the reliable measurement of Ca(2+) concentration and dynamics in different sub-cellular compartments. The main advantages of aequorin are its proteinaceous nature, which allows attachment of a signal peptide for targeting aequorin to virtually any sub-cellular compartment; its low Ca(2+)-binding capacity; the wide range of Ca(2+) concentrations that can be measured, ranging from sub-micromolar to millimolar; its robust performance in aggressive environments, e.g., the strong acidic pH of the lysosomal lumen. Lentiviral vectors represent a popular tool to transduce post-mitotic or hard-to-transfect cells both in vitro and in vivo. Furthermore, it has great potential for gene therapy. Last generation lentiviral vectors represent a perfect compromise for combining large insert size, ease of production and handling, and high degree of biosafety. Here, we describe strategies for cloning aequorin probes - targeted to the cytosol, sub-plasma membrane cytosolic domains, the mitochondrial matrix, and the endoplasmic reticulum lumen - into lentiviral vectors. We describe methods for the production of lentiviral particles, and provide examples of measuring Ca(2+) dynamics by such aequorin-encoding lentiviral vectors in sub-cellular compartments of hard-to-transfect cells, including immortalized striatal neurons, primary cerebellar granule neurons and endothelial progenitor cells, which provide suitable in vitro models for the study of different human diseases. PMID:26992273

  11. Restrictions to cross-species transmission of lentiviral infection gleaned from studies of FIV.

    PubMed

    VandeWoude, Sue; Troyer, Jennifer; Poss, Mary

    2010-03-15

    More than 40 species of primates and over 20 species of cats harbor antibodies that sero-react to lentiviral antigens. In nearly all cases where viral genetic analysis has been conducted, each host species is infected with a unique lentivirus. Though lentivirus clades within a species can be substantially divergent, they are typically monophyletic within that species. A notable significant departure from this observation is apparent cross-species transmission of FIV between bobcats (Lynx rufus) and pumas (Puma concolor) in Southern California that has occurred at least three times; evidence from one bobcat sequence suggests this cross-over may have also occurred in Florida between bobcats and the endangered Florida panther. Several other isolated reports demonstrate cross-species transmission of FIV isolates among captive animals housed in close proximity, and it is well established that HIV-1 and HIV-2 arose from human contact with SIV-infected non-human primates. Using an experimental model, we have determined that domestic cats (Felis catus) are susceptible to FIVs originating from pumas or lions. While infections are initially replicative, and animals seroconvert, within a relatively short period of time circulating virus is reduced to nearly undetectable levels in a majority of animals. This diminution of viral load is proportional to initial viral peak. Although viral reservoirs can be identified in gastrointestinal tissues, most viral genomes recovered peripherally are highly mutated, suggesting that the non-adapted host successfully inhibits normal viral replication, leading to replication incompetent viral progeny. Mechanisms possible for such restriction of cross-species infections in natural settings include: (1) Lack of contact conducive to lentiviral transmission between infected and shedding animals of different species; (2) Lack of suitable receptor repertoire to allow viral entry to susceptible cells of a new species; (3) Cellular machinery in the

  12. The effect of myostatin silencing by lentiviral-mediated RNA interference on goat fetal fibroblasts.

    PubMed

    Lu, Jian; Wei, Caihong; Zhang, Xiaoning; Xu, Lingyang; Zhang, Shifang; Liu, Jiasen; Cao, Jiaxue; Zhao, Fuping; Zhang, Li; Li, Bichun; Du, Lixin

    2013-06-01

    Myostatin is a transforming growth factor-β family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may promote muscle growth, we used RNA interference mediated by a lentiviral vector to knockdown myostatin in goat fetal fibroblast cells. We also investigated the expression changes in relevant myogenic regulatory factors (MRFs) and adipogenic regulatory factors in the absence of myostatin in goat fetal fibroblasts. Quantitative RT-PCR revealed that myostatin transcripts were significantly reduced by 75 % (P < 0.01). Western blot showed that myostatin protein expression was reduced by 95 % (P < 0.01). We also found that the mRNA expression of activin receptor IIB (ACVR2B) significantly increased by 350 % (P < 0.01), and p21 increased 172 % (P < 0.01). Furthermore, myostatin inhibition decreased Myf5 and increased MEF2C mRNA expression in goat fetal fibroblasts, suggesting that myostatin regulates MRFs differently in fibroblasts compared to muscle. In addition, the expression of adipocyte marker genes peroxisome proliferator-activated receptor (PPAR) γ and leptin, but not CCAAT/enhance-binding protein (C/EBP) α and C/EBPβ, were upregulated at the transcript level after myostatin silencing. These results suggest that we have generated a novel way to block myostatin in vitro, which could be used to improve livestock meat production and gene therapy of musculoskeletal diseases. This also suggests that myostatin plays a negative role in regulating the expression of adipogenesis related genes in goat fetal fibroblasts. PMID:23604693

