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Sample records for efficient gene transfer

  1. Gene Transfer Efficiency in Gonococcal Biofilms: Role of Biofilm Age, Architecture, and Pilin Antigenic Variation

    PubMed Central

    Kouzel, Nadzeya; Oldewurtel, Enno R.

    2015-01-01

    ABSTRACT Extracellular DNA is an important structural component of many bacterial biofilms. It is unknown, however, to which extent external DNA is used to transfer genes by means of transformation. Here, we quantified the acquisition of multidrug resistance and visualized its spread under selective and nonselective conditions in biofilms formed by Neisseria gonorrhoeae. The density and architecture of the biofilms were controlled by microstructuring the substratum for bacterial adhesion. Horizontal transfer of antibiotic resistance genes between cocultured strains, each carrying a single resistance, occurred efficiently in early biofilms. The efficiency of gene transfer was higher in early biofilms than between planktonic cells. It was strongly reduced after 24 h and independent of biofilm density. Pilin antigenic variation caused a high fraction of nonpiliated bacteria but was not responsible for the reduced gene transfer at later stages. When selective pressure was applied to dense biofilms using antibiotics at their MIC, the double-resistant bacteria did not show a significant growth advantage. In loosely connected biofilms, the spreading of double-resistant clones was prominent. We conclude that multidrug resistance readily develops in early gonococcal biofilms through horizontal gene transfer. However, selection and spreading of the multiresistant clones are heavily suppressed in dense biofilms. IMPORTANCE Biofilms are considered ideal reaction chambers for horizontal gene transfer and development of multidrug resistances. The rate at which genes are exchanged within biofilms is unknown. Here, we quantified the acquisition of double-drug resistance by gene transfer between gonococci with single resistances. At early biofilm stages, the transfer efficiency was higher than for planktonic cells but then decreased with biofilm age. The surface topography affected the architecture of the biofilm. While the efficiency of gene transfer was independent of the

  2. An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

    PubMed Central

    Chicaybam, Leonardo; Sodre, Andressa Laino; Curzio, Bianca Azevedo; Bonamino, Martin Hernan

    2013-01-01

    Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits. Aims We herein report a convenient and affordable method based on in house made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes. Results This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41–59%), the hard to transfect murine T cells (mean 38%, 36–42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as in vitro expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis. Conclusions We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs. PMID:23555950

  3. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    PubMed Central

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR) can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium. PMID:25520733

  4. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    SciTech Connect

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

  5. Efficient Non-Viral Ocular Gene Transfer with Compacted DNA Nanoparticles

    PubMed Central

    Farjo, Rafal; Skaggs, Jeff; Quiambao, Alexander B.; Cooper, Mark J.; Naash, Muna I.

    2006-01-01

    Background The eye is an excellent candidate for gene therapy as it is immune privileged and much of the disease-causing genetics are well understood. Towards this goal, we evaluated the efficiency of compacted DNA nanoparticles as a system for non-viral gene transfer to ocular tissues. The compacted DNA nanoparticles examined here have been shown to be safe and effective in a human clinical trial, have no theoretical limitation on plasmid size, do not provoke immune responses, and can be highly concentrated. Methods and Findings Here we show that these nanoparticles can be targeted to different tissues within the eye by varying the site of injection. Almost all cell types of the eye were capable of transfection by the nanoparticle and produced robust levels of gene expression that were dose-dependent. Most impressively, subretinal delivery of these nanoparticles transfected nearly all of the photoreceptor population and produced expression levels almost equal to that of rod opsin, the highest expressed gene in the retina. Conclusions As no deleterious effects on retinal function were observed, this treatment strategy appears to be clinically viable and provides a highly efficient non-viral technology to safely deliver and express nucleic acids in the retina and other ocular tissues. PMID:17183666

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

    PubMed

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

    2004-05-15

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

  7. Guanidinylated block copolymers for gene transfer: A comparison with amine-based materials for in vitro and in vivo gene transfer efficiency

    PubMed Central

    Choi, Jennifer L.; Tan, James-Kevin Y.; Sellers, Drew L.; Wei, Hua; Horner, Philip J.; Pun, Suzie H.

    2015-01-01

    There is currently no cure for neuron loss in the brain, which can occur due to traumatic injury or neurodegenerative disease. One method proposed to enhance neurogenesis in the brain is gene transfer to neural progenitor cells. In this work, a guanidine-based copolymer was synthesized and compared to an amine-based copolymer analog previously shown to effectively deliver genes in the murine brain. The guanidine-based copolymer was more efficient at gene transfer to immortalized, cultured cell lines; however, the amine-based copolymer was more effective at gene transfer in the brain. DNA condensation studies revealed that the nucleic acid complexes formed with the guanidine-based copolymer were more susceptible to unpackaging in the presence of heparin sulfate proteoglycans compared to complexes formed with the amine-based copolymer. Therefore, polyplexes formed from the amine-based copolymer may be more resistant to destabilization by the heparan sulfate proteoglycans present in the stem cell niches of the brain. PMID:25907042

  8. The efficiency of genetic transformation of mammalian cells by transfection and microinjection depends on the transferred gene.

    PubMed

    Strauss, M; Kiessling, U; Zavision, B A; Povitza, O N; Tikhonenko, T I; Geissler, E

    1983-01-01

    The efficiency of genetic transformation of mammalian cells was analysed with respect to the kind of the transferred gene and the selective system. Plasmids pAGO and pAG60 harboring the thymidine kinase gene of Herpes simplex virus type 1 and the bacterial neomycin resistance gene, respectively, were compared concerning their ability to transform mouse Ltk-aprt- cells. Using the calcium phosphate technique the neomycin resistance gene transformed at least ten times more efficiently than the thymidine kinase gene (3 X 10(-3) versus 2 X 10(-4] whereas the difference is even more impressive following microinjection of the plasmids into the nuclei (2 X 10(-1) versus 2.5 X 10(-3]. The neomycin system also proved to be more effective in secondary gene transfer experiments and, thus, seems to be the most convenient marker for cotransfer experiments.

  9. SAFETY AND EFFICIENCY OF MODULATING PARACELLULAR PERMEABILITY TO ENHANCE AIRWAY EPITHELIAL GENE TRANSFER IN VIVO

    EPA Science Inventory


    ABSTRACT

    We evaluated the safety of agents that enhance gene transfer by modulating paracellular permeability. Lactate dehydrogenase (LDH) and cytokine release were measured in polarized primary human airway epithelial (HAE) cells after luminal application of vehicle, ...

  10. Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition

    PubMed Central

    Fu, Jun; Wenzel, Silke C.; Perlova, Olena; Wang, Junping; Gross, Frank; Tang, Zhiru; Yin, Yulong; Stewart, A. Francis; Zhang, Youming

    2008-01-01

    Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes. PMID:18701643

  11. An Efficient Algorithm for Gene/Species Trees Parsimonious Reconciliation with Losses, Duplications and Transfers

    NASA Astrophysics Data System (ADS)

    Doyon, Jean-Philippe; Scornavacca, Celine; Gorbunov, K. Yu.; Szöllősi, Gergely J.; Ranwez, Vincent; Berry, Vincent

    Tree reconciliation methods aim at estimating the evolutionary events that cause discrepancy between gene trees and species trees. We provide a discrete computational model that considers duplications, transfers and losses of genes. The model yields a fast and exact algorithm to infer time consistent and most parsimonious reconciliations. Then we study the conditions under which parsimony is able to accurately infer such events. Overall, it performs well even under realistic rates, transfers being in general less accurately recovered than duplications. An implementation is freely available at http://www.atgc-montpellier.fr/MPR.

  12. Inclusion of high molecular weight dextran in calcium phosphate-mediated transfection significantly improves gene transfer efficiency.

    PubMed

    Wu, C; Lu, Y

    2007-05-15

    Calcium phosphate-based mammalian cell transfection is a widely used gene transfer technology. To facilitate the efficiency of this gene transfer method, several polysaccharide compounds were tested and evaluated for their effectiveness in enhancing DNA transfection. Using a HIV-1-derived lentivirus vector plasmid as a gene transfer indicator, we demonstrated that the addition of high molecular weight dextran-500 at 0.6-1.2% in the 2x Hepes buffered saline (HBS) increased transfection efficiency by over 50% (as reflected by the number of GFP-positive cells) and increased the titer of resulting lentivirus vector particles even more (up to 4-fold). This enhancement of transfection efficiency was further increased when higher molecular weight dextran formulations were used in place of dextran-500, and also when dextran was used in combination with polybrene, another polycationic chemical compound. Examination of transfected cells showed that dextran had no apparent adverse effect on cell viability and growth. Our data represent the first report showing that dextran can be used to enhance calcium phosphate-mediated gene transfer; this may be useful in applications for the generation of high-titer virus vector stocks using transient transfection technology.

  13. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation

    PubMed Central

    Lavitrano, Marialuisa; Bacci, Maria Laura; Forni, Monica; Lazzereschi, Davide; Di Stefano, Carla; Fioretti, Daniela; Giancotti, Paola; Marfé, Gabriella; Pucci, Loredana; Renzi, Luigina; Wang, Hongjun; Stoppacciaro, Antonella; Stassi, Giorgio; Sargiacomo, Massimo; Sinibaldi, Paola; Turchi, Valeria; Giovannoni, Roberto; Della Casa, Giacinto; Seren, Eraldo; Rossi, Giancarlo

    2002-01-01

    A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models. PMID:12393815

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

    PubMed

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

    2003-03-15

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

  15. Transendocardial delivery of AAV6 results in highly efficient and global cardiac gene transfer in rhesus macaques.

    PubMed

    Gao, Guangping; Bish, Lawrence T; Sleeper, Meg M; Mu, Xin; Sun, Lan; Lou, You; Duan, Jiachuan; Hu, Chunyan; Wang, Li; Sweeney, H Lee

    2011-08-01

    Heart disease is the leading cause of morbidity and mortality, and cardiac gene transfer has potential as a novel therapeutic approach. We previously demonstrated safe and efficient gene transfer to the canine heart using a percutaneous transendocardial injection procedure to deliver self-complementary (sc) adeno-associated virus 6 (AAV6) vector. In the present study, we proceed with our vertical translation study to evaluate cardiac gene transfer in nonhuman primates (NHPs). We screened approximately 30 adult male rhesus macaques for the presence of neutralizing antibodies against AAV6, AAV8, and AAV9, and then selected seven monkeys whose antibody titers against these three serotypes were lower than 1/5. The animals were then randomized to receive either scAAV6 (n=3), scAAV8 (n=1), or scAAV9 (n=3) vector expressing the enhanced green fluorescent protein (EGFP) reporter gene at a dose of 5.4×10(12) genome copies/kg, which was administered according to a modified version of our previously developed transendocardial injection procedure. One animal treated with scAAV6 died secondary to esophageal intubation. The remaining animals were euthanized 7 days after gene transfer, at which time tissue was collected for analysis of EGFP expression, histopathology, and biodistribution of the vector genome. We found that (i) transendocardial delivery of AAV is safe in the NHP, (ii) AAV6 and AAV8 provide efficient cardiac gene transfer at similar levels and are superior to AAV9, and (iii) AAV6 is more cardiac-specific than AAV8 and AAV9. The results of this NHP study may help guide the development AAV vectors for the treatment of cardiovascular disease in humans.

  16. Optimization of vesicular stomatitis virus-G pseudotyped feline immunodeficiency virus vector for minimized cytotoxicity with efficient gene transfer.

    PubMed

    Song, Jae Jin; Lee, Boyoung; Chang, Jin Woo; Kim, Joo-Hang; Kwon, Yunhee Kim; Lee, Heuiran

    2003-05-01

    FIV-based lentiviral vector has shown a unique opportunity as an efficient gene delivery vehicle, especially to nondividing human cells. Here, we genetically reconstructed the FIV-based vector by serially deleting residual virus genes of gag and vif, leading to minimized cytotoxicity together with efficient virus production and gene transfer. The modified FIV- based vector was generated by transiently transfecting 293T cells with three plasmids of the gene transfer vector with minimal gag region, the packaging plasmid without vif and the VSV-G-expressing plasmid. The vector was routinely generated as many as 1 x 10(7) transducing particles per ml and easily concentrated by simple centrifugation. The cytotoxic effect significantly decreased in sensitive cells to FIV infection even at high multiplicity of infection (MOI), such as 500. Moreover, the transduction efficiency was consistently retained after cell cycle was arrested in a variety of human cells. Taken together, our results suggest that the modified VSV-G pseudotyped FIV-based vector efficiently transduce dividing and nondividing human cells with minimal cytotoxicity.

  17. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

    PubMed

    Nakajima, Keisuke; Yaoita, Yoshio

    2015-01-16

    Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  18. A Cytoplasmic RNA Vector Derived from Nontransmissible Sendai Virus with Efficient Gene Transfer and Expression

    PubMed Central

    Li, Hai-Ou; Zhu, Ya-Feng; Asakawa, Makoto; Kuma, Hidekazu; Hirata, Takahiro; Ueda, Yasuji; Lee, Yun-Sik; Fukumura, Masayuki; Iida, Akihiro; Kato, Atsushi; Nagai, Yoshiyuki; Hasegawa, Mamoru

    2000-01-01

    We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of the Paramyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 × 108 to 1.0 × 108 cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems. PMID:10864670

  19. Efficient in vivo gene transfer by intraperitoneal injection of plasmid DNA and calcium carbonate microflowers in mice.

    PubMed

    Fumoto, Shintaro; Nakajima, Sayuri; Mine, Toyoharu; Yoshikawa, Naoki; Kitahara, Takashi; Sasaki, Hitoshi; Miyamoto, Hirotaka; Nishida, Koyo

    2012-07-02

    Gene transfer to intraperitoneal organs is thought to be a promising approach to treat such conditions as peritoneal fibrosis and peritoneal dissemination of cancers. We previously discovered that simple instillation of naked plasmid DNA (pDNA) onto intraperitoneal organs such as the liver and stomach could effectively transfer foreign genes in mice. In this study, we developed a novel nonviral method to enhance transfection efficiency of naked pDNA to intraperitoneal organs using a calcium carbonate suspension containing pDNA. Using commercially available calcium carbonate, we successfully transfected pDNA to the stomach. Handling of commercially available calcium carbonate, however, was troublesome owing to rapid precipitation and caking. To obtain slowly settling particles of calcium carbonate, we tried to synthesize novel versions of such particles and succeeded in creating flower-shaped particles, named calcium carbonate microflowers. Sedimentation of calcium carbonate microflowers was sufficiently slow for in vivo experiments. Moreover, the transfection efficiency of the suspension of calcium carbonate microflowers to the stomach was more effective than that of commercially available calcium carbonate, especially at low concentrations. Intraperitoneal injection of the suspension of calcium carbonate microflowers containing pDNA greatly enhanced naked pDNA transfer to whole intraperitoneal organs in mice. Furthermore, lactate dehydrogenase activities in intraperitoneal fluid and plasma were not raised by the suspension of calcium carbonate microflowers.

  20. Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins.

    PubMed

    Klein, Ronald L; Dayton, Robert D; Leidenheimer, Nancy J; Jansen, Karen; Golde, Todd E; Zweig, Richard M

    2006-03-01

    Adeno-associated virus (AAV) serotype 8 appears to be the strongest of the natural serotypes reported to date for gene transfer in liver and muscle. In this study, we evaluated AAV8 in the brain by several methods, including biophotonic imaging of green fluorescent protein (GFP). In the adult rat hippocampus, levels of GFP expressed were clearly greater with AAV8 than with AAV2 or AAV5 by Western blot and biophotonic imaging and slightly but significantly greater than AAV1 by Western blot. In the substantia nigra, the GFP expression conferred by AAV8 was toxic to dopamine neurons, although toxicity could be avoided with dose titration. At the low dose at which there was no GFP toxicity from the GFP vector, another AAV8 vector for a disease-related (P301L) form of the microtubule-associated protein tau caused a 78% loss of dopamine neurons and significant amphetamine-stimulated rotational behavior. The AAV8 tau vector-induced cell loss was greater than that from AAV2 or AAV5 tau vectors, demonstrating that the increased gene transfer was functional. While the toxicity observed with GFP expression warrants great caution, the efficient AAV8 is promising for animal models of neurodegenerative diseases and potentially as well for gene therapy of brain diseases.

  1. Targeted modifications in adeno-associated virus serotype 8 capsid improves its hepatic gene transfer efficiency in vivo.

    PubMed

    Sen, Dwaipayan; Gadkari, Rupali A; Sudha, Govindarajan; Gabriel, Nishanth; Kumar, Yesupatham Sathish; Selot, Ruchita; Samuel, Rekha; Rajalingam, Sumathi; Ramya, V; Nair, Sukesh C; Srinivasan, Narayanaswamy; Srivastava, Alok; Jayandharan, Giridhara R

    2013-04-01

    Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (~9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in

  2. High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation.

    PubMed

    Lavertu, Marc; Méthot, Stephane; Tran-Khanh, Nicolas; Buschmann, Michael D

    2006-09-01

    Chitosan is a biodegradable natural polysaccharide that has shown potential for gene delivery, although the ideal molecular weight (MW) and degree of deacetylation (DDA) for this application have not been elucidated. To examine the influence of these parameters on gene transfer, we produced chitosans with different DDAs (98%, 92%, 80% and 72%) and depolymerized them with nitrous acid to obtain different MWs (150, 80, 40 and 10 kDa). We produced 64 formulations of chitosan/pDNA complexes (16 chitosans, 2 amine-to-phosphate (N:P) ratios of 5:1 and 10:1 and 2 transfection media pH of 6.5 and 7.1), characterized them for size and surface charge, and tested them for gene transfection in HEK 293 cells in vitro. Several formulations produced high levels of transgene expression while two conditions, 92-10-5 and 80-10-10 [DDA-MW-N:P ratio] at pH 6.5, showed equivalence to our best positive control. The results also revealed an important coupling between DDA and MW of chitosan in determining transgene expression. Maximum expression was obtained with a certain combination of DDA and MW that depended on N:P ratio and the pH, but similar expression levels could be achieved by simultaneously lowering MW and increasing DDA or lowering DDA and increasing MW, suggesting a predominant role of particle stability, through co-operative electrostatic binding, in determining transfection efficiency.

  3. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids.

    PubMed

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H; Haydon, Rex C; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.

  4. A nuclear localization signal in the matrix of spleen necrosis virus (SNV) does not allow efficient gene transfer into quiescent cells with SNV-derived vectors

    SciTech Connect

    Caron, Marie-Christine; Caruso, Manuel . E-mail: manuel.caruso@crhdq.ulaval.ca

    2005-08-01

    A major limitation in gene therapy for vectors derived from Moloney murine leukemia virus (MLV) is that they only deliver genes into dividing cells. In this study, a careful comparison of spleen necrosis virus (SNV)-derived vectors with MLV and human immunodeficiency virus (HIV)-1 retroviral vectors indicated that SNV vectors can deliver genes 4-fold more efficiently than MLV vectors into aphidicolin-arrested cells, although at a 25-fold lower efficiency than HIV-1-derived vectors. Furthermore, the addition of a NLS in the SNV matrix (MA) that mimics the one located in HIV-1 MA did not increase the ability of SNV vectors to transfer genes into arrested cells. Also, we found that the RD114 envelope was able to pseudotype SNV viral particles in a very efficient manner.

  5. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    SciTech Connect

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.; Miller, A.D.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

  6. Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

    PubMed

    White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

    2008-12-01

    Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

  7. Highly efficient lentiviral gene transfer in CD34+ and CD34+/38-/lin- cells from mobilized peripheral blood after cytokine prestimulation.

    PubMed

    Géronimi, Fabien; Richard, Emmanuel; Redonnet-Vernhet, Isabelle; Lamrissi-Garcia, Isabelle; Lalanne, Magalie; Ged, Cécile; Moreau-Gaudry, François; De Verneuil, Hubert

    2003-01-01

    Because mobilized peripheral blood (mPB) represents an attractive source of cells for gene therapy, we investigated lentiviral gene transfer in CD34(+) cells and the stem/progenitor-cell-enriched CD34(+)/38(-)/lin(-) cell subset isolated from mPB. In this study, we used an optimized third-generation self-inactivating lentiviral vector containing both the central polypurine tract and the woodchuck hepatitis posttranscriptional regulatory element sequences and encoding enhanced green fluorescent protein (EGFP) under the control of the elongation factor lalpha promoter. This lentivector was first used to compare multiplicity of infection (MOI)-dependent gene transfer efficiency in cord blood (CB) versus mPB CD34(+)-derived cells, colony-forming cells (CFCs), and long-term culture-initiating cells (LTC-ICs). Results showed a difference in the percentage of transduced cells particularly significant at low MOIs. A plateau was reached where 15% and 25% of CB and mPB cells, respectively, remained refractory to lentiviral trans-duction. Effects of a cytokine prestimulation period (18 hours) with interleukin-3, stem cell factor, Flt-3 ligand, and thrombopoietin were then analyzed in total cells, CFCs, and LTC-ICs derived from mPB CD34(+) cells. Transduction levels in those conditions demonstrated a two- and fourfold increase in CFCs and LTC-ICs, respectively, compared with unstimulated (<3 hours) control cells. Moreover, using the same transduction protocol, we were able to efficiently transduce CD34(+)/38(-)/lin(-) cells isolated from mPB, with up to >85% of colonies derived from LTC-ICs expressing EGFP and gene transfer levels remaining stable for 10 weeks in liquid culture. We therefore demonstrate a highly efficient gene transfer in this therapeutically relevant target cell population.

  8. IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential.

    PubMed

    Ficara, Francesca; Superchi, Daniela B; Hernández, Raisa Jofra; Mocchetti, Cristina; Carballido-Perrig, Nicole; Andolfi, Grazia; Deola, Sara; Colombo, Augusto; Bordignon, Claudio; Carballido, José M; Roncarolo, Maria Grazia; Aiuti, Alessandro

    2004-12-01

    To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.

  9. Highly efficient and minimally invasive in-vivo gene transfer to the mouse uterus using haemagglutinating virus of Japan (HVJ) envelope vector.

    PubMed

    Nakamura, Hitomi; Kimura, Tadashi; Ikegami, Hiroyuki; Ogita, Kazuhide; Koyama, Shinsuke; Shimoya, Koichiro; Tsujie, Tomoko; Koyama, Masayasu; Kaneda, Yasufumi; Murata, Yuji

    2003-10-01

    The uterus is obviously critical in implantation, development of the fetus and parturition. Endometrial cancer derived from endometrial epithelium is one of the common malignancies in the female reproductive tract. In order to clarify the local mechanisms of reproductive physiology and establish a non-systemic therapeutic strategy for reproductive failure as well as for endometrial cancer, we applied haemagglutinating virus of Japan envelope (HVJ-E) vector to in-vivo gene transfer into the uterine cavity of IVCS mice. Injection of HVJ-E vector into mouse uterine cavity on day 1.5 post coitum (p.c.) introduced a reporter gene approximately 120-fold more efficiently than introduction using the cationic liposome method. The expression of the introduced gene continued for at least 3 days. The plasmid vector was localized in the endometrial epithelium, whereas oligo deoxynucleotides were distributed throughout the epithelium, stromal cells and myometrium. HVJ-E vector did not affect the pregnancy rate, course of pregnancy, litter size, fetal growth in utero or parturition, and did not transfect the exogenous gene to the fetus. These results indicate that gene transfer into the uterus using HVJ-E vector is highly efficient and safe during pregnancy, and results in a well controlled distribution of the exogenous DNA. We believe that this procedure should be widely applicable for investigations of reproductive physiology as well as for methods of local gene therapy in the uterus.