  13. Intraosseous Delivery of Lentiviral Vectors Targeting Factor VIII Expression in Platelets Corrects Murine Hemophilia A

    PubMed Central

    Wang, Xuefeng; Shin, Simon C; Chiang, Andy F J; Khan, Iram; Pan, Dao; Rawlings, David J; Miao, Carol H

    2015-01-01

    Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage-Sca1+c-Kit+ hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency. PMID:25655313

  14. Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.

    PubMed

    Brunger, Jonathan M; Huynh, Nguyen P T; Guenther, Caitlin M; Perez-Pinera, Pablo; Moutos, Franklin T; Sanchez-Adams, Johannah; Gersbach, Charles A; Guilak, Farshid

    2014-03-01

    The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering. PMID:24550481

  15. Functional validation of a human CAPN5 exome variant by lentiviral transduction into mouse retina.

    PubMed

    Wert, Katherine J; Skeie, Jessica M; Bassuk, Alexander G; Olivier, Alicia K; Tsang, Stephen H; Mahajan, Vinit B

    2014-05-15

    Exome sequencing indicated that the gene encoding the calpain-5 protease, CAPN5, is the likely cause of retinal degeneration and autoimmune uveitis in human patients with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). To explore the mechanism of ADNIV, a human CAPN5 disease allele was expressed in mouse retinas with a lentiviral vector created to express either the wild-type human (h) CAPN5 or the ADNIV mutant hCAPN5-R243L allele under a rhodopsin promoter with tandem green fluorescent protein (GFP) expression. Vectors were injected into the subretinal space of perinatal mice. Mouse phenotypes were analyzed using electroretinography, histology and inflammatory gene expression profiling. Mouse calpain-5 showed high homology to its human ortholog with >98% sequence identity that includes the ADNIV mutant residue. Calpain-5 protein was expressed in the inner and outer segments of the photoreceptors and in the outer plexiform layer. Expression of the hCAPN5-R243L allele caused loss of the electroretinogram b-wave, photoreceptor degeneration and induction of immune cell infiltration and inflammatory genes in the retina, recapitulating major features of the ADNIV phenotype. Intraocular neovascularization and fibrosis were not observed during the study period. Our study shows that expression of the hCAPN5-R243L disease allele elicits an ADNIV-like disease in mice. It further suggests that ADNIV is due to CAPN5 gain-of-function rather than haploinsufficiency, and retinal expression may be sufficient to generate an autoimmune response. Genetic models of ADNIV in the mouse can be used to explore protease mechanisms in retinal degeneration and inflammation as well as preclinical therapeutic testing. PMID:24381307

  16. Functional validation of a human CAPN5 exome variant by lentiviral transduction into mouse retina

    PubMed Central

    Wert, Katherine J.; Skeie, Jessica M.; Bassuk, Alexander G.; Olivier, Alicia K.; Tsang, Stephen H.; Mahajan, Vinit B.

    2014-01-01

    Exome sequencing indicated that the gene encoding the calpain-5 protease, CAPN5, is the likely cause of retinal degeneration and autoimmune uveitis in human patients with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). To explore the mechanism of ADNIV, a human CAPN5 disease allele was expressed in mouse retinas with a lentiviral vector created to express either the wild-type human (h) CAPN5 or the ADNIV mutant hCAPN5-R243L allele under a rhodopsin promoter with tandem green fluorescent protein (GFP) expression. Vectors were injected into the subretinal space of perinatal mice. Mouse phenotypes were analyzed using electroretinography, histology and inflammatory gene expression profiling. Mouse calpain-5 showed high homology to its human ortholog with >98% sequence identity that includes the ADNIV mutant residue. Calpain-5 protein was expressed in the inner and outer segments of the photoreceptors and in the outer plexiform layer. Expression of the hCAPN5-R243L allele caused loss of the electroretinogram b-wave, photoreceptor degeneration and induction of immune cell infiltration and inflammatory genes in the retina, recapitulating major features of the ADNIV phenotype. Intraocular neovascularization and fibrosis were not observed during the study period. Our study shows that expression of the hCAPN5-R243L disease allele elicits an ADNIV-like disease in mice. It further suggests that ADNIV is due to CAPN5 gain-of-function rather than haploinsufficiency, and retinal expression may be sufficient to generate an autoimmune response. Genetic models of ADNIV in the mouse can be used to explore protease mechanisms in retinal degeneration and inflammation as well as preclinical therapeutic testing. PMID:24381307