  10. Inferring Horizontal Gene Transfer

    PubMed Central

    Lassalle, Florent; Dessimoz, Christophe

    2015-01-01

    Horizontal or Lateral Gene Transfer (HGT or LGT) is the transmission of portions of genomic DNA between organisms through a process decoupled from vertical inheritance. In the presence of HGT events, different fragments of the genome are the result of different evolutionary histories. This can therefore complicate the investigations of evolutionary relatedness of lineages and species. Also, as HGT can bring into genomes radically different genotypes from distant lineages, or even new genes bearing new functions, it is a major source of phenotypic innovation and a mechanism of niche adaptation. For example, of particular relevance to human health is the lateral transfer of antibiotic resistance and pathogenicity determinants, leading to the emergence of pathogenic lineages [1]. Computational identification of HGT events relies upon the investigation of sequence composition or evolutionary history of genes. Sequence composition-based ("parametric") methods search for deviations from the genomic average, whereas evolutionary history-based ("phylogenetic") approaches identify genes whose evolutionary history significantly differs from that of the host species. The evaluation and benchmarking of HGT inference methods typically rely upon simulated genomes, for which the true history is known. On real data, different methods tend to infer different HGT events, and as a result it can be difficult to ascertain all but simple and clear-cut HGT events. PMID:26020646

  11. A myeloid cell-binding adenovirus efficiently targets gene transfer to the lung and escapes liver tropism

    PubMed Central

    Alberti, Michael O.; Deshane, Jessy S.; Chaplin, David D.; Pereboeva, Larisa; Curiel, David T.; Roth, Justin C.

    2013-01-01

    Specific and efficient gene delivery to the lung has been hampered by liver sequestration of adenovirus serotype 5 (Ad5) vectors. The complexity of Ad5 liver tropism has largely been unraveled, permitting improved efficacy of Ad5 gene delivery. However, Kupffer cell (KC) scavenging and elimination of Ad5 still represent major obstacles to lung gene delivery strategies. KC uptake substantially reduces bioavailability of Ad5 for target tissues and compensatory dose escalation leads to acute hepatotoxicity and a potent innate immune response. Here we report a novel lung-targeting strategy through redirection of Ad5 binding to the concentrated leukocyte pool within the pulmonary microvasculature. We demonstrate that this leukocyte-binding approach retargets Ad5 specifically to lung endothelial cells and prevents KC uptake and hepatocyte transduction, resulting in 165 000-fold enhanced lung-targeting, compared to Ad5. Additionally, myeloid cell-specific binding is preserved in single cell lung suspensions and only Ad.MBP-coated myeloid cells achieved efficient endothelial cell transduction ex vivo. These findings demonstrate that KC sequestration of Ad5 can be prevented through more efficient uptake of virions in target tissues and suggest endothelial transduction is achieved by leukocyte-mediated “hand-off” of Ad. PMID:23171918

  12. Efficient adenovirus-mediated gene transfer into primary T cells and thymocytes in a new coxsackie/adenovirus receptor transgenic model

    PubMed Central

    Hurez, Vincent; Dzialo-Hatton, Robin; Oliver, James; Matthews, R James; Weaver, Casey T

    2002-01-01

    Background Gene transfer studies in primary T cells have suffered from the limitations of conventional viral transduction or transfection techniques. Replication-defective adenoviral vectors are an attractive alternative for gene delivery. However, naive lymphocytes are not readily susceptible to infection with adenoviruses due to insufficient expression of the coxsackie/adenovirus receptor. Results To render T cells susceptible to adenoviral gene transfer, we have developed three new murine transgenic lines in which expression of the human coxsackie/adenovirus receptor (hCAR) with a truncated cytoplasmic domain (hCARΔcyt) is limited to thymocytes and lymphocytes under direction of a human CD2 mini-gene. hCARΔcyt.CD2 transgenic mice were crossed with DO11.10 T cell receptor transgenic mice (DO11.hCARΔcyt) to allow developmental studies in a defined, clonal T cell population. Expression of hCARΔcyt enabled adenoviral transduction of resting primary CD4+ T cells, differentiated effector T cells and thymocytes from DO11.hCARΔcyt with high efficiency. Expression of hCARΔcyt transgene did not perturb T cell development in these mice and adenoviral transduction of DO11.hCARΔcyt T cells did not alter their activation status, functional responses or differentiative potential. Adoptive transfer of the transduced T cells into normal recipients did not modify their physiologic localization. Conclusion The DO11.hCARΔcyt transgenic model thus allows efficient gene transfer in primary T cell populations and will be valuable for novel studies of T cell activation and differentiation. PMID:12019030

  13. Efficient transfer of francium atoms

    NASA Astrophysics Data System (ADS)

    Aubin, Seth; Behr, John; Gorelov, Alexander; Pearson, Matt; Tandecki, Michael; Collister, Robert; Gwinner, Gerald; Shiells, Kyle; Gomez, Eduardo; Orozco, Luis; Zhang, Jiehang; Zhao, Yanting; FrPNC Collaboration

    2016-05-01

    We report on the progress of the FrPNC collaboration towards Parity Non Conservation Measurements (PNC) using francium atoms at the TRIUMF accelerator. We demonstrate efficient transfer (higher than 40%) to the science vacuum chamber where the PNC measurements will be performed. The transfer uses a downward resonant push beam from the high-efficiency capture magneto optical trap (MOT) towards the science chamber where the atoms are recaptured in a second MOT. The transfer is very robust with respect to variations in the parameters (laser power, detuning, alignment, etc.). We accumulate a growing number of atoms at each transfer pulse (limited by the lifetime of the MOT) since the push beam does not eliminate the atoms already trapped in the science MOT. The number of atoms in the science MOT is on track to meet the requirements for competitive PNC measurements when high francium rates (previously demonstrated) are delivered to our apparatus. The catcher/neutralizer for the ion beam has been tested reliably to 100,000 heating/motion cycles. We present initial tests on the direct microwave excitation of the ground hyperfine transition at 45 GHz. Support from NSERC and NRC from Canada, NSF and Fulbright from USA, and CONACYT from Mexico.

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

    PubMed

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

    2002-08-01

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

  15. Characterization of tailor-made copolymers of oligo(ethylene glycol) methyl ether methacrylate and N,N-dimethylaminoethyl methacrylate as nonviral gene transfer agents: influence of macromolecular structure on gene vector particle properties and transfection efficiency.

    PubMed

    Uzgün, Senta; Akdemir, Ozgür; Hasenpusch, Günther; Maucksch, Christof; Golas, Monika M; Sander, Bjoern; Stark, Holger; Imker, Rabea; Lutz, Jean-François; Rudolph, Carsten

    2010-01-11

    Oligo(ethylene glycol) methyl ether methacrylates (OEGMA) of various chain lengths (i.e., 9, 23, or 45 EG units) and N,N-dimethylaminoethyl methacrylate (DMAEMA) were copolymerized by atom transfer radical polymerization (ATRP), yielding well-defined P(DMAEMA-co-OEGMA) copolymers with increasing OEGMA molar fractions (F(OEGMA)) but a comparable degree of polymerization (DP approximately 120). Increase of both F(OEGMA) and OEGMA chain lengths correlated inversely with gene vector size, morphology, and zeta potential. P(DMAEMA-co-OEGMA) copolymers prevented gene vector aggregation at high plasmid DNA (pDNA) concentrations in isotonic solution and did not induce cytotoxicity even at high concentrations. Transfection efficiency of the most efficient P(DMAEMA-co-OEGMA) copolymers was found to be >10-fold lower compared with branched polyethylenimine (PEI) 25 kDa. Although OEGMA copolymerization largely reduced gene vector binding with the cell surface, cellular internalization of the bound complexes was less affected. These observations suggest that inefficient endolysosomal escape limits transfection efficiency of P(DMAEMA-co-OEGMA) copolymer gene vectors. Despite this observation, optimized p(DMAEMA-co-OEGMA) gene vectors remained stable under conditions for in vivo application leading to 7-fold greater gene expression in the lungs compared with PEI. Tailor-made P(DMAEMA-co-OEGMA) copolymers are promising nonviral gene transfer agents that fulfill the requirements for successful in vivo gene delivery.

  16. Therapeutic option of plasmid-DNA based gene transfer.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Kunugiza, Yasuo; Iekushi, Kazuma; Rakugi, Hiromi; Morishita, Ryuichi

    2012-01-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common method in clinical cases because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid deoxyribonucleic acid (DNA)-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a phase III clinical trial did not show sufficient efficiency. In this situation, more efficient plasmid DNA gene transfer is needed all over the world. This review focuses on plasmid DNA gene transfer and its enhancement, including ultrasound with microbubbles, electroporation, hydrodynamic method, gene gun, jet injection, cationic lipids and cationic polymers.

  17. Efficient, Long-term Hepatic Gene Transfer Using Clinically Relevant HDAd Doses by Balloon Occlusion Catheter Delivery in Nonhuman Primates

    PubMed Central

    Brunetti-Pierri, Nicola; Stapleton, Gary E; Law, Mark; Breinholt, John; Palmer, Donna J; Zuo, Yu; Grove, Nathan C; Finegold, Milton J; Rice, Karen; Beaudet, Arthur L; Mullins, Charles E; Ng, Philip

    2008-01-01

    Helper-dependent adenoviral vectors (HDAd) are devoid of all viral coding sequences and are thus an improvement over early generation Ad because they can provide long-term transgene expression in vivo without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic intravenous injection, and this unfortunately results in dose-dependent acute toxicity. To overcome this important obstacle, we have developed a minimally invasive method to preferentially deliver HDAd into the liver of nonhuman primates. Briefly, a balloon occlusion catheter was percutaneously positioned in the inferior vena cava to occlude hepatic venous outflow. HDAd was injected directly into the occluded liver via a percutaneously placed hepatic artery catheter. Compared to systemic vector injection, this approach resulted in substantially higher hepatic transduction efficiency using clinically relevant low vector doses and was accompanied by mild-to-moderate acute but transient toxicities. Transgene expression was sustained for up to 964 days. These results suggest that our minimally invasive method of delivery can significantly improve the vector's therapeutic index and may be a first step toward clinical application of HDAd for liver-directed gene therapy. PMID:19050700

  18. Cytotoxic immune response blunts long-term transgene expression after efficient retroviral-mediated hepatic gene transfer in rat.

    PubMed

    Aubert, Dominique; Ménoret, Séverine; Chiari, Estelle; Pichard, Virginie; Durand, Sophie; Tesson, Laurent; Moullier, Philippe; Anegon, Ignacio; Ferry, Nicolas

    2002-04-01

    Vectors derived from oncoretroviruses can transduce a small proportion of hepatocytes when injected in the regenerating liver. Transgene expression may be sustained for months without immune response. In striking contrast, we observed a rapid extinction when the intravenous injection of a high input of nuclear beta-galactosidase (beta-gal) expression vector, one day after partial hepatectomy, led to a significant proportion of transduced cells in the liver. Extinction was associated with liver inflammation on tissue sections and appearance of antibodies against the transgene product, while vector genomes became undetectable in liver tissue by PCR. These observations suggested the elimination of transduced cells by an immune response. Transgenic rats tolerant for cytoplasmic beta-gal, or normal rats depleted in CD8 T lymphocytes, steadily expressed the beta-gal vector. In the spleen of normal rats, we detected cytotoxic cells directed against cells expressing beta-gal after the injection of the beta-gal vector. In jaundiced Gunn rats deficient in bilirubin glucuronosyl transferase (BGT1) and treated with a human BGT1 cDNA expression vector, we observed the same kinetics of extinction as well as the appearance of anti-BGT1 antibodies. This study demonstrates that retrovirus-mediated gene transfer may induce cytotoxic T lymphocytes specifically directed against transgene-expressing cells.

  19. Polycationic diblock and random polyethylene glycol- or tris(hydroxymethyl)methyl-grafted (co)telomers for gene transfer: synthesis and evaluation of their in vitro transfection efficiency.

    PubMed

    Le Bon, Bertrand; Van Craynest, Nathalie; Boussif, Otmane; Vierling, Pierre

    2002-01-01

    We report on the synthesis of a series of polycationic telomers, polycationic diblock and random polyethylene glycol (PEG)-grafted (co)telomers, and polycationic random tris(hydroxymethyl)methyl (THM) cotelomers, and on their in vitro gene transfer capability. These compounds were obtained by a telomerization process of various amino-, tetraethylene glycol-, or THM-acrylamide taxogens with thiols which might derive from PEG2000. For N/P ratios [N is the number of (co)telomer amine equivalents; P is the number of DNA phosphate equivalents] from 0.8 to 10, these (co)telomers condensed DNA, forming (co)teloplexes with mean sizes in the 85-330 nm range, even for an N/P ratio of 0.8 or 1.25. Some structure-transfection efficiency relationships were established. Among the new polycationic derivatives that were synthesized and investigated for their transfection efficiency, the (i)Bu-[NH](75) telomers and the diblock polyethylene glycol-conjugated PEG2000-[NH](n) telomers are very promising candidates for gene transfer purposes.

  20. Efficient intrathymic gene transfer following in situ administration of a rAAV serotype 8 vector in mice and nonhuman primates.

    PubMed

    Moreau, Aurélie; Vicente, Rita; Dubreil, Laurence; Adjali, Oumeya; Podevin, Guillaume; Jacquet, Chantal; Deschamps, Jack Yves; Klatzmann, David; Cherel, Yan; Taylor, Naomi; Moullier, Philippe; Zimmermann, Valérie S

    2009-03-01

    The thymus is the primary site of T-cell development and plays a key role in the induction of self-tolerance. We previously showed that the intrathymic (i.t.) injection of a transgene-expressing lentiviral vector (LV) in mice can result in the correction of a T cell-specific genetic defect. Nevertheless, the efficiency of thymocyte transduction did not exceed 0.1-0.3% and we were unable to detect any thymus transduction in macaques. As such, we initiated studies to assess the capacity of recombinant adeno-associated virus (rAAV) vectors to transduce murine and primate thymic cells. In vivo administration of AAV serotype 2-derived single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors pseudotyped with capsid proteins of serotypes 1, 2, 4, 5, and 8 demonstrated that murine thymus transduction was significantly enhanced by scAAV2/8. Transgene expression was detected in 5% of thymocytes and, notably, transduced cells represented 1% of peripheral T lymphocytes. Moreover, i.t. administration of scAAV2/8 particles in macaques, by endoscopic-mediated guidance, resulted in significant gene transfer. Thus, in healthy animals, where thymic gene transfer does not provide a selective advantage, scAAV2/8 is a unique tool promoting the in situ transduction of thymocytes with the subsequent export of gene-modified lymphocytes to the periphery.

  1. Lateral gene transfer, rearrangement, reconciliation

    PubMed Central

    2013-01-01

    Background Models of ancestral gene order reconstruction have progressively integrated different evolutionary patterns and processes such as unequal gene content, gene duplications, and implicitly sequence evolution via reconciled gene trees. These models have so far ignored lateral gene transfer, even though in unicellular organisms it can have an important confounding effect, and can be a rich source of information on the function of genes through the detection of transfers of clusters of genes. Result We report an algorithm together with its implementation, DeCoLT, that reconstructs ancestral genome organization based on reconciled gene trees which summarize information on sequence evolution, gene origination, duplication, loss, and lateral transfer. DeCoLT optimizes in polynomial time on the number of rearrangements, computed as the number of gains and breakages of adjacencies between pairs of genes. We apply DeCoLT to 1099 gene families from 36 cyanobacteria genomes. Conclusion DeCoLT is able to reconstruct adjacencies in 35 ancestral bacterial genomes with a thousand gene families in a few hours, and detects clusters of co-transferred genes. DeCoLT may also be used with any relationship between genes instead of adjacencies, to reconstruct ancestral interactions, functions or complexes. Availability http://pbil.univ-lyon1.fr/software/DeCoLT/ PMID:24564205

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

    PubMed

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

    2017-02-28

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

  3. Detecting highways of horizontal gene transfer.

    PubMed

    Bansal, Mukul S; Banay, Guy; Gogarten, J Peter; Shamir, Ron

    2011-09-01

    In a horizontal gene transfer (HGT) event, a gene is transferred between two species that do not have an ancestor-descendant relationship. Typically, no more than a few genes are horizontally transferred between any two species. However, several studies identified pairs of species between which many different genes were horizontally transferred. Such a pair is said to be linked by a highway of gene sharing. We present a method for inferring such highways. Our method is based on the fact that the evolutionary histories of horizontally transferred genes disagree with the corresponding species phylogeny. Specifically, given a set of gene trees and a trusted rooted species tree, each gene tree is first decomposed into its constituent quartet trees and the quartets that are inconsistent with the species tree are identified. Our method finds a pair of species such that a highway between them explains the largest (normalized) fraction of inconsistent quartets. For a problem on n species and m input quartet trees, we give an efficient O(m + n(2))-time algorithm for detecting highways, which is optimal with respect to the quartets input size. An application of our method to a dataset of 1128 genes from 11 cyanobacterial species, as well as to simulated datasets, illustrates the efficacy of our method.

  4. Plant transformation via pollen tube-mediated gene transfer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT ...

  5. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  6. Viral Vectors for in Vivo Gene Transfer

    NASA Astrophysics Data System (ADS)

    Thévenot, E.; Dufour, N.; Déglon, N.

    The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

  7. Plasmid DNA-based gene transfer with ultrasound and microbubbles.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Rakugi, Hiromi; Morishita, Ryuichi

    2011-12-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.

  8. High‐throughput screening of clinically approved drugs that prime polyethylenimine transfection reveals modulation of mitochondria dysfunction response improves gene transfer efficiencies

    PubMed Central

    Nguyen, Albert; Beyersdorf, Jared; Riethoven, Jean‐Jack

    2016-01-01

    Abstract Nonviral gene delivery methods are advantageous over viral vectors in terms of safety, cost, and flexibility in design and application, but suffer from lower gene transfer efficiency. In addition to modifications to nucleic acid design and nonviral carriers, new tools are sought to enhance transfection. Priming is the pharmacological modulation of transfection efficiency and transgene expression, and has demonstrated transfection increase in several compounds, for example, chloroquine and glucocorticoids. To develop a library of transfection priming compounds, a high‐throughput screen was performed of the NIH Clinical Collection (NCC) to identify clinical compounds that prime polyethylenimine (PEI) transfection. HEK293T cells were treated with priming compounds, then transfected with enhanced green fluorescent protein (EGFP)‐encoding plasmid by PEI. After 48‐hr culture, primed and transfected cells were assayed for transfection, cell proliferation, and cell viability by fluorescence measurement of EGFP reporter, Hoechst 33342 nuclei stain, and resazurin metabolic assay. From the microscope image analysis and microplate measurements, transfection fold‐changes were determined, and compounds resulting in statistically significant transfection fold‐change were identified. NCC compounds were clustered using PubChem fingerprint similarity by Tanimoto coefficients in ChemmineTools. Fold‐changes for each compound were linked to drug clusters, from which drug classes that prime transfection were identified. Among the identified drugs classes that primed transfection increases were antioxidants, GABAA receptor modulators, and glucocorticoids. Resveratrol and piceid, stilbenoid antioxidants found in grapes, and zolpidem, a GABAA modulator, increased transfection nearly three‐fold. Literature indicate interaction of the identified transfection priming drug clusters with mitochondria, which may modulate mitochondrial dysfunction known to be associated

  9. T cell receptor (TCR) gene transfer with lentiviral vectors allows efficient redirection of tumor specificity in naive and memory T cells without prior stimulation of endogenous TCR.

    PubMed

    Circosta, Paola; Granziero, Luisa; Follenzi, Antonia; Vigna, Elisa; Stella, Stefania; Vallario, Antonella; Elia, Angela Rita; Gammaitoni, Loretta; Vitaggio, Katiuscia; Orso, Francesca; Geuna, Massimo; Sangiolo, Dario; Todorovic, Maja; Giachino, Claudia; Cignetti, Alessandro

    2009-12-01

    We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

  10. Panspermia and horizontal gene transfer

    NASA Astrophysics Data System (ADS)

    Klyce, Brig

    2009-08-01

    Evidence that extremophiles are hardy and ubiquitous is helping to make panspermia a respectable theory. But even if life on Earth originally came from space, biologists assume that the subsequent evolution of life is still governed by the darwinian paradigm. In this review we show how panspermia could amend darwinism and point to a cosmic source for, not only extremophiles but, all of life. This version of panspermia can be called "strong panspermia." To support this theory we will discuss recent evidence pertaining to horizontal gene transfer, viruses, genes apparently older than the Earthly evolution of the features they encode, and primate-specific genes without identifiable precursors.

  11. High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

    PubMed Central

    Lattanzi, L; Salvatori, G; Coletta, M; Sonnino, C; Cusella De Angelis, M G; Gioglio, L; Murry, C E; Kelly, R; Ferrari, G; Molinaro, M; Crescenzi, M; Mavilio, F; Cossu, G

    1998-01-01

    Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to beta-galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo. PMID:9593768

  12. Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide.

    PubMed

    Maurice, Marielle; Verhoeyen, Els; Salmon, Patrick; Trono, Didier; Russell, Stephen J; Cosset, François-Loïc

    2002-04-01

    In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1-derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G(0) to G(1) phases of the cell cycle. Compared to unmodified HIV-1-based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

  13. High efficiency pump for space helium transfer

    NASA Technical Reports Server (NTRS)

    Hasenbein, Robert; Izenson, Michael G.; Swift, Walter L.; Sixsmith, Herbert

    1991-01-01

    A centrifugal pump was developed for the efficient and reliable transfer of liquid helium in space. The pump can be used to refill cryostats on orbiting satellites which use liquid helium for refrigeration at extremely low temperatures. The pump meets the head and flow requirements of on-orbit helium transfer: a flow rate of 800 L/hr at a head of 128 J/kg. The overall pump efficiency at the design point is 0.45. The design head and flow requirements are met with zero net positive suction head, which is the condition in an orbiting helium supply Dewar. The mass transfer efficiency calculated for a space transfer operation is 0.99. Steel ball bearings are used with gas fiber-reinforced teflon retainers to provide solid lubrication. These bearings have demonstrated the longest life in liquid helium endurance tests under simulated pumping conditions. Technology developed in the project also has application for liquid helium circulation in terrestrial facilities and for transfer of cryogenic rocket propellants in space.

  14. Development of a hamster kidney cell line expressing stably T7 RNA polymerase using retroviral gene transfer technology for efficient rescue of infectious foot-and-mouth disease virus.

    PubMed

    Zheng, Haixue; Tian, Hong; Jin, Ye; Wu, Jinyan; Shang, Youjun; Yin, Shuanghui; Liu, Xiangtao; Xie, Qingge

    2009-03-01

    Reverse genetics systems, with the ability to manipulate viral genomes at the DNA molecular level, are an important platform for study of the assembly and function of viruses. Genome manipulation, such as gene recombination, mosaicism, and mutation may interfere with replication, assembly and release of viruses. An efficient, convenient and economical method of virus rescue is undoubtedly required for increasing the efficiency of rescuing recombinant viruses. To develop an efficient, helper virus-free viral recovery system (reverse genetics), a retroviral gene transfer technology was used to establish a stable BHK-21 cell line (designated as BHKT7) which expressed constitutively bacteriophage T7 RNA polymerase (T7 RNAP). An improved method for rescue of infectious foot-and-mouth disease virus (FMDV) was then developed. FMDV full-length cDNA under control of a T7 promotor, was transfected into BHKT7 of differing passages. FMDV virus was rescued efficiently from the BHKT7 cells, the passage number not having an effect on the efficiency of recovery. As a result, the cell line was stable even after multiple passages, expressing sufficient T7 RNAP to support ex vivo transcription and efficient rescue. The reverse genetics system described below is efficient, stable, and convenient. The system could provide not only the basis of gene function research into FMDV, but could also be used for reverse genetics research into other positive-strand RNA viruses, without the need for helper viruses.