  17. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors

    PubMed Central

    Hart, Bryan E.; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D.; Lukose, Regy; Souther, Sommer J. R.; Rayasam, Swati D. G.; Saelens, Joseph W.; Chen, Ching-ju; Seay, Sarah A.; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E.; Ng, Tony W.; Tobin, David M.; Porcelli, Steven A.; Larsen, Michelle H.; Schmitz, Joern E.; Haynes, Barton F.; Jacobs, William R.; Lee, Sunhee

    2015-01-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. PMID:25924766

  18. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    NASA Astrophysics Data System (ADS)

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2014-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

  19. Fibrin hydrogels for lentiviral gene delivery in vitro and in vivo

    PubMed Central

    Kidd, Martha; Shin, Seungjin; Shea, Lonnie D.

    2011-01-01

    Gene delivery from hydrogels represents a versatile approach for localized expression of tissue inductive factors than can promote cellular processes that lead to regeneration. Lentiviral gene therapy vectors were entrapped within fibrin hydrogels, either alone or complexes with hydroxylapatite (HA) nanoparticles. The inclusion of HA into the hydrogel led to the formation of small aggregates distributed throughout the hydrogel, with no obvious alteration of the pore structure outside the aggregates. The presence of HA slowed hydrogel degradation by collagenase and plasmin relative to fibrin alone, and also decreased the rate of cell migration. Lentivirus had similar release from the fibrin hydrogels formed with or without HA. The altered hydrogel properties suggest an interaction between the nanoparticle and fibrin, which may displace the virus from the particle leading to similar release profiles. Transgene expression by cells migrating into the hydrogel in vitro was reduced in the presence of HA, consistent with the role of cell migration on transgene expression. In vivo, lentivirus loaded fibrin hydrogels promoted localized transgene expression that increased through day 9 and decreased through day 14. For the fibrin only hydrogels, expression continued to decline after day 14. However, hydrogels with HA maintained this transgene expression level for an additional two weeks before declining. Immunostaining identified transgene primarily outside the fibrin-HA gel at day 9; however, at day 21, transgene expression was observed primarily within the fibrin-HA gel. The localized delivery of lentivirus provides an opportunity to enhance the bioactivity of fibrin hydrogels for a wide range of applications in regenerative medicine. PMID:21907251

  20. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

    PubMed

    Hart, Bryan E; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D; Lukose, Regy; Souther, Sommer J R; Rayasam, Swati D G; Saelens, Joseph W; Chen, Ching-Ju; Seay, Sarah A; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E; Ng, Tony W; Tobin, David M; Porcelli, Steven A; Larsen, Michelle H; Schmitz, Joern E; Haynes, Barton F; Jacobs, William R; Lee, Sunhee; Frothingham, Richard

    2015-07-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. PMID:25924766

  1. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    PubMed Central

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2013-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits HIV-1 infection of myeloid-lineage cells 1,2 as well as resting CD4+ T cells 3,4 by reducing the cellular dNTP concentration to a level where the viral reverse transcriptase cannot function 5,6. In other lentiviruses, including HIV-2 and related SIVs, SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation 7,8. The molecular mechanism by which these viral proteins are able to usurp the host cell’s ubiquitination machinery to destroy the cell’s protection against these viruses has not been defined. We present here the crystal structure of a ternary complex of Vpx with the host cell’s E3 ligase substrate adaptor DCAF1 and the C-terminal region of SAMHD1. Vpx is made up of a three-helical bundle, stabilised by a zinc finger motif and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C-terminus making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure provides the first description of how a lentiviral accessory protein is able to subvert the cell’s normal protein degradation pathway to inactivate the cellular viral defence system. PMID:24336198

  2. Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer.

    PubMed

    Mostoslavsky, Gustavo; Fabian, Attila J; Rooney, Sean; Alt, Frederick W; Mulligan, Richard C

    2006-10-31

    Artemis gene mutations are responsible for the development of a severe combined immunodeficiency [radiation-sensitive (RS) SCID] characterized by a severe B and T cell deficiency and a normal natural killer cell population. To establish the feasibility of a gene therapy approach to the treatment of RS-SCID, we generated a series of lentiviral vectors expressing human Artemis from different promoters and used them to transduce highly purified hematopoietic stem cells (HSCs) from Artemis knockout mice. HSCs transduced by the different viruses were transplanted into either lethally irradiated Rag-1-deficient animals or Artemis knockout mice treated with a nonmyeloablative dose of Busulfan. In both models, transplantation of HSCs transduced by a vector that used a murine phosphoglycerate kinase (PGK) promoter led to a complete functional correction of the immunodeficiency. Corrected animals displayed rescue of mature B cells with normal levels of serum immunoglobulins, together with complete rescue of the T cell compartment as evidenced by the presence of mature T lymphocytes in peripheral blood as well as normal values of thymocytes in thymus. Those B and T cells were capable of activation, as shown both by in vitro stimulation responses and in vivo after immune challenge. Overall, the results indicate that a gene therapy approach for RS-SCID involving the transplantation of genetically modified HSCs is indeed feasible. Furthermore, our studies suggest the possibility that nonmyeloablative conditioning regimens might be effectively used to promote engraftment of genetically modified cells in the case of diseases where standard irradiation-based myeloablative bone marrow transplantation protocols may prove problematic. PMID:17062750

  3. Investigation of the Mesenchymal Stem Cell Compartment by Means of a Lentiviral Barcode Library.

    PubMed

    Bigildeev, A E; Cornils, K; Aranyossy, T; Sats, N V; Petinati, N A; Shipounova, I N; Surin, V L; Pshenichnikova, O S; Riecken, K; Fehse, B; Drize, N I

    2016-04-01

    The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic "barcodes" has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells. PMID:27293094

  4. Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer

    PubMed Central

    Mostoslavsky, Gustavo; Fabian, Attila J.; Rooney, Sean; Alt, Frederick W.; Mulligan, Richard C.

    2006-01-01

    Artemis gene mutations are responsible for the development of a severe combined immunodeficiency [radiation-sensitive (RS) SCID] characterized by a severe B and T cell deficiency and a normal natural killer cell population. To establish the feasibility of a gene therapy approach to the treatment of RS-SCID, we generated a series of lentiviral vectors expressing human Artemis from different promoters and used them to transduce highly purified hematopoietic stem cells (HSCs) from Artemis knockout mice. HSCs transduced by the different viruses were transplanted into either lethally irradiated Rag-1-deficient animals or Artemis knockout mice treated with a nonmyeloablative dose of Busulfan. In both models, transplantation of HSCs transduced by a vector that used a murine phosphoglycerate kinase (PGK) promoter led to a complete functional correction of the immunodeficiency. Corrected animals displayed rescue of mature B cells with normal levels of serum immunoglobulins, together with complete rescue of the T cell compartment as evidenced by the presence of mature T lymphocytes in peripheral blood as well as normal values of thymocytes in thymus. Those B and T cells were capable of activation, as shown both by in vitro stimulation responses and in vivo after immune challenge. Overall, the results indicate that a gene therapy approach for RS-SCID involving the transplantation of genetically modified HSCs is indeed feasible. Furthermore, our studies suggest the possibility that nonmyeloablative conditioning regimens might be effectively used to promote engraftment of genetically modified cells in the case of diseases where standard irradiation-based myeloablative bone marrow transplantation protocols may prove problematic. PMID:17062750

  5. Tropical forest response to elevated CO2: Model-experiment integration at the AmazonFACE site.

    NASA Astrophysics Data System (ADS)

    Frankenberg, C.; Berry, J. A.; Guanter, L.; Joiner, J.