  15. Efficient stream distributions in radiative transfer theory

    NASA Technical Reports Server (NTRS)

    Whitney, C.

    1974-01-01

    This paper discusses a new, computationally-efficient method for approximating the integro-differential equation of radiative transfer with a finite set of coupled differential equations for discrete streams. The method uses recommended spatial distributions of streams that are quite different from those typically used in that they are based on the symmetry of several regular Platonic solids. To facilitate the use of such distributions, an explicit, one-parameter relationship between the physical radiance and the abstract stream is formulated. The parameter is used to determine the minimum number of streams required in the radiative transfer model. Accuracy and computational efficiency are shown to be served best by choosing a stream distribution that is invariant to a large number of three space rotations. For various values of the above-mentioned parameter, the resulting recommended stream distribution is shown to be more computationally efficient than more conventional stream distributions. Finally, the incorporation of polarization in the stream definition is described.

  16. Targeting Radiotherapy to Cancer by Gene Transfer

    PubMed Central

    2003-01-01

    Targeted radionuclide therapy is an alternative method of radiation treatment which uses a tumor-seeking agent carrying a radioactive atom to deposits of tumor, wherever in the body they may be located. Recent experimental data signifies promise for the amalgamation of gene transfer with radionuclide targeting. This review encompasses aspects of the integration of gene manipulation and targeted radiotherapy, highlighting the possibilities of gene transfer to assist the targeting of cancer with low molecular weight radiopharmaceuticals. PMID:12721515

  17. Lateral Gene Transfer from the Dead

    PubMed Central

    Szöllősi, Gergely J.; Tannier, Eric; Lartillot, Nicolas; Daubin, Vincent

    2013-01-01

    In phylogenetic studies, the evolution of molecular sequences is assumed to have taken place along the phylogeny traced by the ancestors of extant species. In the presence of lateral gene transfer, however, this may not be the case, because the species lineage from which a gene was transferred may have gone extinct or not have been sampled. Because it is not feasible to specify or reconstruct the complete phylogeny of all species, we must describe the evolution of genes outside the represented phylogeny by modeling the speciation dynamics that gave rise to the complete phylogeny. We demonstrate that if the number of sampled species is small compared with the total number of existing species, the overwhelming majority of gene transfers involve speciation to and evolution along extinct or unsampled lineages. We show that the evolution of genes along extinct or unsampled lineages can to good approximation be treated as those of independently evolving lineages described by a few global parameters. Using this result, we derive an algorithm to calculate the probability of a gene tree and recover the maximum-likelihood reconciliation given the phylogeny of the sampled species. Examining 473 near-universal gene families from 36 cyanobacteria, we find that nearly a third of transfer events (28%) appear to have topological signatures of evolution along extinct species, but only approximately 6% of transfers trace their ancestry to before the common ancestor of the sampled cyanobacteria. [Gene tree reconciliation; lateral gene transfer; macroevolution; phylogeny.] PMID:23355531

  18. Biased gene transfer in microbial evolution.

    PubMed

    Andam, Cheryl P; Gogarten, J Peter

    2011-06-13

    Horizontal gene transfer (HGT) is an important evolutionary process that allows the spread of innovations between distantly related organisms. We present evidence that prokaryotes (bacteria and archaea) are more likely to transfer genetic material with their close relatives than with distantly related lineages. This bias in transfer partners can create phylogenetic signals that are difficult to distinguish from the signal created through shared ancestry. Preferences for transfer partners can be revealed by studying the distribution patterns of divergent genes with identical functions. In many respects, these genes are similar to alleles in a population, except that they coexist only in higher taxonomic groupings and are acquired by a species through HGT. We also discuss the role of biased gene transfer in the formation of taxonomically recognizable natural groups in the tree or net of life.

  19. Efficient Radiative Transfer Computations in the Atmosphere.

    DTIC Science & Technology

    1981-01-01

    absorptance, A = 1 - r , the net flux at level Z is given by equation (5) Net Flux, F (Z) = I - I, = B(Zsfc) -B(Ztop) A (ZtopZ) Zsfc - sft A (Z’,Z)dB(Z’) (5) ztop 11... F . Alyea, N. Phillips and R . Prinn, 1975; A three dimensional dynamical-chemical model of atmos- pheric ozone, J. Atmos. Sci., 32:170-194. 4...AD-ADO? 289 AIR FORCE INST OF TECH WRIGHT-PATTERSON AFB OH F /0 41/I EFFICIENT RADIATIVE TRANSFER COMPUTATIONS IN THE ATNOSI*ERE.fUI JAN 81 C R POSEY

  20. Optical gene transfer by femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Konig, Karsten; Riemann, Iris; Tirlapur, Uday K.

    2003-07-01

    Targeted transfection of cells is an important technique for gene therapy and related biomedical applications. We delineate how high-intensity (1012 W/cm2) near-infrared (NIR) 80 MHz nanojoule femtosecond laser pulses can create highly localised membrane perforations within a minute focal volume, enabling non-invasive direct transfection of mammalian cells with DNA. We suspended Chinese hamster ovarian (CHO), rat kangaroo kidney epithelial (PtK2) and rat fibroblast cells in 0.5 ml culture medium in a sterile miniaturized cell chamber (JenLab GmbH, Jena, Germany) containing 0.2 μg plasmid DNA vector pEGFP-N1 (4.7 kb), which codes for green fluorescent protein (GFP). The NIR laser beam was introduced into a femtosecond laser scanning microscope (JenLab GmbH, Jena, Germany; focussed on the edge of the cell membrane of a target cell for 16 ms. The integration and expression efficiency of EGFP were assessed in situ by two-photon fluorescence-lifetime imaging using time-correlated single photon counting. The unique capability to transfer foreign DNA safely and efficiently into specific cell types (including stem cells), circumventing mechanical, electrical or chemical means, will have many applications, such as targeted gene therapy and DNA vaccination.

  1. Detecting Highways of Horizontal Gene Transfer

    NASA Astrophysics Data System (ADS)

    Bansal, Mukul S.; Gogarten, J. Peter; Shamir, Ron

    In a horizontal gene transfer (HGT) event a gene is transferred between two species that do not share an ancestor-descendant relationship. Typically, no more than a few genes are horizontally transferred between any two species. However, several studies identified pairs of species between which many different genes were horizontally transferred. Such a pair is said to be linked by a highway of gene sharing. We present a method for inferring such highways. Our method is based on the fact that the evolutionary histories of horizontally transferred genes disagree with the corresponding species phylogeny. Specifically, given a set of gene trees and a trusted rooted species tree, each gene tree is first decomposed into its constituent quartet trees and the quartets that are inconsistent with the species tree are identified. Our method finds a pair of species such that a highway between them explains the largest (normalized) fraction of inconsistent quartets. For a problem on n species, our method requires O(n 4) time, which is optimal with respect to the quartets input size. An application of our method to a dataset of 1128 genes from 11 cyanobacterial species, as well as to simulated datasets, illustrates the efficacy of our method.

  2. Gene Transfers Between Distantly Related Organisms

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.

    2003-01-01

    With the completion of numerous microbial genome sequences, reports of individual gene transfers between distantly related prokaryotes have become commonplace. On the other hand, transfers between prokaryotes and eukaryotes still excite the imagination. Many of these claims may be premature, but some are certainly valid. In this chapter, the kinds of supporting data needed to propose transfers between distantly related organisms and cite some interesting examples are considered.

  3. Novel gene transfer systems: intelligent gene transfer vectors for gene medicines.

    PubMed

    Nakajima, Toshihiro

    2012-01-01

    Drug delivery systems for gene transfer are called 'vectors'. These systems were originally invented as a delivery system for the transfection in vitro or in vivo. Several vectors are then developed for clinical use of gene medicines and currently some of them are approved as animal drugs. Conventional drug delivery system generally consists of approved (existing) materials to avoid additional pre-clinical or clinical studies. However, current vectors contain novel materials to improve an efficacy of gene medicines. Thus, these vectors have functions more than a mere delivery of active ingredients. For example some vectors have immunological functions such as adjuvants in vaccines. These new types of vectors are called 'intelligent' or 'innovative' vector system', since the concept or strategy for the development is completely different from conventional drug delivery systems. In this article, we described a current status of 'intelligent gene transfer vectors and discussed on the potentials of them.

  4. Antibody Gene Transfer for HIV Immunoprophylaxis

    PubMed Central

    Balazs, Alejandro B.; West, Anthony P.

    2015-01-01

    Antibody gene transfer, which involves the delivery of genes that encode potent, broadly neutralizing anti-HIV antibodies, is a promising new strategy to prevent HIV infection. A satellite symposium at the AIDS Vaccine 2012 conference brought together many of the groups working in this field. PMID:23238748

  5. Antibody gene transfer for HIV immunoprophylaxis.

    PubMed

    Balazs, Alejandro B; West, Anthony P

    2013-01-01

    Antibody gene transfer, which involves the delivery of genes that encode potent, broadly neutralizing antibodies to human immunodeficiency virus (HIV), is a promising new strategy for preventing HIV infection. A satellite symposium at the AIDS Vaccine 2012 conference brought together many of the groups working in this field.

  6. Efficient transfer of images over networks

    NASA Technical Reports Server (NTRS)

    Percival, J. W.; White, R. L.

    1992-01-01

    Effective remote observing requires sending large images over long distances. The usual approach to the transfer problem is to require high bandwidth transmission links, which are expensive to install and operate. An alternative approach is to use existing low-bandwidth connections, such as phone lines or the Internet, in a highly efficient manner by compressing the images. The combined use of existing low-cost infrastructure and standard networking software means that remote observing can be made practical even for small observatories with limited network resources. The authors have implemented such a scheme based on the H-transform compression method developed for astronomical images, which are often resistant to compression because they are noisy. The H-transform can be used for either lossy or lossless compression, and compression factors of at least 10 can be achieved with no noticeable losses in the astrometric or photometric properties of the compressed images. The H-transform allows us to organize the information in an image so that the 'useful' information can be sent first, followed by the noise, which makes up the bulk of the transmission. The receiver can invert a partially received set of H-coefficients, creating an image that improves with time. The H-transform is particularly well-suited to this style of incremental reconstruction, because the spatially localized nature of the basis functions of the H-transorm prevents the appearance of artifacts such as ringing around point sources and edges. The authors' implementation uses the WIYN Telescope Control System's TCP-based communications protocol. An 800x800 16-bit astronomical image was sent over a 2400 baud connection, which would normally take about 71 minutes; after only 60 seconds, the partially received H-transform produced an image that did not differ appreciably from the original. This poster presents a quantification of the efficiencies, as well as examples of images reconstructed from partial

  7. Molecular Transfer of Nematode Resistance Genes

    PubMed Central

    Williamson, V. M.; Ho, J.-Y.; Ma, H. M.

    1992-01-01

    Recombinant DNA techniques have been used to introduce agronomically valuable traits, including resistance to viruses, herbicides, and insects, into crop plants. Introduction of these genes into plants frequently involves Agrobacterium-mediated gene transfer. The potential exists for applying this technology to nematode control by introducing genes conferring resistance to nematodes. Transferred genes could include those encoding products detrimental to nematode development or reproduction as well as cloned host resistance genes. Host genes that confer resistance to cyst or root-knot nematode species have been identified in many plants. The best characterized is Mi, a gene that confers resistance to root-knot nematodes in tomato. A map-based cloning approach is being used to isolate the gene. For development of a detailed map of the region of the genome surrounding Mi, DNA markers genetically linked to Mi have been identified and analyzed in tomato lines that have undergone a recombination event near Mi. The molecular map will be used to identify DNA corresponding to Mi. We estimate that a clone of Mi will be obtained in 2-5 years. An exciting prospect is that introduction of this gene will confer resistance in plant species without currently available sources of resistance. PMID:19282989

  8. A quantitative multistandard reverse transcriptase-polymerase chain reaction assay of the cystic fibrosis transmembrane conductance regulator: its usefulness in studying efficiency of gene transfer.

    PubMed

    Marchand-Pinatel, S; Planells, R; Merten, M D; Kammouni, W; Figarella, C

    2000-08-01

    Procedures to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels have already been described but are not universally accepted, and many investigators are skeptical about quantification. To be able to accurately monitor gene therapy, we developed a quantitative multistandard RT-PCR method. This was based on the observation that the CFTR and ribosomal phosphoprotein PO (PR-PO) genes have retained important sequence homologies between rat and human species, allowing the use of rat RNA as an internal standard. A mixture of rat and human RNAs is simultaneously reverse-transcribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by restriction analysis. PR-PO is analyzed similarly and serves as a control of template loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) incubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. This method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cystic fibrosis patients.

  9. Unsupervised Learning in Detection of Gene Transfer

    PubMed Central

    Hamel, L.; Nahar, N.; Poptsova, M. S.; Zhaxybayeva, O.; Gogarten, J. P.

    2008-01-01

    The tree representation as a model for organismal evolution has been in use since before Darwin. However, with the recent unprecedented access to biomolecular data, it has been discovered that, especially in the microbial world, individual genes making up the genome of an organism give rise to different and sometimes conflicting evolutionary tree topologies. This discovery calls into question the notion of a single evolutionary tree for an organism and gives rise to the notion of an evolutionary consensus tree based on the evolutionary patterns of the majority of genes in a genome embedded in a network of gene histories. Here, we discuss an approach to the analysis of genomic data of multiple genomes using bipartition spectral analysis and unsupervised learning. An interesting observation is that genes within genomes that have evolutionary tree topologies, which are in substantial conflict with the evolutionary consensus tree of an organism, point to possible horizontal gene transfer events which often delineate significant evolutionary events. PMID:18509479

  10. Viral vectors for gene transfer: current status of gene therapeutics.

    PubMed

    Heilbronn, Regine; Weger, Stefan

    2010-01-01

    Gene therapy for the correction of inherited or acquired disease has gained increasing importance in recent years. Successful treatment of children suffering from severe combined immunodeficiency (SCID) was achieved using retrovirus vectors for gene transfer. Encouraging improvements of vision were reported in a genetic eye disorder (LCA) leading to early childhood blindness. Adeno-associated virus (AAV) vectors were used for gene transfer in these trials. This chapter gives an overview of the design and delivery of viral vectors for the transport of a therapeutic gene into a target cell or tissue. The construction and production of retrovirus, lentivirus, and AAV vectors are covered. The focus is on production methods suitable for biopharmaceutical upscaling and for downstream processing. Quality control measures and biological safety considerations for the use of vectors in clinical trials are discussed.

  11. Efficient exploration of the space of reconciled gene trees.

    PubMed

    Szöllõsi, Gergely J; Rosikiewicz, Wojciech; Boussau, Bastien; Tannier, Eric; Daubin, Vincent

    2013-11-01

    Gene trees record the combination of gene-level events, such as duplication, transfer and loss (DTL), and species-level events, such as speciation and extinction. Gene tree-species tree reconciliation methods model these processes by drawing gene trees into the species tree using a series of gene and species-level events. The reconstruction of gene trees based on sequence alone almost always involves choosing between statistically equivalent or weakly distinguishable relationships that could be much better resolved based on a putative species tree. To exploit this potential for accurate reconstruction of gene trees, the space of reconciled gene trees must be explored according to a joint model of sequence evolution and gene tree-species tree reconciliation. Here we present amalgamated likelihood estimation (ALE), a probabilistic approach to exhaustively explore all reconciled gene trees that can be amalgamated as a combination of clades observed in a sample of gene trees. We implement the ALE approach in the context of a reconciliation model (Szöllősi et al. 2013), which allows for the DTL of genes. We use ALE to efficiently approximate the sum of the joint likelihood over amalgamations and to find the reconciled gene tree that maximizes the joint likelihood among all such trees. We demonstrate using simulations that gene trees reconstructed using the joint likelihood are substantially more accurate than those reconstructed using sequence alone. Using realistic gene tree topologies, branch lengths, and alignment sizes, we demonstrate that ALE produces more accurate gene trees even if the model of sequence evolution is greatly simplified. Finally, examining 1099 gene families from 36 cyanobacterial genomes we find that joint likelihood-based inference results in a striking reduction in apparent phylogenetic discord, with respectively. 24%, 59%, and 46% reductions in the mean numbers of duplications, transfers, and losses per gene family. The open source

  12. Rescuing the Failing Heart by Targeted Gene Transfer

    PubMed Central

    Kawase, Yoshiaki; Ladage, Dennis; Hajjar, Roger J.

    2011-01-01

    Congestive heart failure is a major cause of morbidity and mortality in the US. While progress in conventional treatments is making steady and incremental gains to reduce heart failure mortality, there is a critical need to explore new therapeutic approaches. Gene therapy was initially applied in the clinical setting for inherited monogenic disorders. It is now apparent that gene therapy has broader potential that also includes acquired polygenic diseases, such as congestive heart failure. Recent advances in understanding of the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology, has placed heart failure within reach of gene-based therapy. Furthermore, the recent successful and safe completion of a phase 2 trial targeting the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) along with the start of more recent phase 1 trials usher a new era for gene therapy for the treatment of heart failure. PMID:21371634

  13. Lateral gene transfer in the subsurface

    SciTech Connect

    Barkay, Tamar; Sobecky, Patricia

    2007-08-27

    Lateral gene transfer (LGT) is an important adaptive mechanism among prokaryotic organisms. This mechanism is particularly important for the response of microorganisms to changing environmental conditions because it facilitates the transfer of a large number of genes and their rapid expression. Together the transferred genes promote rapid genetic and metabolic changes that may enhance survival to newly established and sometimes hostile environmental conditions. The goal of our project was to examine if and how LGT enhances microbial adaptation to toxic heavy metals in subsurface environments that had been contaminated by mixed wastes due to activities associated with the production of nuclear energy and weapons. This task has been accomplished by dividing the project to several sub-tasks. Thus, we: (1) Determined the level of resistance of subsurface bacterial isolates to several toxic metals, all identified as pollutants of concern in subsurface environments; (2) Designed, tested, and applied, a molecular approach that determined whether metal resistance genes had evolved by LGT among subsurface bacteria; and (3) Developed a DNA hybridization array for the identification of broad host range plasmids and of metal resistance plasmids. The results are briefly summarized below with references to published papers and manuscripts in preparation where details about our research can be found. Additional information may be found in copies of our published manuscripts and conference proceedings, and our yearly reports that were submitted through the RIMS system.

  14. Clinical Applications Involving CNS Gene Transfer

    PubMed Central

    Kantor, Boris; McCown, Thomas; Leone, Paola; Gray, Steven J.

    2015-01-01

    Diseases of the central nervous system (CNS) have traditionally been the most difficult to treat by traditional pharmacological methods, due mostly to the blood–brain barrier and the difficulties associated with repeated drug administration targeting the CNS. Viral vector gene transfer represents a way to permanently provide a therapeutic protein within the nervous system after a single administration, whether this be a gene replacement strategy for an inherited disorder or a disease-modifying protein for a disease such as Parkinson's. Gene therapy approaches for CNS disorders has evolved considerably over the last two decades. Although a breakthrough treatment has remained elusive, current strategies are now considerably safer and potentially much more effective. This chapter will explore the past, current, and future status of CNS gene therapy, focusing on clinical trials utilizing adeno-associated virus and lentiviral vectors. PMID:25311921

  15. Perinatal Gene Transfer to the Liver

    PubMed Central

    McKay, Tristan R; Rahim, Ahad A; Buckley, Suzanne M.K; Ward, Natalie J; Chan, Jerry K.Y; Howe, Steven J; Waddington, Simon N

    2011-01-01

    The liver acts as a host to many functions hence raising the possibility that any one may be compromised by a single gene defect. Inherited or de novo mutations in these genes may result in relatively mild diseases or be so devastating that death within the first weeks or months of life is inevitable. Some diseases can be managed using conventional medicines whereas others are, as yet, untreatable. In this review we consider the application of early intervention gene therapy in neonatal and fetal preclinical studies. We appraise the tools of this technology, including lentivirus, adenovirus and adeno-associated virus (AAV)-based vectors. We highlight the application of these for a range of diseases including hemophilia, urea cycle disorders such as ornithine transcarbamylase deficiency, organic acidemias, lysosomal storage diseases including mucopolysaccharidoses, glycogen storage diseases and bile metabolism. We conclude by assessing the advantages and disadvantages associated with fetal and neonatal liver gene transfer. PMID:21774770

  16. Methamphetamine residue dermal transfer efficiencies from household surfaces.

    PubMed

    Van Dyke, Mike; Martyny, John W; Serrano, Kate A

    2014-01-01

    Methamphetamine contamination from illegal production operations poses a potential health concern for emergency responders, child protective services, law enforcement, and children living in contaminated structures. The objective of this study was to evaluate dermal transfer efficiencies of methamphetamine from contaminated household surfaces. These transfer efficiencies are lacking for methamphetamine, and would be beneficial for use in exposure models. Surfaces were contaminated using a simulated smoking method in a stainless steel chamber. Household surfaces were carpet, painted drywall, and linoleum. Dermal transfer efficiencies were obtained using cotton gloves for two hand conditions, dry or saliva moistened (wet). In addition, three contact scenarios were evaluated for both hand conditions: one, two, or three contacts with contaminated surfaces. Dermal transfer efficiencies were calculated for both hand conditions and used as inputs in a Stochastic Human Exposure and Dose Simulation model (SHEDS-Multimedia, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, N.C.). Results of this study showed that average dermal transfer efficiencies of methamphetamine ranged from 11% for dry hands to 26% for wet hands. There was a significantly higher wet transfer as compared to dry transfer for all surfaces. For wet hands, dermal transfer depended on surface type with higher transfer from carpet and linoleum as compared to drywall. Based on our estimates of dermal transfer efficiency, a surface contamination clearance level of 1.5 μg/100 cm(2) may not ensure absorbed doses remain below the level associated with adverse health effects in all cases. Additional dermal transfer studies should be performed using skin surrogates that may better predict actual skin transfer.

  17. Energy Efficient Storage and Transfer of Cryogens

    NASA Technical Reports Server (NTRS)

    Fesmire, James E.

    2013-01-01

    Cryogenics is globally linked to energy generation, storage, and usage. Thermal insulation systems research and development is an enabling part of NASA's technology goals for Space Launch and Exploration. New thermal testing methodologies and materials are being transferred to industry for a wide range of commercial applications.

  18. Efficient Exploration of the Space of Reconciled Gene Trees

    PubMed Central

    Szöllősi, Gergely J.; Rosikiewicz, Wojciech; Boussau, Bastien; Tannier, Eric; Daubin, Vincent

    2013-01-01

    Gene trees record the combination of gene-level events, such as duplication, transfer and loss (DTL), and species-level events, such as speciation and extinction. Gene tree–species tree reconciliation methods model these processes by drawing gene trees into the species tree using a series of gene and species-level events. The reconstruction of gene trees based on sequence alone almost always involves choosing between statistically equivalent or weakly distinguishable relationships that could be much better resolved based on a putative species tree. To exploit this potential for accurate reconstruction of gene trees, the space of reconciled gene trees must be explored according to a joint model of sequence evolution and gene tree–species tree reconciliation. Here we present amalgamated likelihood estimation (ALE), a probabilistic approach to exhaustively explore all reconciled gene trees that can be amalgamated as a combination of clades observed in a sample of gene trees. We implement the ALE approach in the context of a reconciliation model (Szöllősi et al. 2013), which allows for the DTL of genes. We use ALE to efficiently approximate the sum of the joint likelihood over amalgamations and to find the reconciled gene tree that maximizes the joint likelihood among all such trees. We demonstrate using simulations that gene trees reconstructed using the joint likelihood are substantially more accurate than those reconstructed using sequence alone. Using realistic gene tree topologies, branch lengths, and alignment sizes, we demonstrate that ALE produces more accurate gene trees even if the model of sequence evolution is greatly simplified. Finally, examining 1099 gene families from 36 cyanobacterial genomes we find that joint likelihood-based inference results in a striking reduction in apparent phylogenetic discord, with respectively. 24%, 59%, and 46% reductions in the mean numbers of duplications, transfers, and losses per gene family. The open source

  19. Human gene transfer: Characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man

    SciTech Connect

    Kasid, A.; Morecki, S.; Aebersold, P.; Cornetta, K.; Culver, K.; Freeman, S.; Director, E.; Lotze, M.T.; Blaese, R.M.; Anderson, W.F.; Rosenberg, S.A. )

    1990-01-01

    Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (Neo{sup R}) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo{sup R} gene integration and expressed high levels of neomycin phosphotransferase activity. The Neo{sup R} gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for {beta}- and {gamma}-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the {beta} and {gamma} chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors ({alpha} and {beta}) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.

  20. Horizontal Gene Transfer, Dispersal and Haloarchaeal Speciation

    PubMed Central

    Papke, R. Thane; Corral, Paulina; Ram-Mohan, Nikhil; de la Haba, Rafael R.; Sánchez-Porro, Cristina; Makkay, Andrea; Ventosa, Antonio

    2015-01-01

    The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria. PMID:25997110

  1. Efficient methods for finding transfer function zeros of power systems

    SciTech Connect

    Martins, N.; Pinto, H.J.C.P. ); Lima, L.T.G. )

    1992-08-01

    This paper is probably the first to describe algorithms suited to the efficient calculation of both proper and non-proper transfer function zeros of linearized dynamic models for large interconnected power systems. The paper also describes an improvement to the well known AESOPS algorithm, formulating it as an exact transfer function zero finding problem which is efficiently solved by a Newton-Raphson iterative scheme. Large power system results are presented in this paper.

  2. AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

    PubMed

    Yu, Hongwei; Fischer, Gregory; Hogan, Quinn H

    2016-01-01

    Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

  3. Gene Therapy Inhibiting Neointimal Vascular Lesion: In vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene

    NASA Astrophysics Data System (ADS)

    von der Leyen, Heiko E.; Gibbons, Gary H.; Morishita, Ryuichi; Lewis, Neil P.; Zhang, Lunan; Nakajima, Masatoshi; Kaneda, Yasufumi; Cooke, John P.; Dzau, Victor J.

    1995-02-01

    It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as β-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessel. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.

  4. Adenovirus dodecahedron, a new vector for human gene transfer.

    PubMed

    Fender, P; Ruigrok, R W; Gout, E; Buffet, S; Chroboczek, J

    1997-01-01

    Recombinant adenovirus is one of most efficient delivery vehicles for gene therapy. However, the initial enthusiasm for the use of recombinant adenovirus for gene therapy has been tempered by strong immune responses that develop to the virus and virus-infected cells. Even though recombinant adenoviruses are replication-defective, they introduce into the recipient cell, together with the gene of interest, viral genetes that might lead to fortuitous recombination if the recipient is infected by wild-type adenovirus. We propose the use of a dodecahedron made of adenovirus pentons or penton bases as an alternative vector for human gene therapy. The penton is a complex of two oligomeric proteins, a penton base and fiber, involved in the cell attachment, internalization, and liberation of virus into the cytoplasm. The dodecahedron retains many of the advantages of adenovirus for gene transfer such as efficiency of entry, efficient release of DNA from endosomes, and wide range of cell and tissue targets. Because it consists of only one or two adenovirus proteins instead of the 11 contained in an adenovirus virion and it does not contain the viral genome, it is potentially a safer alternative to recombinant adenovirus.

  5. Efficiency Of Information Transfer In Diagnostic Radiology

    NASA Astrophysics Data System (ADS)

    Brodie, I.; Gutcheck, R. A.

    1981-11-01

    Pictorial information theory is restated in a form that predicts, with fair precision, the minimum radiation required to detect a given feature size in diagnostic radiology. In contrast to conventional approaches, the probability of detection is made a visible variable in the calculation--an aspect of prime importance for diagnostic interpretation. An accurate knowledge of the absorption coefficients of the tissue concerned and the sizes one wishes to detect is required, but the calculation is relatively easy to carry out with a programmable calculator or microcomputer. The concept of "viewing efficiency" is introduced to compare the performance of a real radiographic system with that of a perfect radiographic system. The theory is applied to mammography, where the viewing efficiencies of film, film/ screen combinations, and xeroradiography are compared. It is shown that a more efficient method for detecting the latent charge image in xeroradiography, coupled with digital data storage and appropriate viewing methods, should result in substantial reductions in exposure compared to the present toning methods.

  6. Horizontal gene transfer in parasitic plants.

    PubMed

    Davis, Charles C; Xi, Zhenxiang

    2015-08-01

    Horizontal gene transfer (HGT) between species has been a major focus of plant evolutionary research during the past decade. Parasitic plants, which establish a direct connection with their hosts, have provided excellent examples of how these transfers are facilitated via the intimacy of this symbiosis. In particular, phylogenetic studies from diverse clades indicate that parasitic plants represent a rich system for studying this phenomenon. Here, HGT has been shown to be astonishingly high in the mitochondrial genome, and appreciable in the nuclear genome. Although explicit tests remain to be performed, some transgenes have been hypothesized to be functional in their recipient species, thus providing a new perspective on the evolution of novelty in parasitic plants.

  7. Center for fetal monkey gene transfer for heart, lung, and blood diseases: an NHLBI resource for the gene therapy community.

    PubMed

    Tarantal, Alice F; Skarlatos, Sonia I

    2012-11-01

    The goals of the National Heart, Lung, and Blood Institute (NHLBI) Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases are to conduct gene transfer studies in monkeys to evaluate safety and efficiency; and to provide NHLBI-supported investigators with expertise, resources, and services to actively pursue gene transfer approaches in monkeys in their research programs. NHLBI-supported projects span investigators throughout the United States and have addressed novel approaches to gene delivery; "proof-of-principle"; assessed whether findings in small-animal models could be demonstrated in a primate species; or were conducted to enable new grant or IND submissions. The Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases successfully aids the gene therapy community in addressing regulatory barriers, and serves as an effective vehicle for advancing the field.

  8. [Gene transfer agent--a novel and widespread occurrence mechanism of gene exchange in ocean-a review].

    PubMed

    Cai, Haiyuan

    2012-01-01

    Gene Transfer Agent (GTA) particles are released by bacteria and resemble small, tailed bacteriophages. GTA particles contain small, random pieces of host DNA rather than GTA structural genes or a phage genome. Gene transfer mediated by GTA is efficient and species specific based on knowledge of currently best studied GTAs produced by 4 anaerobes. Genome sequencing projects have revealed a remarkable distribution of GTA gene clusters in the genomes of marine bacterioplankton, implying GTA may be an important mechanism for horizontal gene transfer in ocean. On basis of characterization of the 4 best studied GTAs, this review described GTAs released by numerically dominant marine bacteria, discussed their properties that were important for horizontal gene transfer in ocean, and gave future perspectives to advance GTA research.

  9. Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

    PubMed Central

    2011-01-01

    Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods. Results Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that

  10. Thixotropic solutions enhance viral-mediated gene transfer to airway epithelia.

    PubMed

    Seiler, Michael P; Luner, Paul; Moninger, Thomas O; Karp, Philip H; Keshavjee, Shaf; Zabner, Joseph

    2002-08-01

    Adenovirus-mediated gene transfer to airway epithelia is inefficient in part because its receptor is absent on the apical surface of the airways. Targeting adenovirus to other receptors, increasing the viral concentration, and even prolonging the incubation time with adenovirus vectors can partially overcome the lack of receptors and facilitate gene transfer. Unfortunately, mucociliary clearance would prevent prolonged incubation time in vivo. Thixotropic solutions (TS) are gels that upon a vigorous shearing force reversibly become liquid. We hypothesized that formulating recombinant adenoviruses in TS would decrease virus clearance and thus enhance gene transfer to the airway epithelia. We found that clearance of virus-sized fluorescent beads by human airway epithelia in vitro and by monkey trachea in vivo were markedly decreased when the beads were formulated in TS compared with phosphate-buffered saline (PBS). Adenovirus formulated in TS significantly increased adenovirus-mediated gene transfer of a reporter gene in human airway epithelia in vitro and in murine airway epithelia in vivo. Furthermore, an adenovirus encoding the cystic fibrosis transmembrane regulator (CFTR) gene (AdCFTR) formulated in TS was more efficient in correcting the chloride transport defect in cystic fibrosis airway epithelia than AdCFTR formulated in PBS. These data indicate a novel strategy to augment the efficiency of gene transfer to the airways that may be applicable to a number of different gene transfer vectors and could be of value in gene transfer to cystic fibrosis (CF) airway epithelia in vivo.

  11. Reducible cationic lipids for gene transfer.

    PubMed Central

    Wetzer, B; Byk, G; Frederic, M; Airiau, M; Blanche, F; Pitard, B; Scherman, D

    2001-01-01

    One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA--disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA--lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA--lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA--lipid complexes after intracellular reduction and represent a tool for improved vectorization. PMID:11389682

  12. Reducible cationic lipids for gene transfer.

    PubMed

    Wetzer, B; Byk, G; Frederic, M; Airiau, M; Blanche, F; Pitard, B; Scherman, D

    2001-06-15

    One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA--disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA--lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA--lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA--lipid complexes after intracellular reduction and represent a tool for improved vectorization.

  13. Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

    PubMed Central

    Geguchadze, Ramaz; Wang, Zhimin; Zourelias, Lee; Perez-Riveros, Paola; Edwards, Paul C; Machen, Laurie; Passineau, Michael J

    2014-01-01

    In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis. PMID:25414909

  14. Horizontal gene transfer from Agrobacterium to plants

    PubMed Central

    Matveeva, Tatiana V.; Lutova, Ludmila A.

    2014-01-01

    Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named “cellular T-DNA” (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role. PMID:25157257

  15. Ultrasound Gene Transfer into Fibroblast Cells using Microbubbles

    NASA Astrophysics Data System (ADS)

    Nakamura, Yoji; Hirayama, Kota; Yoshinaka, Kiyoshi; Tei, Yuichi; Takagi, Shu; Matsumoto, Yoichiro

    2009-04-01

    Ultrasound is widely applied in the medical field and offers the strong advantages of non-invasiveness and high-selectivity. Gene transfer using ultrasound, which is called sonoporation, is one application. Ultrasound has the potential to deliver therapeutic materials such as genes, drugs or proteins into cells. Microbubbles are known to be able to improve delivery efficiency. This is attributed to therapeutic materials passing through the cell membrane after permeability is increased by destruction or oscillation of microbubbles. The present study tried to deliver the GFP plasmids into fibroblast cells. Cells were cultured in 6-well culture plates and exposed to ultrasound (frequency, 2.1 MHz; wave pattern, duty cycle 10%; intensity, 0-26 W/cm2; time, 0-200 s) transmitted through medium containing microbubbles (Levovist® (void fraction, 8×10-5) or Sonazoid® (void fraction, 0-24×10-4)) and GFP plasmids at a concentration of 15 μg/mL. Density of microbubbles after ultrasound irradiation was measured. When ultrasound intensity was increased with Levovist® 8×10-4, transfection efficiency increased, cell viability decreased and microbubbles disappeared. With Sonazoid®, transfection efficiency and cell viability were basically unchanged and microbubbles decreased, but did not disappear. Transfection efficiency also improved with increased ultrasound irradiation time or microbubble density. Microbubble destruction appeared to have the main effect on gene transfection under Levovist® and microbubble oscillation had the main effect under Sonazoid®.

  16. Studies of DEAE-dextran-mediated gene transfer.

    PubMed

    Yang, Y W; Yang, J C

    1997-02-01

    DEAE-dextran-mediated gene transfer was studied for the introduction of pSV2neo DNA into Fisher-rat 3T3 (FR3T3) cells. Zeta (zeta) potentials of the DEAE-dextran-DNA complexes and FR3T3 cells were found to be dependent on the concentration of DEAE-dextran in the medium. The maximum transfection efficiency occurred at a DEAE-dextran/DNA ratio of 50:1 or thereabouts. The interaction between DNA and cells is determined by the adsorption process. The results obtained, along with the correlation between the kinetic adsorption behaviour of 3H-labelled DNA and the transfection efficiency, indicated that adsorption of DEAE-dextran-DNA complexes to the negatively charged cell surfaces, due to electrostatic and dispersion attraction, plays the decisive role in determining the DNA transfection efficiencies.

  17. Ultrasound -Assisted Gene Transfer to Adipose Tissue-Derived Stem/Progenitor Cells (ASCs)

    NASA Astrophysics Data System (ADS)

    Miyamoto, Yoshitaka; Ueno, Hitomi; Hokari, Rei; Yuan, Wenji; Kuno, Shuichi; Kakimoto, Takashi; Enosawa, Shin; Negishi, Yoichi; Yoshinaka, Kiyoshi; Matsumoto, Yoichiro; Chiba, Toshio; Hayashi, Shuji

    2011-09-01

    In recent years, multilineage adipose tissue-derived stem cells (ASCs) have become increasingly attractive as a promising source for cell transplantation and regenerative medicine. Particular interest has been expressed in the potential to make tissue stem cells, such as ASCs and marrow stromal cells (MSCs), differentiate by gene transfection. Gene transfection using highly efficient viral vectors such as adeno- and sendai viruses have been developed for this purpose. Sonoporation, or ultrasound (US)-assisted gene transfer, is an alternative gene manipulation technique which employs the creation of a jet stream by ultrasonic microbubble cavitation. Sonoporation using non-viral vectors is expected to be a much safer, although less efficient, tool for prospective clinical gene therapy. In this report, we assessed the efficacy of the sonoporation technique for gene transfer to ASCs. We isolated and cultured adipocyets from mouse adipose tissue. ASCs that have the potential to differentiate with transformation into adipocytes or osteoblasts were obtained. Using the US-assisted system, plasmid DNA containing beta-galactosidase (beta-Gal) and green fluorescent protein (GFP) genes were transferred to the ASCs. For this purpose, a Sonopore 4000 (NEPAGENE Co.) and a Sonazoid (Daiichi Sankyo Co.) instrument were used in combination. ASCs were subjected to US (3.1 MHz, 50% duty cycle, burst rate 2.0 Hz, intensity 1.2 W/cm2, exposure time 30 sec). We observed that the gene was more efficiently transferred with increased concentrations of plasmid DNA (5-150 μg/mL). However, further optimization of the US parameters is required, as the gene transfer efficiency was still relatively low. In conclusion, we herein demonstrate that a gene can be transferred to ASCs using our US-assisted system. In regenerative medicine, this system might resolve the current issues surrounding the use of viral vectors for gene transfer.

  18. Horizontal Gene Transfer Contributes to the Evolution of Arthropod Herbivory.

    PubMed

    Wybouw, Nicky; Pauchet, Yannick; Heckel, David G; Van Leeuwen, Thomas

    2016-06-27

    Within animals, evolutionary transition toward herbivory is severely limited by the hostile characteristics of plants. Arthropods have nonetheless counteracted many nutritional and defensive barriers imposed by plants and are currently considered as the most successful animal herbivores in terrestrial ecosystems. We gather a body of evidence showing that genomes of various plant feeding insects and mites possess genes whose presence can only be explained by horizontal gene transfer (HGT). HGT is the asexual transmission of genetic information between reproductively isolated species. Although HGT is known to have great adaptive significance in prokaryotes, its impact on eukaryotic evolution remains obscure. Here, we show that laterally transferred genes into arthropods underpin many adaptations to phytophagy, including efficient assimilation and detoxification of plant produced metabolites. Horizontally acquired genes and the traits they encode often functionally diversify within arthropod recipients, enabling the colonization of more host plant species and organs. We demonstrate that HGT can drive metazoan evolution by uncovering its prominent role in the adaptations of arthropods to exploit plants.

  19. Horizontal Gene Transfer Contributes to the Evolution of Arthropod Herbivory

    PubMed Central

    Wybouw, Nicky; Pauchet, Yannick; Heckel, David G.; Van Leeuwen, Thomas

    2016-01-01

    Within animals, evolutionary transition toward herbivory is severely limited by the hostile characteristics of plants. Arthropods have nonetheless counteracted many nutritional and defensive barriers imposed by plants and are currently considered as the most successful animal herbivores in terrestrial ecosystems. We gather a body of evidence showing that genomes of various plant feeding insects and mites possess genes whose presence can only be explained by horizontal gene transfer (HGT). HGT is the asexual transmission of genetic information between reproductively isolated species. Although HGT is known to have great adaptive significance in prokaryotes, its impact on eukaryotic evolution remains obscure. Here, we show that laterally transferred genes into arthropods underpin many adaptations to phytophagy, including efficient assimilation and detoxification of plant produced metabolites. Horizontally acquired genes and the traits they encode often functionally diversify within arthropod recipients, enabling the colonization of more host plant species and organs. We demonstrate that HGT can drive metazoan evolution by uncovering its prominent role in the adaptations of arthropods to exploit plants. PMID:27307274

  20. Gene duplication and transfer events in plant mitochondria genome

    SciTech Connect

    Xiong Aisheng Peng Rihe; Zhuang Jing; Gao Feng; Zhu Bo; Fu Xiaoyan; Xue Yong; Jin Xiaofen; Tian Yongsheng; Zhao Wei; Yao Quanhong

    2008-11-07

    Gene or genome duplication events increase the amount of genetic material available to increase the genomic, and thereby phenotypic, complexity of organisms during evolution. Gene duplication and transfer events have been important to molecular evolution in all three domains of life, and may be the first step in the emergence of new gene functions. Gene transfer events have been proposed as another accelerator of evolution. The duplicated gene or genome, mainly nuclear, has been the subject of several recent reviews. In addition to the nuclear genome, organisms have organelle genomes, including mitochondrial genome. In this review, we briefly summarize gene duplication and transfer events in the plant mitochondrial genome.

  1. Dynamical tuning of energy transfer efficiency on a graphene monolayer

    NASA Astrophysics Data System (ADS)

    Karanikolas, Vasilios D.; Marocico, Cristian A.; Bradley, A. Louise

    2015-03-01

    In this contribution we present a theoretical investigation of the energy transfer efficiency between quantum systems placed in proximity of a monolayer of conducting graphene. We calculate the spontaneous emission rate of a quantum system and the energy transfer rate between a donor-acceptor pair, and thus the energy transfer efficiency, using a Green's tensor formalism. The direct interaction between the donor and acceptor dominates when they are close to each other, but is modified from its free-space behavior due to the presence of the graphene monolayer and its interaction with the donor and acceptor. We report on a very large influence of the graphene monolayer on the energy transfer efficiency due to both the Förster mechanism and the propagating graphene plasmon mode. In particular, the Förster radius R0 is modified from its free-space value of 20 nm and can reach values of 120 nm when close to a graphene monolayer. As the donor-acceptor separation is increased, their direct interaction is overshadowed by the interaction via the surface plasmon mode. Due to the large propagation length of the surface plasmon mode on graphene, an energy transfer efficiency as high as 50% can still be achieved for distances as large as 300 nm. The interaction via the surface plasmon mode is tunable via the doping of the graphene monolayer and the surface plasmon channel can also be switched off this way.

  2. Horizontal gene transfer: A critical view

    PubMed Central

    Kurland, C. G.; Canback, B.; Berg, Otto G.

    2003-01-01

    It has been suggested that horizontal gene transfer (HGT) is the “essence of phylogeny.” In contrast, much data suggest that this is an exaggeration resulting in part from a reliance on inadequate methods to identify HGT events. In addition, the assumption that HGT is a ubiquitous influence throughout evolution is questionable. Instead, rampant global HGT is likely to have been relevant only to primitive genomes. In modern organisms we suggest that both the range and frequencies of HGT are constrained most often by selective barriers. As a consequence those HGT events that do occur most often have little influence on genome phylogeny. Although HGT does occur with important evolutionary consequences, classical Darwinian lineages seem to be the dominant mode of evolution for modern organisms. PMID:12902542

  3. The influence of substrate on DNA transfer and extraction efficiency.

    PubMed

    Verdon, Timothy J; Mitchell, R John; van Oorschot, Roland A H

    2013-01-01

    The circumstances surrounding deposition of DNA profiles are increasingly becoming an issue in court proceedings, especially whether or not the deposit was made by primary transfer. In order to improve the currently problematic evaluation of transfer scenarios in court proceedings, we examined the influence a variety of nine substrate types (six varieties of fabric, plywood, tarpaulin, and plastic sheets) has on DNA transfer involving blood. DNA transfer percentages were significantly higher (p=0.03) when the primary substrate was of non-porous material (such as tarpaulin, plastic or, to a lesser degree, wood) and the secondary substrate porous (such as fabrics). These findings on transfer percentages confirm the results of previous studies. Fabric composition was also shown to have a significant (p=0.03) effect on DNA transfer; when experiments were performed with friction from a variety of fabrics to a specific weave of cotton, transfer percentages ranged from 4% (flannelette) to 94% (acetate). The propensity for the same nine substrates to impact upon the efficiency of DNA extraction procedures was also examined. Significant (p=0.03) differences were found among the extraction efficiencies from different materials. When 15μL of blood was deposited on each of the substrates, the lowest quantity of DNA was extracted from plastic (20ng) and the highest quantities extracted from calico and flannelette (650ng). Significant (p<0.05) differences also exist among the DNA extraction yield from different initial blood volumes from all substrates. Also, significantly greater (p<0.05) loss of DNA was seen during concentration of extracts with higher compared to lower initial quantities of DNA. These findings suggest that the efficiency of extraction and concentration impacts upon the final amount of DNA available for analysis and that consideration of these effects should not be ignored. The application of correction factors to adjust for any variation among extraction and

  4. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  5. Bacterial gene transfer by natural genetic transformation in the environment.

    PubMed Central

    Lorenz, M G; Wackernagel, W

    1994-01-01

    Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation. PMID:7968924

  6. Gene Transfer & Hybridization Studies in Hyperthermophilic Species

    SciTech Connect

    Nelson, Karen E.