    2014-12-01

    The terrestrial biosphere's response to current and future elevated atmospheric carbon dioxide (eCO2) is a large source of uncertainty in future projections of the C cycle, climate and ecosystem functioning. In particular, the sensitivity of tropical rainforest ecosystems to eCO­2 is largely unknown even though the importance of tropical forests for biodiversity, carbon storage and regional and global climate feedbacks is unambiguously recognized. The AmazonFACE (Free-Air Carbon Enrichment) project will be the first ecosystem scale eCO2 experiment undertaken in the tropics, as well as the first to be undertaken in a mature forest. AmazonFACE provides the opportunity to integrate ecosystem modeling with experimental observations right from the beginning of the experiment, harboring a two-way exchange, i.e. models provide hypotheses to be tested, and observations deliver the crucial data to test and improve ecosystem models. We present preliminary exploration of observed and expected process responses to eCO2 at the AmazonFACE site from the dynamic global vegetation model LPJ-GUESS, highlighting opportunities and pitfalls for model integration of tropical FACE experiments. The preliminary analysis provides baseline hypotheses, which are to be further developed with a follow-up multiple model inter-comparison. The analysis builds on the recently undertaken FACE-MDS (Model-Data Synthesis) project, which was applied to two temperate FACE experiments and exceeds the traditional focus on comparing modeled end-target output. The approach has proven successful in identifying well (and less well) represented processes in models, which are separated for six clusters also here; (1) Carbon fluxes, (2) Carbon pools, (3) Energy balance, (4) Hydrology, (5) Nutrient cycling, and (6) Population dynamics. Simulation performance of observed conditions at the AmazonFACE site (a.o. from Manaus K34 eddy flux tower) will highlight process-based model deficiencies, and aid the separation

  6. Integration of landslide hazard maps into probabilistic risk assessment in context of global changes: an alpine test site

    NASA Astrophysics Data System (ADS)

    Vandromme, Rosalie; Desramaut, Nicolas; Baills, Audrey; Fontaine, Mélanie; Hohmann, Audrey; Grandjean, Gilles; Sedan, Olivier; Puissant, Anne; Malet, Jean-Philippe

    2013-04-01

    The aim of this work is to develop a methodology to integrate global changes scenarios into quantitative risk assessment. This paper describes a methodology to take into account effects of changing climate on landslides activity and impacts of social changes on exposure to provide a complete evaluation of risk for given scenarios. This approach is applied for demonstration purpose on a southern alpine test site. Mechanical approaches represent a solution to quantify landslide susceptibility and to model hazard on unprecedented conditions, as it is likely to occur. However, as the quantity and the quality of data are generally very heterogeneous at a regional scale, it is necessary to take into account their uncertainty in the analysis. In this perspective, a new hazard modeling method has been developed and integrated in a GIS-based software called ALICE®. To go further, climate change scenarios have been computed for the alpine test site (Barcelonnette area, France) using the REMO-COSMO-LM. From the precipitation time series, a daily index of the soil water content has been computed thanks to a reservoir-based model (GARDENIA®). Hence, the program classifies hazard zones depending on the several spatial data (lithological, DEM, etc…) and different hydrological contexts varying in time. The probabilistically initiated landslides are then propagated thank to a semi-empirical model (BORA) to provide real hazard maps. Different scenarios of land-use have been developed using an automate cellular model to cover the probable range of development of potential elements at risks in the future. These exposure maps are then combined with the aforementioned hazard maps to obtain risk maps for the different periods and the different land-use development scenarios. Potential evolutions of landslide risks are then evaluated, with a general increase in the 7 communes. This methodology also allows the analysis of the contributions of both considered global changes (climate and

  7. Tropical forest response to elevated CO2: Model-experiment integration at the AmazonFACE site.

    NASA Astrophysics Data System (ADS)

    Fleischer, K.

    2015-12-01

    The terrestrial biosphere's response to current and future elevated atmospheric carbon dioxide (eCO2) is a large source of uncertainty in future projections of the C cycle, climate and ecosystem functioning. In particular, the sensitivity of tropical rainforest ecosystems to eCO­2 is largely unknown even though the importance of tropical forests for biodiversity, carbon storage and regional and global climate feedbacks is unambiguously recognized. The AmazonFACE (Free-Air Carbon Enrichment) project will be the first ecosystem scale eCO2 experiment undertaken in the tropics, as well as the first to be undertaken in a mature forest. AmazonFACE provides the opportunity to integrate ecosystem modeling with experimental observations right from the beginning of the experiment, harboring a two-way exchange, i.e. models provide hypotheses to be tested, and observations deliver the crucial data to test and improve ecosystem models. We present preliminary exploration of observed and expected process responses to eCO2 at the AmazonFACE site from the dynamic global vegetation model LPJ-GUESS, highlighting opportunities and pitfalls for model integration of tropical FACE experiments. The preliminary analysis provides baseline hypotheses, which are to be further developed with a follow-up multiple model inter-comparison. The analysis builds on the recently undertaken FACE-MDS (Model-Data Synthesis) project, which was applied to two temperate FACE experiments and exceeds the traditional focus on comparing modeled end-target output. The approach has proven successful in identifying well (and less well) represented processes in models, which are separated for six clusters also here; (1) Carbon fluxes, (2) Carbon pools, (3) Energy balance, (4) Hydrology, (5) Nutrient cycling, and (6) Population dynamics. Simulation performance of observed conditions at the AmazonFACE site (a.o. from Manaus K34 eddy flux tower) will highlight process-based model deficiencies, and aid the separation