    2005-10-14

    A. ABSTRACT The importance of lateral gene transfer (LGT) in the evolution of microbial species has become increasingly evident with each completed microbial genome sequence. Most significantly, the genome of Thermotoga maritima MSB8, a hyperthermophilic bacterium isolated by Karl Stetter and workers from Vulcano Italy in 1986, and sequenced at The Institute for Genomic Research (TIGR) in Rockville Maryland in 1999, revealed extensive LGT between % . this bacterium and members of the archaeal domain (in particular Archaeoglobus fulgidus, and Pyracoccus frcriosus species). Based on whole genome comparisons, it was estimated that 24% of the genetic information in this organism was acquired by genetic exchange with archaeal species, Independent analyses including periodicity analysis of the T. maritimu genomic DNA sequence, phylogenetic reconstruction based on genes that appear archaeal-like, and codon and amino acid usage, have provided additional evidence for LGT between T. maritima and the archaea. More recently, DiRuggiero and workers have identified a very recent LGT event between two genera of hyperthermophilic archaea, where a nearly identical DNA fragment of 16 kb in length flanked by insertion sequence (IS) elements, exists. Undoubtedly, additional examples of LGT will be identified as more microbial genomes are completed. For the present moment however, the genome sequence of T. maritima and other hyperthermophiles including P. furiosus, Pyrococcus horikoshii, Pyrococcus abyssi, A. fulgidus, and Aquifex aeolicus, have significantly increased out awareness of evolution being a web of life rather than a tree of life, as suggested by single gene phylogenies. In this proposal, we will aim to determine the extent of LGT across the hyperthemophiles, employing iY maritima as the model organism. A variety of biochemical techniques and phylogenetic reconstructions will allow for a detailed and thorough characterization of the extent of LGT in this species. The

  7. Stable transformation of maize after gene transfer by electroporation.

    PubMed

    Fromm, M E; Taylor, L P; Walbot, V

    The graminaceous monocots, including the economically important cereals, seem to be refractory to infection by Agrobacterium tumefaciens, a natural gene transfer system that has been successfully exploited for transferring foreign genes into higher plants. Therefore, direct transfer techniques that are potentially applicable to all plant species have been developed using a few dicot and monocot species as model systems. One of these techniques, electroporation, uses electrical pulses of high field strength to permeabilize cell membranes reversibly so as to facilitate the transfer of DNA into cells. Electroporation-mediated gene transfer has resulted in stably transformed animal cells and transient gene expression in monocot and dicot plant cells. Here we report that electroporation-mediated DNA transfer of a chimaeric gene encoding neomycin phosphotransferase results in stably transformed maize cells that are resistant to kanamycin.

  8. Estimation of hand-to-mouth transfer efficiency of lead.

    PubMed

    Sahmel, Jennifer; Hsu, Elleen I; Avens, Heather J; Beckett, Evan M; Devlin, Kathryn D

    2015-03-01

    There are currently no published empirical data that characterize hand-to-mouth transfer efficiencies for metallic lead. The purpose of this study was to quantify the hand-to-mouth transfer efficiency of lead in adult volunteers (n = 6) using human saliva as a surrogate for the mouth and commercially available, 100% lead fishing weights as the source of lead for dermal loading. Study volunteers' saliva was collected and subsequently poured onto a sheet of wax paper placed on a balance scale. The volunteers handled lead fishing weights with both hands for approximately 15 s and then pressed three fingers from the right hand (test hand) into their saliva 10 times, with ~0.45kg of pressure. The left hand (control hand) was used as a comparison for dermal loading of lead and had no contact with saliva. SKC Full Disclosure® wipes were used to collect lead from the saliva and skin surfaces. Samples were analyzed using the NIOSH 7300 method, which was modified for wipes. The mean lead skin-to-saliva transfer efficiency was 24% (range: 12-34%). These data will be useful for more accurately characterizing lead hand-to-mouth transfer efficiencies and are likely to be helpful in exposure assessments or human health risk assessments.

  9. Efficient vector radiative transfer calculations in vertically inhomogeneous cloudy atmospheres.

    PubMed

    van Diedenhoven, Bastiaan; Hasekamp, Otto P; Landgraf, Jochen

    2006-08-10

    Accurate radiative transfer calculations in cloudy atmospheres are generally time consuming, limiting their practical use in satellite remote sensing applications. We present a model to efficiently calculate the radiative transfer of polarized light in atmospheres that contain homogeneous cloud layers. This model combines the Gauss-Seidel method, which is efficient for inhomogeneous cloudless atmospheres, with the doubling method, which is efficient for homogeneous cloud layers. Additionally to reduce the computational effort for radiative transfer calculations in absorption bands, the cloud reflection and transmission matrices are interpolated over the absorption and scattering optical thicknesses within the cloud layer. We demonstrate that the proposed radiative transfer model in combination with this interpolation technique is efficient for the simulation of satellite measurements for inhomogeneous atmospheres containing one homogeneous cloud layer. For example, the Scanning Imaging Absorption Spectrometer for Atmospheric Cartography (SCIAMACHY) measurements in the oxygen A band (758-773 nm) and the Hartley-Huggins ozone band (295-335 nm) with a spectral resolution of 0.4 nm can be simulated for these atmospheres within 1 min on a 2.8 GHz PC with an accuracy better than 0.1%.

  10. Alkoxyamine-cyanoborane adducts: efficient cyanoborane transfer agents.

    PubMed

    Márquez, José M; Martínez-Castro, Elisa; Gabrielli, Serena; López, Óscar; Maya, Inés; Angulo, Manuel; Álvarez, Eleuterio; Fernández-Bolaños, José G

    2011-05-21

    We report the synthesis of the hitherto unknown zwitterionic alkoxyamino cyanoboranes by reduction of O-alkyloximes with sodium cyanoborohydride; unprecedented cyanoboronated N-alkoxyformamidines were also isolated as by-products. Boronated alkoxyamines were found to be efficient cyanoborane transfer agents towards more basic amines, including aminosugars; they were also successfully transformed into neoglycoconjugates by the neoglycorandomization reaction with reducing sugars.

  11. Horizontal Gene Transfer of Pectinases from Bacteria Preceded the Diversification of Stick and Leaf Insects

    PubMed Central

    Shelomi, Matan; Danchin, Etienne G. J.; Heckel, David; Wipfler, Benjamin; Bradler, Sven; Zhou, Xin; Pauchet, Yannick

    2016-01-01

    Genes acquired by horizontal transfer are increasingly being found in animal genomes. Understanding their origin and evolution requires knowledge about the phylogenetic relationships from both source and recipient organisms. We used RNASeq data and respective assembled transcript libraries to trace the evolutionary history of polygalacturonase (pectinase) genes in stick insects (Phasmatodea). By mapping the distribution of pectinase genes on a Polyneoptera phylogeny, we identified the transfer of pectinase genes from known phasmatodean gut microbes into the genome of an early euphasmatodean ancestor that took place between 60 and 100 million years ago. This transfer preceded the rapid diversification of the suborder, enabling symbiont-free pectinase production that would increase the insects’ digestive efficiency and reduce dependence on microbes. Bacteria-to-insect gene transfer was thought to be uncommon, however the increasing availability of large-scale genomic data may change this prevailing notion. PMID:27210832

  12. Methods for Gene Transfer to the Central Nervous System

    PubMed Central

    Kantor, Boris; Bailey, Rachel M.; Wimberly, Keon; Kalburgi, Sahana N.; Gray, Steven J.

    2015-01-01

    Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed. PMID:25311922

  13. Tumor specific ultrasound enhanced gene transfer in vivo with novel liposomal bubbles.

    PubMed

    Suzuki, Ryo; Takizawa, Tomoko; Negishi, Yoichi; Utoguchi, Naoki; Sawamura, Kaori; Tanaka, Kumiko; Namai, Eisuke; Oda, Yusuke; Matsumura, Yasuhiro; Maruyama, Kazuo

    2008-01-22

    Bubble liposomes (liposomes which entrap an ultrasound imaging gas) may constitute a unique system for delivering various molecules efficiently into mammalian cells in vitro. In this study, Bubble liposomes were compared with cationic lipid (CL)-DNA complexes as potential gene delivery carriers into tumor in vivo. The delivery of genes by Bubble liposomes depended on the intensity of the applied ultrasound. Transfection efficiency plateaued at 0.7 W/cm(2) ultrasound intensity. Bubble liposomes efficiently transferred genes into cultured cells even when the cells were exposed to ultrasound for only 1 s. In addition, Bubble liposomes could introduce the luciferase gene more effectively than CL-DNA complexes into mouse ascites tumor cells and solid tumor tissue. We conclude that the combination of Bubble liposomes and ultrasound is a minimally-invasive and tumor specific gene transfer method in vivo.

  14. Optimizing A Lipocomplex-Based Gene Transfer Method into HeLa Cell Line

    PubMed Central

    Asgharian, Alimohammad; Banan, Mehdi; Najmabadi, Hossein

    2014-01-01

    One of the most significant steps in gene expression studies is transferring genes into cell cultures. Despite there are different methods for gene delivery such as viral and non-viral producers, some cationic lipid reagents have recently developed to transfect into mam- malian cell lines. The main aim of this study was optimizing and improving lipocomplex based transient transfection procedures into HeLa cell line which is being used widely as a typical cell in biological studies. This study was an experimental research. In this work, pCMV β-Gal DNA plasmid was used as a reporter DNA for determining the rate of gene transfection into HeLa cells. To accomplish the highest gene delivery into HeLa cells, optimizing experiments were carried out in different volumes of FuGENE-HD, LipofectamineTM2000 and X-tremeGENE. Also, we investigated tranasfection efficiency in presence of various cell densities of HeLa cells. Then, transfection efficiency and cell toxicity were measured by beta gal staining and trypan blue methods, respectively. Using FuGENE-HD in volume of 4µl along with 105 HeLa cells, transfection efficiency was higher (43.66 ± 1.52%) in comparison with the cationic lipids LipofectamineTM2000 and X-tremeGENE. In addition, the rate of cell toxicity in presence of FuGENE-HD was less than 5%. In summary, the cationic lipid FuGENE-HD indicates a suitable potential to transfer DNA into HeLa cells and it can be an efficient reagent for gene delivery for HeLa cells in vitro. Moreover, it is worth designing and optimizing gene transfer experiments for other cell lines with FuGENE-HD due to its low toxicity and high efficiency. PMID:24381863

  15. Optimizing A Lipocomplex-Based Gene Transfer Method into HeLa Cell Line.

    PubMed

    Asgharian, Alimohammad; Banan, Mehdi; Najmabadi, Hossein

    2014-01-01

    One of the most significant steps in gene expression studies is transferring genes into cell cultures. Despite there are different methods for gene delivery such as viral and non-viral producers, some cationic lipid reagents have recently developed to transfect into mam- malian cell lines. The main aim of this study was optimizing and improving lipocomplex based transient transfection procedures into HeLa cell line which is being used widely as a typical cell in biological studies. This study was an experimental research. In this work, pCMV β-Gal DNA plasmid was used as a reporter DNA for determining the rate of gene transfection into HeLa cells. To accomplish the highest gene delivery into HeLa cells, optimizing experiments were car- ried out in different volumes of FuGENE-HD, Lipofectamine(TM)2000 and X-tremeGENE. Also, we investigated tranasfection efficiency in presence of various cell densities of HeLa cells. Then, transfection efficiency and cell toxicity were measured by beta gal staining and trypan blue methods, respectively. Using FuGENE-HD in volume of 4µl along with 10(5) HeLa cells, transfection efficiency was higher (43.66 ± 1.52%) in comparison with the cationic lipids Lipofectamine(TM)2000 and X-tremeGENE. In addition, the rate of cell toxicity in presence of FuGENE-HD was less than 5%. In summary, the cationic lipid FuGENE-HD indicates a suitable potential to transfer DNA into HeLa cells and it can be an efficient reagent for gene delivery for HeLa cells in vitro. Moreover, it is worth designing and optimizing gene transfer experiments for other cell lines with FuGENE-HD due to its low toxicity and high efficiency.

  16. Transfer of nonselectable genes into mouse teratocarcinoma cells and transcription of the transferred human. beta. -globin gene

    SciTech Connect

    Wagner, E.F.; Mintz, B.

    1982-02-01

    Teratocarcinoma (TCC) stem cells can function as vehicles for the introduction of specific recombinant genes into mice. Because most genes do not code for a selectable marker, the authors investigated the transformation efficiency of vectors with a linked selectable gene. In one series, TCC cells first selected for thymidine kinase deficiency were treated with DNA from the plasmid vector PtkH..beta..1 containing the human genomic ..beta..-globin gene and the thymidine kinase gene of herpes simplex virus. A high transformation frequency was obtained after selection in hypoxanthine-aminopterin-thymidine medium. Hybridization tests revealed that the majority of transformants had intact copies of the human gene among three to six total copies per cell. These were associated with cellular DNA sequences as judged from the presence of additional new restriction fragments and from stability of the sequences in tumors produced by injecting the cells subcutaneously. Total polyadenylate-containing RNA from cell cultures of two out of four transformants examined showed hybridization to the human gene probe: one RNA species resembled mature human ..beta..-globin mRNA transcripts; the others were of larger size. In differentiating tumors, various tissues, including hematopoietic cells of TCC provenance could be found. In a second model set of experiments, wild-type TCC cells were used to test a dominant-selection scheme with pSV-gpt vectors. Numerous transformants were isolated, and their transfected DNA was apparently stably integrated. Thus, any gene of choice can be transferred into TCC stem cells even without mutagenesis of the cells, and selected cell clones can be characterized. Cells of interest may then be introduced into early embryos to produce new mouse strains with predetermined genetic changes.

  17. Problems associated with gene transfer and opportunities for microgravity environments

    SciTech Connect

    Tennessen, D.J.

    1997-01-01

    The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of {ital Agrobacterium} mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed. {copyright} {ital 1997 American Institute of Physics.}

  18. Problems associated with gene transfer and opportunities for microgravity environments

    NASA Astrophysics Data System (ADS)

    Tennessen, Daniel J.

    1997-01-01

    The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of Agrobacterium mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed.

  19. Light Increases Energy Transfer Efficiency in a Boreal Stream

    PubMed Central

    Lesutienė, Jūratė; Gorokhova, Elena; Stankevičienė, Daiva; Bergman, Eva; Greenberg, Larry

    2014-01-01

    Periphyton communities of a boreal stream were exposed to different light and nutrient levels to estimate energy transfer efficiency from primary to secondary producers using labeling with inorganic 13C. In a one-day field experiment, periphyton grown in fast-flow conditions and dominated by opportunistic green algae were exposed to light levels corresponding to sub-saturating (forest shade) and saturating (open stream section) irradiances, and to N and P nutrient additions. In a two-week laboratory experiment, periphyton grown in low-flow conditions and dominated by slowly growing diatoms were incubated under two sub-saturating light and nutrient enrichment levels as well as grazed and non-grazed conditions. Light had significant positive effect on 13C uptake by periphyton. In the field experiment, P addition had a positive effect on 13C uptake but only at sub-saturating light levels, whereas in the laboratory experiment nutrient additions had no effect on the periphyton biomass, 13C uptake, biovolume and community composition. In the laboratory experiment, the grazer (caddisfly) effect on periphyton biomass specific 13C uptake and nutrient content was much stronger than the effects of light and nutrients. In particular, grazers significantly reduced periphyton biomass and increased biomass specific 13C uptake and C:nutrient ratios. The energy transfer efficiency, estimated as a ratio between 13C uptake by caddisfly and periphyton, was positively affected by light conditions, whereas the nutrient effect was not significant. We suggest that the observed effects on energy transfer were related to the increased diet contribution of highly palatable green algae, stimulated by higher light levels. Also, high heterotrophic microbial activity under low light levels would facilitate energy loss through respiration and decrease overall trophic transfer efficiency. These findings suggest that even a small increase in light intensity could result in community-wide effects on

  20. Light increases energy transfer efficiency in a boreal stream.

    PubMed

    Lesutienė, Jūratė; Gorokhova, Elena; Stankevičienė, Daiva; Bergman, Eva; Greenberg, Larry

    2014-01-01

    Periphyton communities of a boreal stream were exposed to different light and nutrient levels to estimate energy transfer efficiency from primary to secondary producers using labeling with inorganic (13)C. In a one-day field experiment, periphyton grown in fast-flow conditions and dominated by opportunistic green algae were exposed to light levels corresponding to sub-saturating (forest shade) and saturating (open stream section) irradiances, and to N and P nutrient additions. In a two-week laboratory experiment, periphyton grown in low-flow conditions and dominated by slowly growing diatoms were incubated under two sub-saturating light and nutrient enrichment levels as well as grazed and non-grazed conditions. Light had significant positive effect on (13)C uptake by periphyton. In the field experiment, P addition had a positive effect on (13)C uptake but only at sub-saturating light levels, whereas in the laboratory experiment nutrient additions had no effect on the periphyton biomass, (13)C uptake, biovolume and community composition. In the laboratory experiment, the grazer (caddisfly) effect on periphyton biomass specific (13)C uptake and nutrient content was much stronger than the effects of light and nutrients. In particular, grazers significantly reduced periphyton biomass and increased biomass specific (13)C uptake and C:nutrient ratios. The energy transfer efficiency, estimated as a ratio between (13)C uptake by caddisfly and periphyton, was positively affected by light conditions, whereas the nutrient effect was not significant. We suggest that the observed effects on energy transfer were related to the increased diet contribution of highly palatable green algae, stimulated by higher light levels. Also, high heterotrophic microbial activity under low light levels would facilitate energy loss through respiration and decrease overall trophic transfer efficiency. These findings suggest that even a small increase in light intensity could result in community

  1. Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer.

    PubMed

    Sinn, Patrick L; Burnight, Erin R; Hickey, Melissa A; Blissard, Gary W; McCray, Paul B

    2005-10-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

  2. Intrapleural 'outside-in' gene therapy: therapeutics for organs of the chest via gene transfer to the pleura.

    PubMed

    Heguy, Adriana; Crystal, Ronald G

    2005-10-01

    The pleural space is an attractive site for using viral vectors to deliver gene products to the lung parenchyma, other thoracic structures and the systemic circulation. The advantages of intrapleural gene transfer using viral vectors include: (i) easy accessibility; (ii) large surface area; (iii) ability to provide high concentrations of secreted gene products to chest structures; (iv) low risk of detrimental effects of possible vector-induced inflammation compared with intravascular delivery; and (v) because it is local, lower vector doses can be used to deliver therapeutic genes to thoracic structures than less efficient systemic routes. Examples of pleural gene transfer include the use of adenovirus vectors to treat mesothelioma by transiently expressing genes that encode toxic proteins, immunomodulatory molecules or anti-angiogenesis factors. Intrapleural delivery of adeno-associated viral vectors represents an efficient strategy to treat alpha1-antitrypsin (alpha1AT) deficiency, achieving high lung and systemic therapeutic levels of alpha1AT. Intrapleural delivery of gene transfer vectors holds promise for the treatment of diseases requiring transient, localized gene expression, as well as sustained expression of genes to correct hereditary disorders requiring localized or systemic expression of the therapeutic protein.

  3. Moving toward a higher efficiency of microcell-mediated chromosome transfer

    PubMed Central

    Liskovykh, Mikhail; Lee, Nicholas CO; Larionov, Vladimir; Kouprina, Natalay

    2016-01-01

    Microcell-mediated chromosome transfer (MMCT) technology enables individual mammalian chromosomes, megabase-sized chromosome fragments, or mammalian artificial chromosomes that include human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) to be transferred from donor to recipient cells. In the past few decades, MMCT has been applied to various studies, including mapping the genes, analysis of chromosome status such as aneuploidy and epigenetics. Recently, MMCT was applied to transfer MACs/HACs carrying entire chromosomal copies of genes for genes function studies and has potential for regenerative medicine. However, a safe and efficient MMCT technique remains an important challenge. The original MMCT protocol includes treatment of donor cells by Colcemid to induce micronucleation, where each chromosome becomes surrounded with a nuclear membrane, followed by disarrangement of the actin cytoskeleton using Cytochalasin B to help induce microcells formation. In this study, we modified the protocol and demonstrated that replacing Colcemid and Cytochalasin B with TN-16 + Griseofulvin and Latrunculin B in combination with a Collage/Laminin surface coating increases the efficiency of HAC transfer to recipient cells by almost sixfold and is possibly less damaging to HAC than the standard MMCT method. We tested the improved MMCT protocol on four recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells that could facilitate the cell engineering by HACs. PMID:27382603

  4. EFFICIENT THREE-DIMENSIONAL NLTE DUST RADIATIVE TRANSFER WITH SKIRT

    SciTech Connect

    Baes, Maarten; Verstappen, Joris; De Looze, Ilse; Fritz, Jacopo; Saftly, Waad; Vidal Perez, Edgardo; Stalevski, Marko; Valcke, Sander

    2011-10-01

    We present an updated version of SKIRT, a three-dimensional (3D) Monte Carlo radiative transfer code developed to simulate dusty galaxies. The main novel characteristics of the SKIRT code are the use of a stellar foam to generate random positions, an efficient combination of eternal forced scattering and continuous absorption, and a new library approach that links the radiative transfer code to the DustEM dust emission library. This approach enables a fast, accurate, and self-consistent calculation of the dust emission of arbitrary mixtures of transiently heated dust grains and polycyclic aromatic hydrocarbons, even for full 3D models containing millions of dust cells. We have demonstrated the accuracy of the SKIRT code through a set of simulations based on the edge-on spiral galaxy UGC 4754. The models we ran were gradually refined from a smooth, two-dimensional, local thermal equilibrium (LTE) model to a fully 3D model that includes non-LTE (NLTE) dust emission and a clumpy structure of the dusty interstellar medium. We find that clumpy models absorb UV and optical radiation less efficiently than smooth models with the same amount of dust, and that the dust in clumpy models is on average both cooler and less luminous. Our simulations demonstrate that, given the appropriate use of optimization techniques, it is possible to efficiently and accurately run Monte Carlo radiative transfer simulations of arbitrary 3D structures of several million dust cells, including a full calculation of the NLTE emission by arbitrary dust mixtures.

  5. Gene transfer in hepatocarcinoma cell lines: in vitro optimization of a virus-free system.

    PubMed

    Ghoumari, A M; Rixe, O; Yarovoi, S V; Zerrouqi, A; Mouawad, R; Poynard, T; Opolon, P; Khayat, D; Soubrane, C

    1996-06-01

    Many approaches exist for hepatic gene delivery, including viral vectors and non-viral vectors. In this study, we tested a panel of liposomes to transfer pAGO, a plasmid containing one copy of herpes simplex virus (HSVtk) gene, and pYED11, a plasmid containing two copies of the HSVtk gene, into a murine hepatocarcinoma cell line (Hepa 1-6) and a human hepatocarcinoma cell line (Hep-G2). The efficiency of gene delivery and expression was characterized by beta-galactosidase staining, flow cytometric analysis and quantitative lacZ activity. Different combinations of liposomes and DNA and the ratio of the concentration of liposome to DNA were tested. The efficient transfer was shown with DOTAP followed by transfectam and lipofectamine. Under these conditions, we tested the cytotoxicity of ganciclovir (GCV) exposure on Hepa 1-6 and Hep-G2 transfected separately with liposome-pAGO and liposome-pYED11 complexes. This study demonstrates the in vitro efficacy of each liposome tested to transduce the HSVtk gene into hepatocarcinoma cell lines. The transfer of two copies of the HSVtk gene rendered cells 1.5 times more sensitive to GCV than cells transduced by pAGO as compared to controls. This was achieved most efficiently by the DOTAP-pYED11 complex. Thus, pYED11 may be considered as an alternative to pAGO as a gene transfer vector.

  6. Therapeutic antibody gene transfer: an active approach to passive immunity.

    PubMed

    Bakker, Joost M; Bleeker, Wim K; Parren, Paul W H I

    2004-09-01

    Advances in gene transfer approaches are enabling the possibility of applying therapeutic antibodies using DNA. In particular gene transfer in combination with electroporation is promising and can result in generating in vivo antibody concentrations in the low therapeutic range. However, several important problems need to be dealt with before antibody gene transfer can become a valuable supplement to the current therapies. As antibody production following gene transfer is difficult to control, the danger of inducing autoimmune conditions or uncontrollable side effects occurs in cases in which autologous antigens are targeted. It is suggested that the most promising area of application therefore appears to be infectious disease in which heterologous antigens are targeted and concerns for long-term antibody exposure are minimal. Finally, genes encoding fully human antibodies will enhance long-term expression and decrease problems linked to immunogenicity.

  7. Direct Gene Transfer into Human Cultured Cells Facilitated by Laser Micropuncture of the Cell Membrane

    NASA Astrophysics Data System (ADS)

    Tao, Wen; Wilkinson, Joyce; Stanbridge, Eric J.; Berns, Michael W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in medium containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual human chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8 × 10-4-3 × 10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  8. The power of phylogenetic approaches to detect horizontally transferred genes

    PubMed Central

    Poptsova, Maria S; Gogarten, J Peter

    2007-01-01

    Background Horizontal gene transfer plays an important role in evolution because it sometimes allows recipient lineages to adapt to new ecological niches. High genes transfer frequencies were inferred for prokaryotic and early eukaryotic evolution. Does horizontal gene transfer also impact phylogenetic reconstruction of the evolutionary history of genomes and organisms? The answer to this question depends at least in part on the actual gene transfer frequencies and on the ability to weed out transferred genes from further analyses. Are the detected transfers mainly false positives, or are they the tip of an iceberg of many transfer events most of which go undetected by current methods? Results Phylogenetic detection methods appear to be the method of choice to infer gene transfers, especially for ancient transfers and those followed by orthologous replacement. Here we explore how well some of these methods perform using in silico transfers between the terminal branches of a gamma proteobacterial, genome based phylogeny. For the experiments performed here on average the AU test at a 5% significance level detects 90.3% of the transfers and 91% of the exchanges as significant. Using the Robinson-Foulds distance only 57.7% of the exchanges and 60% of the donations were identified as significant. Analyses using bipartition spectra appeared most successful in our test case. The power of detection was on average 97% using a 70% cut-off and 94.2% with 90% cut-off for identifying conflicting bipartitions, while the rate of false positives was below 4.2% and 2.1% for the two cut-offs, respectively. For all methods the detection rates improved when more intervening branches separated donor and recipient. Conclusion Rates of detected transfers should not be mistaken for the actual transfer rates; most analyses of gene transfers remain anecdotal. The method and significance level to identify potential gene transfer events represent a trade-off between the frequency of erroneous

  9. Gene Transfer by Guanidinium-Cholesterol Cationic Lipids into Airway Epithelial Cells in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Oudrhiri, Noufissa; Vigneron, Jean-Pierre; Peuchmaur, Michel; Leclerc, Tony; Lehn, Jean-Marie; Lehn, Pierre

    1997-03-01

    Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis (guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.

  10. Transferability of the Electrospray Ionization Efficiency Scale between Different Instruments

    NASA Astrophysics Data System (ADS)

    Liigand, Jaanus; Kruve, Anneli; Liigand, Piia; Laaniste, Asko; Girod, Marion; Antoine, Rodolphe; Leito, Ivo

    2015-11-01

    For the first time, quantitative electrospray (ESI) ionization efficiencies (IE), expressed as log IE values, obtained on different mass-spectrometric setups (four mass analyzers and four ESI sources) are compared for 15 compounds of diverse properties. The general trends of change of IE with molecular structure are the same with all experimental setups. The obtained IE scales could be applied on different setups: there were no statistically significant changes in the order of ionization efficiency and the root mean of squared differences of the log IE values of compounds between the scales compiled on different instruments were found to be between 0.21 and 0.55 log units. The results show that orthogonal ESI source geometry gives better differentiating power and additional pneumatic assistance improves it even more. It is also shown that the ionization efficiency values are transferable between different mass-spectrometric setups by three anchoring points and a linear model. The root mean square error of log IE prediction ranged from 0.24 to 0.72 depending on the instrument. This work demonstrates for the first time the inter-instrument transferability of quantitative electrospray ionization efficiency data.

  11. A recently transferred cluster of bacterial genes in Trichomonas vaginalis - lateral gene transfer and the fate of acquired genes

    PubMed Central

    2014-01-01

    Background Lateral Gene Transfer (LGT) has recently gained recognition as an important contributor to some eukaryote proteomes, but the mechanisms of acquisition and fixation in eukaryotic genomes are still uncertain. A previously defined norm for LGTs in microbial eukaryotes states that the majority are genes involved in metabolism, the LGTs are typically localized one by one, surrounded by vertically inherited genes on the chromosome, and phylogenetics shows that a broad collection of bacterial lineages have contributed to the transferome. Results A unique 34 kbp long fragment with 27 clustered genes (TvLF) of prokaryote origin was identified in the sequenced genome of the protozoan parasite Trichomonas vaginalis. Using a PCR based approach we confirmed the presence of the orthologous fragment in four additional T. vaginalis strains. Detailed sequence analyses unambiguously suggest that TvLF is the result of one single, recent LGT event. The proposed donor is a close relative to the firmicute bacterium Peptoniphilus harei. High nucleotide sequence similarity between T. vaginalis strains, as well as to P. harei, and the absence of homologs in other Trichomonas species, suggests that the transfer event took place after the radiation of the genus Trichomonas. Some genes have undergone pseudogenization and degradation, indicating that they may not be retained in the future. Functional annotations reveal that genes involved in informational processes are particularly prone to degradation. Conclusions We conclude that, although the majority of eukaryote LGTs are single gene occurrences, they may be acquired in clusters of several genes that are subsequently cleansed of evolutionarily less advantageous genes. PMID:24898731

  12. LATERAL GENE TRANSFER AND THE HISTORY OF BACTERIAL GENOMES

    SciTech Connect

    Howard Ochman

    2006-02-22

    The aims of this research were to elucidate the role and extent of lateral transfer in the differentiation of bacterial strains and species, and to assess the impact of gene transfer on the evolution of bacterial genomes. The ultimate goal of the project is to examine the dynamics of a core set of protein-coding genes (i.e., those that are distributed universally among Bacteria) by developing conserved primers that would allow their amplification and sequencing in any bacterial taxa. In addition, we adopted a bioinformatic approach to elucidate the extent of lateral gene transfer in sequenced genome.

  13. Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase

    PubMed Central

    Daley, Daniel O.; Clifton, Rachel; Whelan, James

    2002-01-01

    Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2. PMID:12142462

  14. Efficient estimation of energy transfer efficiency in light-harvesting complexes.

    PubMed

    Shabani, A; Mohseni, M; Rabitz, H; Lloyd, S

    2012-07-01

    The fundamental physical mechanisms of energy transfer in photosynthetic complexes is not yet fully understood. In particular, the degree of efficiency or sensitivity of these systems for energy transfer is not known given their realistic with surrounding photonic and phononic environments. One major problem in studying light-harvesting complexes has been the lack of an efficient method for simulation of their dynamics in biological environments. To this end, here we revisit the second order time-convolution (TC2) master equation and examine its reliability beyond extreme Markovian and perturbative limits. In particular, we present a derivation of TC2 without making the usual weak system-bath coupling assumption. Using this equation, we explore the long-time behavior of exciton dynamics of Fenna-Matthews-Olson (FMO) portein complex. Moreover, we introduce a constructive error analysis to estimate the accuracy of TC2 equation in calculating energy transfer efficiency, exhibiting reliable performance for system-bath interactions with weak and intermediate memory and strength. Furthermore, we numerically show that energy transfer efficiency is optimal and robust for the FMO protein complex of green sulfur bacteria with respect to variations in reorganization energy and bath correlation time scales.

  15. Nonreplicating vaccinia vector efficiently expresses recombinant genes.

    PubMed

    Sutter, G; Moss, B

    1992-11-15

    Modified vaccinia Ankara (MVA), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. MVA has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. Nevertheless, we found that replication of viral DNA appeared normal and that both early and late viral proteins were synthesized in human cells. Proteolytic processing of viral structural proteins was inhibited, however, and only immature virus particles were detected by electron microscopy. We constructed an insertion plasmid with the Escherichia coli lacZ gene under the control of the vaccinia virus late promoter P11, flanked by sequences of MVA DNA, to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome. MVA recombinants were isolated and propagated in permissive avian cells and shown to express the enzyme beta-galactosidase upon infection of nonpermissive human cells. The amount of enzyme made was similar to that produced by a recombinant of vaccinia virus strain Western Reserve, which also had the lacZ gene under control of the P11 promoter, but multiplied to high titers. Since recombinant gene expression is unimpaired in nonpermissive human cells, MVA may serve as a highly efficient and exceptionally safe vector.

  16. DNA Intercalated Psoralen Undergoes Efficient Photoinduced Electron Transfer.

    PubMed

    Fröbel, Sascha; Reiffers, Anna; Torres Ziegenbein, Christian; Gilch, Peter

    2015-04-02

    The interaction of psoralens with DNA has been used for therapeutic and research purposes for decades. Still the photoinduced behavior of psoralens in DNA has never been observed directly. Femtosecond transient absorption spectroscopy is used here to gain direct insight into the photophysics of a DNA-intercalated psoralen (4'-aminomethyl-4,5',8-trimethyl-psoralen (AMT)). Intercalation reduces the excited singlet lifetime of AMT to 4 ps compared with 1400 ps for AMT in water. This singlet quenching prohibits the population of the triplet state that is accessed in free AMT. Instead, a DNA to AMT electron transfer takes place. The resulting radical pair decays primarily via charge recombination with a time constant of 30 ps. The efficient electron transfer observed here reveals a completely new aspect of the psoralen-DNA interaction.

  17. Gene transfer and mutagenesis mediated by Sleeping Beauty transposon in Nile tilapia (Oreochromis niloticus).

    PubMed

    He, Xiaozhen; Li, Jie; Long, Yong; Song, Guili; Zhou, Peiyong; Liu, Qiuxiang; Zhu, Zuoyan; Cui, Zongbin

    2013-10-01

    The success of gene transfer has been demonstrated in many of vertebrate species, whereas the efficiency of producing transgenic animals remains pretty low due to the random integration of foreign genes into a recipient genome. The Sleeping Beauty (SB) transposon is able to improve the efficiency of gene transfer in zebrafish and mouse, but its activity in tilapia (Oreochromis niloticus) has yet to be characterized. Herein, we demonstrate the potential of using the SB transposon system as an effective tool for gene transfer and insertional mutagenesis in tilapia. A transgenic construct pT2/tiHsp70-SB11 was generated by subcloning the promoter of tilapia heat shock protein 70 (tiHsp70) gene, the SB11 transposase gene and the carp β-actin gene polyadenylation signal into the second generation of SB transposon. Transgenic tilapia was produced by microinjection of this construct with in vitro synthesized capped SB11 mRNA. SB11 transposon was detected in 28.89 % of founders, 12.9 % of F1 and 43.75 % of F2. Analysis of genomic sequences flanking integrated transposons indicates that this transgenic tilapia line carries two copies of SB transposon, which landed into two different endogenous genes. Induced expression of SB11 gene after heat shock was detected using reverse transcription PCR in F2 transgenic individuals. In addition, the Cre/loxP system was introduced to delete the SB11 cassette for stabilization of gene interruption and bio-safety. These findings suggest that the SB transposon system is active and can be used for efficient gene transfer and insertional mutagenesis in tilapia.

  18. The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

    PubMed

    Rim, Chol Ho; Fu, Zhixin; Bao, Lei; Chen, Haide; Zhang, Dan; Luo, Qiong; Ri, Hak Chol; Huang, Hefeng; Luan, Zhidong; Zhang, Yan; Cui, Chun; Xiao, Lei; Jong, Ui Myong

    2013-12-01

    To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, p<0.05) and average litter size (4.1 ± 2.3, 7 ± 3.6 vs. 2.5 ± 0.5). In vitro culture of reconstructed embryos for a longer time (40 h vs. 20 h) resulted in higher (p<0.05) pregnancy rate (44 ± 9 vs. 31 ± 3%) and delivery rate (44 ± 9 vs. 25 ± 9%). Furthermore, double oviductal transfer dramatically increased pregnancy rate (83 ± 6 vs. 27+8%, p<0.05), delivery rate (75 ± 2 vs. 27+8%, p<0.05) and average litter size (6.5 ± 2.8 vs. 2.6 ± 1.2) compared to single oviductal transfer. Our study demonstrated that an improvement in pig cloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern.

  19. Efficient inter-trap transfer of cold francium atoms

    NASA Astrophysics Data System (ADS)

    Zhang, J.; Collister, R.; Shiells, K.; Tandecki, M.; Aubin, S.; Behr, J. A.; Gomez, E.; Gorelov, A.; Gwinner, G.; Orozco, L. A.; Pearson, M. R.; Zhao, Y.

    2016-12-01

    We report on the status of the FrPNC experiments and summarize our plans for measurements of parity non-conservation (PNC) in a sample of cold francium. The FrPNC collaboration has commissioned a laser cooling apparatus at the TRIUMF accelerator that collects and cools francium atoms for PNC experiments. We have recently demonstrated the robust, high efficiency transfer (50 %) of laser cooled francium atoms to a second laser cooling apparatus, located 0.7 m below the first, where the PNC experiments will be conducted.

  20. Research on Efficiency of Knowledge Transfer in Technical Innovation Alliances

    NASA Astrophysics Data System (ADS)

    Zhang-sheng, Jiang

    The knowledge transfer efficiency (KTE) is closely relative to the success or failure of technology innovation in strategic alliances. This paper takes the KTE as the essential variable to establish the benefit function model of technology innovations to explore the KTE's influences on partners' innovative decisions under two different modes: independent innovations and alliance innovations. It is found that the higher the KTE, the greater the reducing extent of production costs is. The results could provide some theoretical supports for selections of the optimal competitive-ooperative relationship and managerial flexibility in technical innovation alliances.

  1. Fabrication of DNA-antibody-apatite composite layers for cell-targeted gene transfer

    NASA Astrophysics Data System (ADS)

    Yazaki, Yushin; Oyane, Ayako; Araki, Hiroko; Sogo, Yu; Ito, Atsuo; Yamazaki, Atsushi; Tsurushima, Hideo

    2012-12-01

    Surface-mediated gene transfer systems using apatite (Ap)-based composite layers have received increased attention in tissue engineering applications owing to their safety, biocompatibility and relatively high efficiency. In this study, DNA-antibody-apatite composite layers (DA-Ap layers), in which DNA and antibody molecules are immobilized within a matrix of apatite nanocrystals, were fabricated using a biomimetic coating process. They were then assayed for their gene transfer capability for application in a specific cell-targeted gene transfer. A DA-Ap layer that was fabricated with an anti-CD49f antibody showed a higher gene transfer capability to the CD49f-positive CHO-K1 cells than a DNA-apatite composite layer (D-Ap layer). The antibody facilitated the gene transfer capability of the DA-Ap layer only to the specific cells that were expressing corresponding antigens. When the DA-Ap layer was fabricated with an anti-N-cadherin antibody, a higher gene transfer capability compared with the D-Ap layer was found in the N-cadherin-positive P19CL6 cells, but not in the N-cadherin-negative UV♀2 cells or in the P19CL6 cells that were pre-blocked with anti-N-cadherin. Therefore, the antigen-antibody binding that takes place at the cell-layer interface should be responsible for the higher gene transfer capability of the DA-Ap than D-Ap layer. These results suggest that the DA-Ap layer works as a mediator in a specific cell-targeted gene transfer system.

  2. Fabrication of DNA-antibody-apatite composite layers for cell-targeted gene transfer.

    PubMed

    Yazaki, Yushin; Oyane, Ayako; Araki, Hiroko; Sogo, Yu; Ito, Atsuo; Yamazaki, Atsushi; Tsurushima, Hideo

    2012-12-01

    Surface-mediated gene transfer systems using apatite (Ap)-based composite layers have received increased attention in tissue engineering applications owing to their safety, biocompatibility and relatively high efficiency. In this study, DNA-antibody-apatite composite layers (DA-Ap layers), in which DNA and antibody molecules are immobilized within a matrix of apatite nanocrystals, were fabricated using a biomimetic coating process. They were then assayed for their gene transfer capability for application in a specific cell-targeted gene transfer. A DA-Ap layer that was fabricated with an anti-CD49f antibody showed a higher gene transfer capability to the CD49f-positive CHO-K1 cells than a DNA-apatite composite layer (D-Ap layer). The antibody facilitated the gene transfer capability of the DA-Ap layer only to the specific cells that were expressing corresponding antigens. When the DA-Ap layer was fabricated with an anti-N-cadherin antibody, a higher gene transfer capability compared with the D-Ap layer was found in the N-cadherin-positive P19CL6 cells, but not in the N-cadherin-negative UV♀2 cells or in the P19CL6 cells that were pre-blocked with anti-N-cadherin. Therefore, the antigen-antibody binding that takes place at the cell-layer interface should be responsible for the higher gene transfer capability of the DA-Ap than D-Ap layer. These results suggest that the DA-Ap layer works as a mediator in a specific cell-targeted gene transfer system.

  3. Gene transfer in inner ear cells: a challenging race.

    PubMed

    Sacheli, R; Delacroix, L; Vandenackerveken, P; Nguyen, L; Malgrange, B

    2013-03-01

    Recent advances in human genomics led to the identification of numerous defective genes causing deafness, which represent novel putative therapeutic targets. Future gene-based treatment of deafness resulting from genetic or acquired sensorineural hearing loss may include strategies ranging from gene therapy to antisense delivery. For successful development of gene therapies, a minimal requirement involves the engineering of appropriate gene carrier systems. Transfer of exogenous genetic material into the mammalian inner ear using viral or non-viral vectors has been characterized over the last decade. The nature of inner ear cells targeted, as well as the transgene expression level and duration, are highly dependent on the vector type, the route of administration and the strength of the promoter driving expression. This review summarizes and discusses recent advances in inner ear gene-transfer technologies aimed at examining gene function or identifying new treatment for inner ear disorders.

  4. Gene transfer as a future therapy for rheumatoid arthritis.

    PubMed

    Müller-Ladner, Ulf; Pap, Thomas; Gay, Renate E; Gay, Steffen

    2003-07-01

    Inhibiting key pathogenic processes within the rheumatoid synovium is a most attractive goal to achieve, and the number of potential intra- and extracellular pathways operative in rheumatoid arthritis (RA) that could be used for a gene therapy strategy is increasing continuously. Gene transfer or gene therapy might also be one of the approaches to solve the problem of long-term expression of therapeutic genes, in order to replace the frequent application of recombinant proteins, in the future. However, at present, gene therapy has not reached a realistic clinical stage, which is mainly due to severe side effects in humans, the complexity of RA pathophysiology and the current state of available gene transfer techniques. On the other hand, novel gene delivery systems are not restricted to vectors or certain types of cells, as mobile cells including macrophages, dendritic cells, lymphocytes and multipotent stem cells can also be used as smart gene transfer vehicles. Moreover, the observation in animal models that application of viral vectors into a joint can exert additional therapeutic effects in nearby joints might also facilitate the transfer from animal to human gene therapy. Future strategies will also examine the potential of novel long-term expression vectors such as lentiviruses and cytomegalovirus (CMV)-based viruses as a basis for future clinical trials in RA.

  5. The agricultural antibiotic carbadox induces phage-mediated gene transfer in Salmonella

    PubMed Central

    Bearson, Bradley L.; Allen, Heather K.; Brunelle, Brian W.; Lee, In Soo; Casjens, Sherwood R.; Stanton, Thaddeus B.

    2013-01-01

    Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the US during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli (STEC) and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness genes in the

  6. AAV8-mediated hepatic gene transfer in infant rhesus monkeys (Macaca mulatta).

    PubMed

    Wang, Lili; Bell, Peter; Lin, Jianping; Calcedo, Roberto; Tarantal, Alice F; Wilson, James M

    2011-11-01

    Many genetic metabolic diseases manifest in infancy, therefore, it is important to develop effective treatments that could be implemented at this time. Adeno-associated virus serotype 8 (AAV8) gene transfer has been studied in neonatal mouse, cat, and dog models and shown some efficacy with a single hepatic injection at birth. AAV8-mediated liver gene transfer has also generated sustained therapeutic effects in feline and canine models of lysosomal storage disorders. In these models, delaying the age of vector treatment increased gene transfer stability. The growth rate of infant nonhuman primates is more similar to the growth trajectory of humans, thus infant monkeys provide an excellent model to study AAV gene transfer efficiency, stability, and safety. In this study, we report for the first time that AAV8-mediated hepatic gene transfer in infant monkeys is safe and efficient but less stable when compared to adolescent animals. Infant monkeys administered AAV8 intravenously at 1 week postnatal age achieved up to 98% transduction of hepatocytes within 7 days of injection; however, there was significant dilution of genomes and loss of transgene expression 35 days postadministration. Delaying the injection to 1 month postnatal age did not improve stability of transduction but decreased the antibody response to AAV8 capsid.

  7. Muscle as a target for supplementary factor IX gene transfer.

    PubMed

    Hoffman, Brad E; Dobrzynski, Eric; Wang, Lixin; Hirao, Lauren; Mingozzi, Federico; Cao, Ou; Herzog, Roland W

    2007-07-01

    Immune responses to the factor IX (F.IX) transgene product are a concern in gene therapy for the X-linked bleeding disorder hemophilia B. The risk for such responses is determined by several factors, including the vector, target tissue, and others. Previously, we have demonstrated that hepatic gene transfer with adeno-associated viral (AAV) vectors can induce F.IX-specific immune tolerance. Muscle-derived F.IX expression, however, is limited by a local immune response. Here, skeletal muscle was investigated as a target for supplemental gene transfer. Given the low invasiveness of intramuscular injections, this route would be ideal for secondary gene transfer, thereby boosting levels of transgene expression. However, this is feasible only if immune tolerance established by compartmentalization of expression to the liver extends to other sites. Immune tolerance to human F.IX established by prior hepatic AAV-2 gene transfer was maintained after subsequent injection of AAV-1 or adenoviral vector into skeletal muscle, and tolerized mice failed to form antibodies or an interferon (IFN)-gamma(+) T cell response to human F.IX. A sustained increase in systemic transgene expression was obtained for AAV-1, whereas an increase after adenoviral gene transfer was transient. A CD8(+) T cell response specifically against adenovirus-transduced fibers was observed, suggesting that cytotoxic T cell responses against viral antigens were sufficient to eliminate expression in muscle. In summary, the data demonstrate that supplemental F.IX gene transfer to skeletal muscle does not break tolerance achieved by liver-derived expression. The approach is efficacious, if the vector for muscle gene transfer does not express immunogenic viral proteins.

  8. Stress transfer efficiency in model composites under dynamic loading

    NASA Astrophysics Data System (ADS)

    Koimtzoglou, C.; Kostopoulos, V.; Galiotis, C.

    The micromechanics of tension-tension fatigue loading in model single-fibre composite geometries is investigated in this paper. In an attempt to emulate the conditions encountered in full carbon fibre composites, the fibres were prestrained prior to the curing process to ensure that they were free of high residual compressive stresses as a result of resin shrinkage. The resulting specimens were grouped into two categories depending on the level of the initial fibre prestrain (case A low, case B high). The cyclic load is designed to be well below the endurance fatigue limit of the polymer matrix ( 0.6%), and to have a frequency low enough to avoid unwanted thermal post curing. Throughout the preparation procedure, as well as during fatigue loading, the fibre stress (strain) was constantly monitored by means of laser Raman spectroscopy. The fibre axial stress distributions at each fatigue step were converted to interfacial shear stress (ISS) distributions, from which important parameters such as the maximum ISS the system can accommodate, the transfer length for efficient stress built-up and the length required for the attainment of maximum ISS were obtained. The results showed that, up to 2×106 loading cycles, the main parameters which affected the stress transfer efficiency at the interface were the fibre fracture process itself and the viscoelastic behaviour of the matrix material.