  8. Integrated GPR and ERT as Enhanced Detection for Subsurface Historical Structures Inside Babylonian Houses Site, Uruk City, Southern Iraq

    NASA Astrophysics Data System (ADS)

    Al-Khersan, Emad H.; Al-Ani, Jassim M. T.; Abrahem, Salah N.

    2016-03-01

    Uruk archaeological site, which located in Al-Muthanna Governorate southern Iraq, was investigated by integrated geophysical methods, ground penetration radar (GPR) and electric resistivity tomography (ERT) to image the historical buried structures. The GPR images show large radar attributes characterized by its continuous reflections having different widths. GPR attributes at shallower depth are mainly representing the upper part of Babylonian Houses that can often be found throughout the study area. In addition, radargrams characterized objects such as buried items, buried trenches and pits which were mainly concentrated near the surface. The ERT results show the presence of several anomalies at different depths generally having low resistivities. It is clear that the first upper zone can be found throughout the whole area and it may represent the top zone of the Babylonian houses. This zone is characterized by its dry clay and sandy soil containing surface broken bricks and slag mixed with core boulders. The second one underneath the top shows a prominent lower resistivity zone. It is probably caused by the moisture content that reduces the resistivity. The thickness of this zone is not equal at all parts of the site. The third deeper zone typically represents the archaeological walls. Most of the main anomalies perhaps referred to the buried clay brick walls. The map of the archaeological anomalies distribution and 3D view of the foundations at the study area using GPR and ERT techniques clearly show the characteristics of the Babylonian remains. A contour map and 3D view of Uruk show that the archaeological anomalies are concentrated mainly at the NE part of the district with higher values of wall height that range between 6 and 8 m and reach to more than 10 m. At the other directions, there are fewer walls with lower heights of 4-6 m and reach in some places the wall foot.

  9. [Expression of NANOG gene in acute lymphoblastic leukemia cells and construction of lentiviral vector carrying NANOG specific shRNA].

    PubMed

    Cao, Jiang; Meng, Fan-Jing; Li, Li; Lu, Chao; Zhou, Jun; Cheng, Hai; Chen, Wei; Chen, Chong; Xu, Kai-Lin

    2014-04-01

    The aim of this study was to detect the expression of NANOG gene in acute lymphoblastic leukemia (ALL) cells, and to construct the lentiviral vector carrying NANOG specific shRNA. The expression of NANOG was detected by RT-PCR and Western blot in MOLT-4, CCRF-HSB2, Jurkat cells and bone marrow cells from 15 patients with ALL in our hospital. The lentiviral vector carrying NANOG specific shRNA was constructed. After infection of MOLT-4 cells with the lentivirus constructs, GFP (+) cells were harvested by flow cytometry. The efficiency of RNA interference was detected by real-time quantitative PCR and Western blot. The results showed that the expression of NANOG mRNA and protein was detected in MOLT-4, CCRF-HSB2 cells and 33.3% samples of bone marrow from patients with ALL. The sequencing results demonstrated that the mRNAs amplified from these leukemic cells showed higher homology to NANOGP8 than NANOG1. The lentiviral vector pLB-shNANOG-1, pLB-shNANOG-2 and pLB-shcontrol were constructed. The viral particles were harvested and concentrated by ultracentrifugation. The virus titers were (1.83-3.12) ×10(8) IU/ml. After infection of MOLT-4 cells with the lentivirus, flow cytometry detection indicated that the GFP(+) cells were harvested by real-time quantitative PCR and Western blot, the assays showed that the 2 designed shRNA could significantly down-regulate expression of NANOG gene and protein. It is concluded that NANOGP8 is expressed in various types of ALL cells and in 33.3% of marrow cell samples obtained from ALL patients. After infection with the lentivirus constructs, MOLT-4 cells which stably down-regulate the expression of NANOG mRNA are obtained. PMID:24762991