  9. An ultra-efficient energy transfer beyond plasmonic light scattering

    SciTech Connect

    Fu, Sze-Ming; Zhong, Yan-Kai; Lin, Albert

    2014-11-14

    The energy transfer between nano-particles is of great importance for, solar cells, light-emitting diodes, nano-particle waveguides, and other photonic devices. This study shows through novel design and algorithm optimization, the energy transfer efficiency between plasmonic and dielectric nano-particles can be greatly improved. Using versatile designs including core-shell wrapping, supercells and dielectric mediated plasmonic scattering, 0.05 dB/μm attenuation can be achieved, which is 20-fold reduction over the baseline plasmonic nano-particle chain, and 8-fold reduction over the baseline dielectric nano-particle chain. In addition, it is also found that the dielectric nano-particle chains can actually be more efficient than the plasmonic ones, at their respective optimized geometry. The underlying physics is that although plasmonic nano-particles provide stronger coupling and field emission, the effect of plasmonic absorption loss is actually more dominant resulting in high attenuation. Finally, the group velocity for all design schemes proposed in this work is shown to be maintained above 0.4c, and it is found that the geometry optimization for transmission also boosts the group velocity.

  10. Effect of microfouling on heat-transfer efficiency

    SciTech Connect

    Little, B.; Berger, L.R.

    1980-01-01

    Field experiments, performed at Keahole Point, Hawaii and in the Gulf of Mexico, were designed to determine the relationship between decreased heat transfer efficiency and the accumulation of corrosion and/or biofouling films on heat exchanger surfaces. The sample tubes were maintained under conditions simulating those of an Ocean Thermal Energy Conversion (OTEC) system and data from the two sites have been compared. Seawater flowed through 2.54 (internal diameter) metal tubes at approximately 1.8m sec/sup -1/. Four types of tubes were used: 5052 Aluminum (A1), Grade 2 titanium (Ti), 90-10 copper-nickel (Cu-Ni) and Allegheny-Ludlum 6X stainless ssteel (SS). All surfaces were colonized by microorganisms, though colonization of the Cu-Ni surface was initially retarded. Total film weight was greatest for the Al and Cu-Ni surfaces which were characterized by corrosion as well as microbial fouling. The total organic carbon: total nitrogen ratios of the fouling films from Ti, Al, SS and Cu-Ni, 4.2, 4.0, 4.8 and 7.9 respectively, remained constant throughout the experiment. The degradation of heat transfer efficiency due to the formation of fouling layers on Ti and SS is neither linear nor a simple exponential function. A microfouling model is proposed for corrosion-resistant surfaces that is consistent with field observations.

  11. Global Analysis of Horizontal Gene Transfer in Fusarium verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The co-occurrence of microbes within plants and other specialized niches may facilitate horizontal gene transfer (HGT) affecting host-pathogen interactions. We recently identified fungal-to-fungal HGTs involving metabolic gene clusters. For a global analysis of HGTs in the maize pathogen Fusarium ve...

  12. Evolution of and horizontal gene transfer in the Endornavirus genus.

    PubMed

    Song, Dami; Cho, Won Kyong; Park, Sang-Ho; Jo, Yeonhwa; Kim, Kook-Hyung

    2013-01-01

    The transfer of genetic information between unrelated species is referred to as horizontal gene transfer. Previous studies have demonstrated that both retroviral and non-retroviral sequences have been integrated into eukaryotic genomes. Recently, we identified many non-retroviral sequences in plant genomes. In this study, we investigated the evolutionary origin and gene transfer of domains present in endornaviruses which are double-stranded RNA viruses. Using the available sequences for endornaviruses, we found that Bell pepper endornavirus-like sequences homologous to the glycosyltransferase 28 domain are present in plants, fungi, and bacteria. The phylogenetic analysis revealed the glycosyltransferase 28 domain of Bell pepper endornavirus may have originated from bacteria. In addition, two domains of Oryza sativa endornavirus, a glycosyltransferase sugar-binding domain and a capsular polysaccharide synthesis protein, also exhibited high similarity to those of bacteria. We found evidence that at least four independent horizontal gene transfer events for the glycosyltransferase 28 domain have occurred among plants, fungi, and bacteria. The glycosyltransferase sugar-binding domains of two proteobacteria may have been horizontally transferred to the genome of Thalassiosira pseudonana. Our study is the first to show that three glycome-related viral genes in the genus Endornavirus have been acquired from marine bacteria by horizontal gene transfer.

  13. Evolution of and Horizontal Gene Transfer in the Endornavirus Genus

    PubMed Central

    Park, Sang-Ho; Jo, Yeonhwa; Kim, Kook-Hyung

    2013-01-01

    The transfer of genetic information between unrelated species is referred to as horizontal gene transfer. Previous studies have demonstrated that both retroviral and non-retroviral sequences have been integrated into eukaryotic genomes. Recently, we identified many non-retroviral sequences in plant genomes. In this study, we investigated the evolutionary origin and gene transfer of domains present in endornaviruses which are double-stranded RNA viruses. Using the available sequences for endornaviruses, we found that Bell pepper endornavirus-like sequences homologous to the glycosyltransferase 28 domain are present in plants, fungi, and bacteria. The phylogenetic analysis revealed the glycosyltransferase 28 domain of Bell pepper endornavirus may have originated from bacteria. In addition, two domains of Oryza sativa endornavirus, a glycosyltransferase sugar-binding domain and a capsular polysaccharide synthesis protein, also exhibited high similarity to those of bacteria. We found evidence that at least four independent horizontal gene transfer events for the glycosyltransferase 28 domain have occurred among plants, fungi, and bacteria. The glycosyltransferase sugar-binding domains of two proteobacteria may have been horizontally transferred to the genome of Thalassiosira pseudonana. Our study is the first to show that three glycome-related viral genes in the genus Endornavirus have been acquired from marine bacteria by horizontal gene transfer. PMID:23667703

  14. Gene transfer agents: phage-like elements of genetic exchange

    PubMed Central

    Lang, Andrew S.; Zhaxybayeva, Olga; Beatty, J. Thomas

    2013-01-01

    Horizontal gene transfer is important in the evolution of bacterial and archaeal genomes. An interesting genetic exchange process is carried out by diverse phage-like gene transfer agents (GTAs) that are found in a wide range of prokaryotes. Although GTAs resemble phages, they lack the hallmark capabilities that define typical phages, and they package random pieces of the producing cell’s genome. In this Review, we discuss the defining characteristics of the GTAs that have been identified to date, along with potential functions for these agents and the possible evolutionary forces that act on the genes involved in their production. PMID:22683880

  15. Vector-mediated antibody gene transfer for infectious diseases.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2015-01-01

    This chapter discusses the emerging field of vector-mediated antibody gene transfer as an alternative vaccine for infectious disease, with a specific focus on HIV. However, this methodology need not be confined to HIV-1; the general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets like hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. This approach is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, via a recombinant adeno-associated virus (rAAV) vector; this in turn results in long-term endogenous antibody expression from the injected muscle that confers protective immunity. Vector-mediated antibody gene transfer can rapidly move existing, potent broadly cross-neutralizing HIV-1-specific antibodies into the clinic. The gene transfer products demonstrate a potency and breadth identical to the original product. This strategy eliminates the need for immunogen design and interaction with the adaptive immune system to generate protection, a strategy that so far has shown limited promise.

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

    PubMed Central

    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

  17. 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

    2015-08-20

    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.

  18. High frequency of horizontal gene transfer in the oceans.

    PubMed

    McDaniel, Lauren D; Young, Elizabeth; Delaney, Jennifer; Ruhnau, Fabian; Ritchie, Kim B; Paul, John H

    2010-10-01

    Oceanic bacteria perform many environmental functions, including biogeochemical cycling of many elements, metabolizing of greenhouse gases, functioning in oceanic food webs (microbial loop), and producing valuable natural products and viruses. We demonstrate that the widespread capability of marine bacteria to participate in horizontal gene transfer (HGT) in coastal and oceanic environments may be the result of gene transfer agents (GTAs), viral-like particles produced by α-Proteobacteria. We documented GTA-mediated gene transfer frequencies a thousand to a hundred million times higher than prior estimates of HGT in the oceans, with as high as 47% of the culturable natural microbial community confirmed as gene recipients. These findings suggest a plausible mechanism by which marine bacteria acquire novel traits, thus ensuring resilience in the face of environmental change.

  19. Emerging role of regulatory T cells in gene transfer.

    PubMed

    Cao, Ou; Furlan-Freguia, Christian; Arruda, Valder R; Herzog, Roland W

    2007-10-01

    Induction and maintenance of immune tolerance to therapeutic transgene products are key requirements for successful gene replacement therapies. Gene transfer may also be used to specifically induce immune tolerance and thereby augment other types of therapies. Similarly, gene therapies for treatment of autoimmune diseases are being developed in order to restore tolerance to self-antigens. Regulatory T cells have emerged as key players in many aspects of immune tolerance, and a rapidly increasing body of work documents induction and/or activation of regulatory T cells by gene transfer. Regulatory T cells may suppress antibody formation and cytotoxic T cell responses and may be critical for immune tolerance to therapeutic proteins. In this regard, CD4(+)CD25(+) regulatory T cells have been identified as important components of tolerance in several gene transfer protocols, including hepatic in vivo gene transfer. Augmentation of regulatory T cell responses should be a promising new tool to achieve tolerance and avoid immune-mediated rejection of gene therapy. During the past decade, it has become obvious that immune regulation is an important and integral component of tolerance to self-antigens and of many forms of induced tolerance. Gene therapy can only be successful if the immune system does not reject the therapeutic transgene product. Recent studies provide a rapidly growing body of evidence that regulatory T cells (T(reg)) are involved and often play a crucial role in tolerance to proteins expressed by means of gene transfer. This review seeks to provide an overview of these data and their implications for gene therapy.

  20. Characterization of Two Cysteine Transfer RNA Genes from Xenopus Laevis

    DTIC Science & Technology

    1984-07-12

    author hereby certifies that the use of any copyrighted material in the dissertation manuscript entitled: "Characterization of two cysteine tRNA genes...Uniformed Services University of the Health Sciences 11 ABSTRACT Title of Thesis: Characterization of Two Cysteine Transfer RNA Genes from Xenopus...method after constructing a set of deletions and reclonlng into the plasmid pUC 8. The DNA fragment is 1737 bp long and contains two cysteine tRNA genes

  1. Complexity of genetic sequences modified by horizontal gene transfer and degraded-DNA uptake

    NASA Astrophysics Data System (ADS)

    Tremberger, George; Dehipawala, S.; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.

    2015-09-01

    Horizontal gene transfer has been a major vehicle for efficient transfer of genetic materials among living species and could be one of the sources for noncoding DNA incorporation into a genome. Our previous study of lnc- RNA sequence complexity in terms of fractal dimension and information entropy shows a tight regulation among the studied genes in numerous diseases. The role of sequence complexity in horizontal transferred genes was investigated with Mealybug in symbiotic relation with a 139K genome microbe and Deinococcus radiodurans as examples. The fractal dimension and entropy showed correlation R-sq of 0.82 (N = 6) for the studied Deinococcus radiodurans sequences. For comparison the Deinococcus radiodurans oxidative stress tolerant catalase and superoxide dismutase genes under extracellular dGMP growth condition showed R-sq ~ 0.42 (N = 6); and the studied arsenate reductase horizontal transferred genes for toxicity survival in several microorganisms showed no correlation. Simulation results showed that R-sq < 0.4 would be improbable at less than one percent chance, suggestive of additional selection pressure when compared to the R-sq ~ 0.29 (N = 21) in the studied transferred genes in Mealybug. The mild correlation of R-sq ~ 0.5 for fractal dimension versus transcription level in the studied Deinococcus radiodurans sequences upon extracellular dGMP growth condition would suggest that lower fractal dimension with less electron density fluctuation favors higher transcription level.

  2. The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro.

    PubMed

    Stern, M; Caplen, N J; Browning, J E; Griesenbach, U; Sorgi, F; Huang, L; Gruenert, D C; Marriot, C; Crystal, R G; Geddes, D M; Alton, E W

    1998-01-01

    Trials of gene transfer for cystic fibrosis (CF) are currently underway. However, direct application to the airways may be impeded by the presence of airway secretions. We have therefore assessed the effect of CF sputum on the expression of the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE in a number of cell lines in vitro. Transfection was markedly inhibited in the presence of sputum; the effect was concentration dependent and was only partially ameliorated by removal of sputum with phosphate-buffered saline (PBS) washing before gene transfer. However, treatment of the sputum-covered cells with recombinant human DNase (rhDNase, 50 micrograms/ml) but not with N-acetylcysteine, Nacystelyn, lysine (all 20 mM) or recombinant alginase (0.5 U/ml) significantly (P < 0.005) improved gene transfer. Adenovirus-mediated gene transfer efficiency in the presence of sputum was similarly inhibited, and again, treatment with rhDNase before transfection significantly improved gene transfer (P < 0.005). Transfection of Cos 7 cells in the presence of exogenous genomic DNA alone demonstrated similar inhibition to that observed with sputum and was also ameliorated by pre-treatment of DNA-covered cells with rhDNase. In a separate series of experiments performed in the absence of added sputum or genomic DNA, increasing concentrations of rhDNase resulted in a concentration-related decline in transfection efficiency. However, even at the highest concentration (500 micrograms/ml of rhDNase), transfection efficiency remained more than 50% of control. Thus, pre-treatment of CF airways with rhDNase may be appropriate before liposome or adenovirus-mediated gene therapy.

  3. Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium.

    PubMed

    Ferrari, S; Kitson, C; Farley, R; Steel, R; Marriott, C; Parkins, D A; Scarpa, M; Wainwright, B; Evans, M J; Colledge, W H; Geddes, D M; Alton, E W

    2001-09-01

    Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal.

  4. A novel glutathione modified chitosan conjugate for efficient gene delivery.

    PubMed

    Li, Congxin; Guo, Tianying; Zhou, Dezhong; Hu, Yuling; Zhou, Hao; Wang, Shufang; Chen, Jiatong; Zhang, Zhengpu

    2011-09-05

    A novel non-viral gene vector based on poly[poly(ethylene glycol) methacrylate] (PMPEG) and l-glutathione (GSH) grafted chitosan (CS) has been fabricated. First, well-defined brush-like PMPEG living polymers with dithioester residues were prepared by the reversible addition-fragmentation chain transfer (RAFT) polymerization and grafted onto the allylchitosan via radical coupling method. Then, the tripeptide GSH was introduced onto the end of PMPEG chain to give a CS-PMPEG-GSH conjugate. In comparison with pristine chitosan, CS-PMPEG-GSH conjugate could not only condense plasmid DNA (pDNA) and prevent the condensed CS-PMPEG-GSH/pDNA nanoparticle self-aggregation, but also increase the binding ability to cell membrane efficiently and improve decondensed ability of pDNA from the nanoparticles in cytoplasm which thus has resulted in the higher transfection efficiency in mouse embryonic fibroblast cells (NIH3T3). In addition, cytotoxicity assays showed that the conjugate is less cytotoxic than CS, and still retain the cationic polyelectrolyte characteristic as chitosan. These results indicate that the non-viral vector is a promising candidate for gene therapy in clinical application.

  5. Lateral Transfer of Genes and Gene Fragments in Staphylococcus Extends beyond Mobile Elements ▿ †

    PubMed Central

    Chan, Cheong Xin; Beiko, Robert G.; Ragan, Mark A.

    2011-01-01

    The widespread presence of antibiotic resistance and virulence among Staphylococcus isolates has been attributed in part to lateral genetic transfer (LGT), but little is known about the broader extent of LGT within this genus. Here we report the first systematic study of the modularity of genetic transfer among 13 Staphylococcus genomes covering four distinct named species. Using a topology-based phylogenetic approach, we found, among 1,354 sets of homologous genes examined, strong evidence of LGT in 368 (27.1%) gene sets, and weaker evidence in another 259 (19.1%). Within-gene and whole-gene transfer contribute almost equally to the topological discordance of these gene sets against a reference phylogeny. Comparing genetic transfer in single-copy and in multicopy gene sets, we observed a higher frequency of LGT in the latter, and a substantial functional bias in cases of whole-gene transfer (little such bias was observed in cases of fragmentary genetic transfer). We found evidence that lateral transfer, particularly of entire genes, impacts not only functions related to antibiotic, drug, and heavy-metal resistance, as well as membrane transport, but also core informational and metabolic functions not associated with mobile elements. Although patterns of sequence similarity support the cohesion of recognized species, LGT within S. aureus appears frequently to disrupt clonal complexes. Our results demonstrate that LGT and gene duplication play important parts in functional innovation in staphylococcal genomes. PMID:21622749

  6. RANGE: Gene Transfer of Reversibly Controlled Polycistronic Genes

    PubMed Central

    Chen, Yiwei; Cao, Liji; Luo, Chonglin; Ditzel, Désirée AW; Peter, Jörg; Sprengel, Rolf

    2013-01-01

    We developed a single vector recombinant adeno-associated viral (rAAV) expression system for spatial and reversible control of polycistronic gene expression. Our approach (i) integrates the advantages of the tetracycline (Tet)-controlled transcriptional silencer tTSKid and the self-cleaving 2A peptide bridge, (ii) combines essential regulatory components as an autoregulatory loop, (iii) simplifies the gene delivery scheme, and (iv) regulates multiple genes in a synchronized manner. Controlled by an upstream Tet-responsive element (TRE), both the ubiquitous chicken β-actin promoter (CAG) and the neuron-specific synapsin-1 promoter (Syn) could regulate expression of tTSKid together with two 2A-linked reporter genes. Transduction in vitro exhibited maximally 50-fold regulation by doxycycline (Dox). Determined by gene delivery method as well as promoter, highly specific tissues were transduced in vivo. Bioluminescence imaging (BLI) visualized reversible “ON/OFF” gene switches over repeated “Doxy-Cycling” in living mice. Thus, the reversible rAAV-mediated N-cistronic gene expression system, termed RANGE, may serve as a versatile tool to achieve reversible polycistronic gene regulation for the study of gene function as well as gene therapy. PMID:23571608

  7. Horizontal functional gene transfer from bacteria to fishes

    PubMed Central

    Sun, Bao-Fa; Li, Tong; Xiao, Jin-Hua; Jia, Ling-Yi; Liu, Li; Zhang, Peng; Murphy, Robert W.; He, Shun-Min; Huang, Da-Wei

    2015-01-01

    Invertebrates can acquire functional genes via horizontal gene transfer (HGT) from bacteria but fishes are not known to do so. We provide the first reliable evidence of one HGT event from marine bacteria to fishes. The HGT appears to have occurred after emergence of the teleosts. The transferred gene is expressed and regulated developmentally. Its successful integration and expression may change the genetic and metabolic repertoire of fishes. In addition, this gene contains conserved domains and similar tertiary structures in fishes and their putative donor bacteria. Thus, it may function similarly in both groups. Evolutionary analyses indicate that it evolved under purifying selection, further indicating its conserved function. We document the first likely case of HGT of functional gene from prokaryote to fishes. This discovery certifies that HGT can influence vertebrate evolution. PMID:26691285

  8. Horizontal functional gene transfer from bacteria to fishes.

    PubMed

    Sun, Bao-Fa; Li, Tong; Xiao, Jin-Hua; Jia, Ling-Yi; Liu, Li; Zhang, Peng; Murphy, Robert W; He, Shun-Min; Huang, Da-Wei

    2015-12-22

    Invertebrates can acquire functional genes via horizontal gene transfer (HGT) from bacteria but fishes are not known to do so. We provide the first reliable evidence of one HGT event from marine bacteria to fishes. The HGT appears to have occurred after emergence of the teleosts. The transferred gene is expressed and regulated developmentally. Its successful integration and expression may change the genetic and metabolic repertoire of fishes. In addition, this gene contains conserved domains and similar tertiary structures in fishes and their putative donor bacteria. Thus, it may function similarly in both groups. Evolutionary analyses indicate that it evolved under purifying selection, further indicating its conserved function. We document the first likely case of HGT of functional gene from prokaryote to fishes. This discovery certifies that HGT can influence vertebrate evolution.

  9. Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus

    PubMed Central

    Duan, Dongsheng; Yue, Yongping; Yan, Ziying; Yang, Jusan; Engelhardt, John F.

    2000-01-01

    The restriction of viral receptors and coreceptors to the basolateral surface of airway epithelial cells has been blamed for the inefficient transfer of viral vectors to the apical surface of this tissue. We now report, however, that differentiated human airway epithelia internalize rAAV type-2 virus efficiently from their apical surfaces, despite the absence of known adeno-associated virus–2 (AAV-2) receptors or coreceptors at these sites. The dramatically lower transduction efficiency of rAAV infection from the apical surface of airway cells appears to result instead from differences in endosomal processing and nuclear trafficking of apically or basolaterally internalized virions. AAV capsid proteins are ubiquitinated after endocytosis, and gene transfer can be significantly enhanced by proteasome or ubiquitin ligase inhibitors. Tripeptide proteasome inhibitors increased persistent rAAV gene delivery from the apical surface >200-fold, to a level nearly equivalent to that achieved with basolateral infection. In vivo application of proteasome inhibitor in mouse lung augmented rAAV gene transfer from undetectable levels to a mean of 10.4 ± 1.6% of the epithelial cells in large bronchioles. Proteasome inhibitors also increased rAAV-2–mediated gene transfer to the liver tenfold, but they did not affect transduction of skeletal or cardiac muscle. These findings suggest that tissue-specific ubiquitination of viral capsid proteins interferes with rAAV-2 transduction and provides new approaches to circumvent this barrier for gene therapy of diseases such as cystic fibrosis. PMID:10841516

  10. Occurrence and expression of gene transfer agent genes in marine bacterioplankton.

    PubMed

    Biers, Erin J; Wang, Kui; Pennington, Catherine; Belas, Robert; Chen, Feng; Moran, Mary Ann

    2008-05-01

    Genes with homology to the transduction-like gene transfer agent (GTA) were observed in genome sequences of three cultured members of the marine Roseobacter clade. A broader search for homologs for this host-controlled virus-like gene transfer system identified likely GTA systems in cultured Alphaproteobacteria, and particularly in marine bacterioplankton representatives. Expression of GTA genes and extracellular release of GTA particles ( approximately 50 to 70 nm) was demonstrated experimentally for the Roseobacter clade member Silicibacter pomeroyi DSS-3, and intraspecific gene transfer was documented. GTA homologs are surprisingly infrequent in marine metagenomic sequence data, however, and the role of this lateral gene transfer mechanism in ocean bacterioplankton communities remains unclear.

  11. HST WFC3/UVIS: charge transfer efficiency monitoring and mitigation

    NASA Astrophysics Data System (ADS)

    Baggett, Sylvia M.; Sosey, Megan L.; Anderson, Jay; Gosmeyer, Catherine; Bourque, Matthew; Bajaj, Varun; Khandrika, Harish G.; Martlin, Catherine; Kozhurina-Platais, Vera; Sabbi, Elena; WFC3 Team

    2016-01-01

    The harsh low-earth orbit environment is known to damage CCD devices and the HST WFC3/UVIS camera is no exception. One consequence of the radiation damage is charge-transfer efficiency (CTE) loss over time. We summarize the level of the CTE losses, the effect on science data, and the pre- and post-observation mitigation options available. Among them is the pixel-based CTE correction, which has been incorporated into the HST automatic data processing pipeline. The pipeline now provides both standard and CTE-corrected data products; observers with older data can re-retrieve their images via the the Mikulski Archive for Space Telescopes (MAST) to obtain the new products.

  12. Recent events dominate interdomain lateral gene transfers between prokaryotes and eukaryotes and, with the exception of endosymbiotic gene transfers, few ancient transfer events persist

    PubMed Central

    Katz, Laura A.

    2015-01-01

    While there is compelling evidence for the impact of endosymbiotic gene transfer (EGT; transfer from either mitochondrion or chloroplast to the nucleus) on genome evolution in eukaryotes, the role of interdomain transfer from bacteria and/or archaea (i.e. prokaryotes) is less clear. Lateral gene transfers (LGTs) have been argued to be potential sources of phylogenetic information, particularly for reconstructing deep nodes that are difficult to recover with traditional phylogenetic methods. We sought to identify interdomain LGTs by using a phylogenomic pipeline that generated 13 465 single gene trees and included up to 487 eukaryotes, 303 bacteria and 118 archaea. Our goals include searching for LGTs that unite major eukaryotic clades, and describing the relative contributions of LGT and EGT across the eukaryotic tree of life. Given the difficulties in interpreting single gene trees that aim to capture the approximately 1.8 billion years of eukaryotic evolution, we focus on presence–absence data to identify interdomain transfer events. Specifically, we identify 1138 genes found only in prokaryotes and representatives of three or fewer major clades of eukaryotes (e.g. Amoebozoa, Archaeplastida, Excavata, Opisthokonta, SAR and orphan lineages). The majority of these genes have phylogenetic patterns that are consistent with recent interdomain LGTs and, with the notable exception of EGTs involving photosynthetic eukaryotes, we detect few ancient interdomain LGTs. These analyses suggest that LGTs have probably occurred throughout the history of eukaryotes, but that ancient events are not maintained unless they are associated with endosymbiotic gene transfer among photosynthetic lineages. PMID:26323756

  13. Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation.

    PubMed

    Keen, Eric C; Bliskovsky, Valery V; Malagon, Francisco; Baker, James D; Prince, Jeffrey S; Klaus, James S; Adhya, Sankar L

    2017-01-17

    Bacteriophages infect an estimated 10(23) to 10(25) bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub "superspreaders," releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications.

  14. The interconnection between biofilm formation and horizontal gene transfer.

    PubMed

    Madsen, Jonas Stenløkke; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2012-07-01

    Recent research has revealed that horizontal gene transfer and biofilm formation are connected processes. Although published research investigating this interconnectedness is still limited, we will review this subject in order to highlight the potential of these observations because of their believed importance in the understanding of the adaptation and subsequent evolution of social traits in bacteria. Here, we discuss current evidence for such interconnectedness centred on plasmids. Horizontal transfer rates are typically higher in biofilm communities compared with those in planktonic states. Biofilms, furthermore, promote plasmid stability and may enhance the host range of mobile genetic elements that are transferred horizontally. Plasmids, on the other hand, are very well suited to promote the evolution of social traits such as biofilm formation. This, essentially, transpires because plasmids are independent replicons that enhance their own success by promoting inter-bacterial interactions. They typically also carry genes that heighten their hosts' direct fitness. Furthermore, current research shows that the so-called mafia traits encoded on mobile genetic elements can enforce bacteria to maintain stable social interactions. It also indicates that horizontal gene transfer ultimately enhances the relatedness of bacteria carrying the mobile genetic elements of the same origin. The perspective of this review extends to an overall interconnectedness between horizontal gene transfer, mobile genetic elements and social evolution of bacteria.

  15. [Gene doping: gene transfer and possible molecular detection].

    PubMed

    Argüelles, Carlos Francisco; Hernández-Zamora, Edgar

    2007-01-01

    The use of illegal substances in sports to enhance athletic performance during competition has caused international sports organizations such as the COI and WADA to take anti doping measures. A new doping method know as gene doping is defined as "the non-therapeutic use of genes, genetic elements and/or cells that have the capacity to enhance athletic performance". However, gene doping in sports is not easily identified and can cause serious consequences. Molecular biology techniques are needed in order to distinguish the difference between a "normal" and an "altered" genome. Further, we need to develop new analytic methods and biological molecular techniques in anti-doping laboratories, and design programs that avoid the non therapeutic use of genes.

  16. Antibacterial gene transfer across the tree of life

    PubMed Central

    Metcalf, Jason A; Funkhouser-Jones, Lisa J; Brileya, Kristen; Reysenbach, Anna-Louise; Bordenstein, Seth R

    2014-01-01

    Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group. DOI: http://dx.doi.org/10.7554/eLife.04266.001 PMID:25422936

  17. Phosphatidylserine immobilization of lentivirus for localized gene transfer

    PubMed Central

    Shin, Seungjin; Tuinstra, Hannah M.; Salvay, David M.; Shea, Lonnie D.

    2010-01-01

    Localized and efficient gene transfer can be promoted by exploiting the interaction between the vector and biomaterial. Regulation of the vector-material interaction was investigated by capitalizing on the binding between lentivirus and phosphatidylserine (PS), a component of the plasma membrane. PS was incorporated into microspheres composed of the copolymers of lactide and glycolide (PLG) using an emulsion process. Increasing the weight ratio of PS to PLG led to a greater incorporation of PS. Lentivirus, but not adenovirus, associated with PS-PLG microspheres, and binding was specific to PS relative to PLG alone or PLG modified with phosphatidylcholine. Immobilized lentivirus produced large numbers of transduced cells, and increased transgene expression relative to virus alone. Microspheres were subsequently formed into porous tissue engineering scaffolds, with retention of lentivirus binding. Lentivirus immobilization resulted in long-term and localized expression within a subcutaneously implanted scaffold. Microspheres were also formed into multiple channel bridges for implantation into the spinal cord. Lentivirus delivery from the bridge produced maximal expression at the implant and a gradient of expression rostrally and caudally. This specific binding of lentiviral vectors to biomaterial scaffolds may provide a versatile tool for numerous applications in regenerative medicine or within model systems that investigate tissue development. PMID:20206382

  18. [Polymeric nanoparticles with therapeutic gene for gene therapy: I. Preparation and in vivo gene transfer study].

    PubMed

    Yang, Jing; Song, Cunxian; Sun, Hongfan; Wu, Li; Tang, Lina; Leng, Xigang; Wang, Pengyan; Xu, Yiyao; Li, Yongjun; Guan, Heng

    2005-06-01

    VEGF nanoparticle (VEGF-NP) was prepared by a multi-emulsification technique using a biodegradable poly-dl-lactic-co-glycolic (PLGA) as matrix material. The nanoparticles were characterized for size, VEGF loading capacity, and in vitro release. VEGF-NP and naked VEGF plasmid were intramuscularly injected into the ischemia site of the rabbit chronic hindlimb ischemia model and the efficiency of VEGF-NP as gene delivery carrier for gene therapy in animal model was evaluated. Gene therapuetic effect was assessed evaluated by RT-PCR, immunohistochemistry and angiography assay. The average size of VEGF-NP was around 300 nm. The encapsulation efficiency of VEGF was above 96%. Loading amount of VEGF in the nanoparticles was about 4%. In vitro, nanoparticles maintained sustained-release of VEGF for two weeks. Two weeks post gene injection the capillary density in VEGF-NP group (81.22 per mm2) was significantly higher than that in control group (29.54 mm2). RT-PCR results showed greatly higher VEGF expression in VEGF-NP group (31.79au * mm) than that in naked VEGF group (9.15 au * mm). As a carrier system for gene therapy in animal model, VEGF-NP is much better than naked DNA plasmid. The results demonstrate great possibility of using NP carrier in human gene therapy.

  19. Replacing and Additive Horizontal Gene Transfer in Streptococcus

    PubMed Central

    Choi, Sang Chul; Rasmussen, Matthew D.; Hubisz, Melissa J.; Gronau, Ilan; Stanhope, Michael J.; Siepel, Adam

    2012-01-01

    The prominent role of Horizontal Gene Transfer (HGT) in the evolution of bacteria is now well documented, but few studies have differentiated between evolutionary events that predominantly cause genes in one lineage to be replaced by homologs from another lineage (“replacing HGT”) and events that result in the addition of substantial new genomic material (“additive HGT”). Here in, we make use of the distinct phylogenetic signatures of replacing and additive HGTs in a genome-wide study of the important human pathogen Streptococcus pyogenes (SPY) and its close relatives S. dysgalactiae subspecies equisimilis (SDE) and S. dysgalactiae subspecies dysgalactiae (SDD). Using recently developed statistical models and computational methods, we find evidence for abundant gene flow of both kinds within each of the SPY and SDE clades and of reduced levels of exchange between SPY and SDD. In addition, our analysis strongly supports a pronounced asymmetry in SPY–SDE gene flow, favoring the SPY-to-SDE direction. This finding is of particular interest in light of the recent increase in virulence of pathogenic SDE. We find much stronger evidence for SPY–SDE gene flow among replacing than among additive transfers, suggesting a primary influence from homologous recombination between co-occurring SPY and SDE cells in human hosts. Putative virulence genes are correlated with transfer events, but this correlation is found to be driven by additive, not replacing, HGTs. The genes affected by additive HGTs are enriched for functions having to do with transposition, recombination, and DNA integration, consistent with previous findings, whereas replacing HGTs seen to influence a more diverse set of genes. Additive transfers are also found to be associated with evidence of positive selection. These findings shed new light on the manner in which HGT has shaped pathogenic bacterial genomes. PMID:22617954

  20. Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes

    PubMed Central

    Huddleston, Jennifer R

    2014-01-01

    Bacterial infections are becoming increasingly difficult to treat due to widespread antibiotic resistance among pathogens. This review aims to give an overview of the major horizontal transfer mechanisms and their evolution and then demonstrate the human lower gastrointestinal tract as an environment in which horizontal gene transfer of resistance determinants occurs. Finally, implications for antibiotic usage and the development of resistant infections and persistence of antibiotic resistance genes in populations as a result of horizontal gene transfer in the large intestine will be discussed. PMID:25018641

  1. Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment

    PubMed Central

    JACOBS, WILLIAM R.

    2016-01-01

    Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids—chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages—was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research. “I had to know my enemy in order to prevail against him.”Nelson Mandela PMID:26105819

  2. Plasmid and clonal interference during post horizontal gene transfer evolution.

    PubMed

    Bedhomme, S; Perez Pantoja, D; Bravo, I G

    2017-02-16

    Plasmids are nucleic acid molecules that can drive their own replication in a living cell. They can be transmitted horizontally and can thrive in the host cell to high-copy numbers. Plasmid replication and gene expression consume cellular resources and cells carrying plasmids incur fitness costs. But many plasmids carry genes that can be beneficial under certain conditions, allowing the cell to endure in the presence of antibiotics, toxins, competitors or parasites. Horizontal transfer of plasmid-encoded genes can thus instantaneously confer differential adaptation to local or transient selection conditions. This conflict between cellular fitness and plasmid spread sets the scene for multilevel selection processes. We have engineered a system to study the short-term evolutionary impact of different synonymous versions of a plasmid-encoded antibiotic resistance gene. Applying experimental evolution under different selection conditions and deep sequencing allowed us to show rapid local adaptation to the presence of antibiotic and to the specific version of the resistance gene transferred. We describe the presence of clonal interference at two different levels: at the within-cell level, because a single cell can carry several plasmids, and at the between-cell level, because a bacterial population may contain several clones carrying different plasmids and displaying different fitness in the presence/absence of antibiotic. Understanding the within-cell and between-cell dynamics of plasmids after horizontal gene transfer is essential to unravel the dense network of mobile elements underlying the worldwide threat to public health of antibiotic resistance.

  3. Horizontal gene transfer in eukaryotes: The weak-link model

    PubMed Central

    Huang, Jinling

    2013-01-01

    The significance of horizontal gene transfer (HGT) in eukaryotic evolution remains controversial. Although many eukaryotic genes are of bacterial origin, they are often interpreted as being derived from mitochondria or plastids. Because of their fixed gene pool and gene loss, however, mitochondria and plastids alone cannot adequately explain the presence of all, or even the majority, of bacterial genes in eukaryotes. Available data indicate that no insurmountable barrier to HGT exists, even in complex multicellular eukaryotes. In addition, the discovery of both recent and ancient HGT events in all major eukaryotic groups suggests that HGT has been a regular occurrence throughout the history of eukaryotic evolution. A model of HGT is proposed that suggests both unicellular and early developmental stages as likely entry points for foreign genes into multicellular eukaryotes. PMID:24037739

  4. Horizontal gene transfer in eukaryotes: the weak-link model.

    PubMed

    Huang, Jinling

    2013-10-01

    The significance of horizontal gene transfer (HGT) in eukaryotic evolution remains controversial. Although many eukaryotic genes are of bacterial origin, they are often interpreted as being derived from mitochondria or plastids. Because of their fixed gene pool and gene loss, however, mitochondria and plastids alone cannot adequately explain the presence of all, or even the majority, of bacterial genes in eukaryotes. Available data indicate that no insurmountable barrier to HGT exists, even in complex multicellular eukaryotes. In addition, the discovery of both recent and ancient HGT events in all major eukaryotic groups suggests that HGT has been a regular occurrence throughout the history of eukaryotic evolution. A model of HGT is proposed that suggests both unicellular and early developmental stages as likely entry points for foreign genes into multicellular eukaryotes.

  5. Bacterial Genes in the Aphid Genome: Absence of Functional Gene Transfer from Buchnera to Its Host

    PubMed Central

    Nikoh, Naruo; McCutcheon, John P.; Kudo, Toshiaki; Miyagishima, Shin-ya; Moran, Nancy A.; Nakabachi, Atsushi

    2010-01-01

    Genome reduction is typical of obligate symbionts. In cellular organelles, this reduction partly reflects transfer of ancestral bacterial genes to the host genome, but little is known about gene transfer in other obligate symbioses. Aphids harbor anciently acquired obligate mutualists, Buchnera aphidicola (Gammaproteobacteria), which have highly reduced genomes (420–650 kb), raising the possibility of gene transfer from ancestral Buchnera to the aphid genome. In addition, aphids often harbor other bacteria that also are potential sources of transferred genes. Previous limited sampling of genes expressed in bacteriocytes, the specialized cells that harbor Buchnera, revealed that aphids acquired at least two genes from bacteria. The newly sequenced genome of the pea aphid, Acyrthosiphon pisum, presents the first opportunity for a complete inventory of genes transferred from bacteria to the host genome in the context of an ancient obligate symbiosis. Computational screening of the entire A. pisum genome, followed by phylogenetic and experimental analyses, provided strong support for the transfer of 12 genes or gene fragments from bacteria to the aphid genome: three LD–carboxypeptidases (LdcA1, LdcA2,ψLdcA), five rare lipoprotein As (RlpA1-5), N-acetylmuramoyl-L-alanine amidase (AmiD), 1,4-beta-N-acetylmuramidase (bLys), DNA polymerase III alpha chain (ψDnaE), and ATP synthase delta chain (ψAtpH). Buchnera was the apparent source of two highly truncated pseudogenes (ψDnaE and ψAtpH). Most other transferred genes were closely related to genes from relatives of Wolbachia (Alphaproteobacteria). At least eight of the transferred genes (LdcA1, AmiD, RlpA1-5, bLys) appear to be functional, and expression of seven (LdcA1, AmiD, RlpA1-5) are highly upregulated in bacteriocytes. The LdcAs and RlpAs appear to have been duplicated after transfer. Our results excluded the hypothesis that genome reduction in Buchnera has been accompanied by gene transfer to the host

  6. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  7. Adenoviral-mediated imaging of gene transfer using a somatostatin receptor-cytosine deaminase fusion protein.

    PubMed

    Lears, K A; Parry, J J; Andrews, R; Nguyen, K; Wadas, T J; Rogers, B E

    2015-03-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy owing to the enzyme's ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that both the SSTR2 and yCD were functional in binding assays, conversion assays and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy.

  8. Antibiotics and gene transfer in swine gut bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mammalian gastrointestinal (GI) tract hosts a diverse collection bacteria, most of which are beneficial for host health. This bacterial community also supports a community of viruses that infect bacteria (called bacteriophages or phages). Phages transfer genes between bacteria, and phage-media...

  9. Quasispecies theory for horizontal gene transfer and recombination

    NASA Astrophysics Data System (ADS)

    Muñoz, Enrique; Park, Jeong-Man; Deem, Michael W.

    2008-12-01

    We introduce a generalization of the parallel, or Crow-Kimura, and Eigen models of molecular evolution to represent the exchange of genetic information between individuals in a population. We study the effect of different schemes of genetic recombination on the steady-state mean fitness and distribution of individuals in the population, through an analytic field theoretic mapping. We investigate both horizontal gene transfer from a population and recombination between pairs of individuals. Somewhat surprisingly, these nonlinear generalizations of quasispecies theory to modern biology are analytically solvable. For two-parent recombination, we find two selected phases, one of which is spectrally rigid. We present exact analytical formulas for the equilibrium mean fitness of the population, in terms of a maximum principle, which are generally applicable to any permutation invariant replication rate function. For smooth fitness landscapes, we show that when positive epistatic interactions are present, recombination or horizontal gene transfer introduces a mild load against selection. Conversely, if the fitness landscape exhibits negative epistasis, horizontal gene transfer or recombination introduces an advantage by enhancing selection towards the fittest genotypes. These results prove that the mutational deterministic hypothesis holds for quasispecies models. For the discontinuous single sharp peak fitness landscape, we show that horizontal gene transfer has no effect on the fitness, while recombination decreases the fitness, for both the parallel and the Eigen models. We present numerical and analytical results as well as phase diagrams for the different cases.

  10. "Active" cancer immunotherapy by anti-Met antibody gene transfer.

    PubMed

    Vigna, Elisa; Pacchiana, Giovanni; Mazzone, Massimiliano; Chiriaco, Cristina; Fontani, Lara; Basilico, Cristina; Pennacchietti, Selma; Comoglio, Paolo M

    2008-11-15

    Gene therapy provides a still poorly explored opportunity to treat cancer by "active" immunotherapy as it enables the transfer of genes encoding antibodies directed against specific oncogenic proteins. By a bidirectional lentiviral vector, we transferred the cDNA encoding the heavy and light chains of a monoclonal anti-Met antibody (DN-30) to epithelial cancer cells. In vitro, the transduced cells synthesized and secreted correctly assembled antibodies with the expected high affinity, inducing down-regulation of the Met receptor and strong inhibition of the invasive growth response. The inhibitory activity resulted (a) from the interference of the antibody with the Met receptor intracellular processing ("cell autonomous activity," in cis) and (b) from the antibody-induced cleavage of Met expressed at the cell surface ("bystander effect," in trans). The monoclonal antibody gene transferred into live animals by systemic administration or by local intratumor delivery resulted in substantial inhibition of tumor growth. These data provide proof of concept both for targeting the Met receptor and for a gene transfer-based immunotherapy strategy.

  11. Horizontal gene transfer and the evolution of methanogenic pathways.

    PubMed

    Fournier, Greg

    2009-01-01

    Horizontal gene transfer (HGT) is a driving force in the evolution of metabolic pathways, allowing novel enzymatic functions that provide a selective advantage to be rapidly incorporated into an organism's physiology. Here, the role of two HGT events in the evolution of methanogenesis is described. First, the acetoclastic sub-pathway of methanogenesis is shown to have evolved via a transfer of the ackA and pta genes from a cellulolytic clostridia to a family of methanogenic archaea. Second, the system for encoding the amino acid pyrrolysine, used for the synthesis of enzymes for methanogenesis from methylamines, is shown to likely have evolved via transfer from an ancient, unknown, deeply branching organismal lineage.

  12. Gene transfer in the liver using recombinant adeno-associated virus

    PubMed Central

    Ahmed, Seemin Seher; Li, Jia; Godwin, Jonathan; Gao, Guangping; Zhong, Li

    2013-01-01

    Liver-directed gene transfer and gene therapy are rapidly gaining attention primarily because the liver is centrally involved in a variety of metabolic functions that are affected in various inherited disorders. Recombinant adeno-associated virus (rAAV) is a popular gene delivery vehicle for gene therapy and intravenous delivery of some rAAV serotypes results in very efficient transduction of the liver. rAAV-mediated and liver-directed gene transfer can help in creating somatic transgenic animals or disease models and studying the function of various genes and miRNAs. The liver is the target tissue for gene therapy of many inborn metabolic diseases and may also be exploited as a “bio-factory” for the production of coagulation factors, insulin and growth hormones and other non-hepatic proteins. Hence efficient delivery of transgenes and small RNAs to the liver by rAAV vectors has been of long-standing interest to research scientists and clinicians alike. PMID:23686826

  13. Efficient, crosswise catalytic promiscuity among enzymes that catalyze phosphoryl transfer.

    PubMed

    Mohamed, Mark F; Hollfelder, Florian

    2013-01-01

    The observation that one enzyme can accelerate several chemically distinct reactions was at one time surprising because the enormous efficiency of catalysis was often seen as inextricably linked to specialization for one reaction. Originally underreported, and considered a quirk rather than a fundamental property, enzyme promiscuity is now understood to be important as a springboard for adaptive evolution. Owing to the large number of promiscuous enzymes that have been identified over the last decade, and the increased appreciation for promiscuity's evolutionary importance, the focus of research has shifted to developing a better understanding of the mechanistic basis for promiscuity and the origins of tolerant or restrictive specificity. We review the evidence for widespread crosswise promiscuity amongst enzymes that catalyze phosphoryl transfer, including several members of the alkaline phosphatase superfamily, where large rate accelerations between 10(6) and 10(17) are observed for both native and multiple promiscuous reactions. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.

  14. Biased gene transfer mimics patterns created through shared ancestry

    PubMed Central

    Andam, Cheryl P.; Williams, David; Gogarten, J. Peter

    2010-01-01

    In phylogenetic reconstruction, two types of bacterial tyrosyl-tRNA synthetases (TyrRS) form distinct clades with many bacterial phyla represented in both clades. Very few taxa possess both forms, and maximum likelihood analysis of the distribution of TyrRS types suggests horizontal gene transfer (HGT), rather than an ancient duplication followed by differential gene loss, as the contributor to the evolutionary history of TyrRS in bacteria. However, for each TyrRS type, phylogenetic reconstruction yields phylogenies similar to the ribosomal phylogeny, revealing that frequent gene transfer has not destroyed the expected phylogeny; rather, the expected phylogenetic signal was reinforced or even created by HGT. We show that biased HGT can mimic patterns created through shared ancestry by in silico simulation. Furthermore, in cases where genomic synteny is sufficient to allow comparisons of relative gene positions, both tyrRS types occupy equivalent positions in closely related genomes, rejecting the loss hypothesis. Although the two types of bacterial TyrRS are only distantly related and only rarely coexist in a single genome, they have many features in common with alleles that are swapped between related lineages. We propose to label these functionally similar homologs as homeoalleles. We conclude that the observed phylogenetic pattern reflects both vertical inheritance and biased HGT and that the signal caused by common organismal descent is difficult to distinguish from the signal due to biased gene transfer. PMID:20495090

  15. Biased gene transfer mimics patterns created through shared ancestry.

    PubMed

    Andam, Cheryl P; Williams, David; Gogarten, J Peter

    2010-06-08

    In phylogenetic reconstruction, two types of bacterial tyrosyl-tRNA synthetases (TyrRS) form distinct clades with many bacterial phyla represented in both clades. Very few taxa possess both forms, and maximum likelihood analysis of the distribution of TyrRS types suggests horizontal gene transfer (HGT), rather than an ancient duplication followed by differential gene loss, as the contributor to the evolutionary history of TyrRS in bacteria. However, for each TyrRS type, phylogenetic reconstruction yields phylogenies similar to the ribosomal phylogeny, revealing that frequent gene transfer has not destroyed the expected phylogeny; rather, the expected phylogenetic signal was reinforced or even created by HGT. We show that biased HGT can mimic patterns created through shared ancestry by in silico simulation. Furthermore, in cases where genomic synteny is sufficient to allow comparisons of relative gene positions, both tyrRS types occupy equivalent positions in closely related genomes, rejecting the loss hypothesis. Although the two types of bacterial TyrRS are only distantly related and only rarely coexist in a single genome, they have many features in common with alleles that are swapped between related lineages. We propose to label these functionally similar homologs as homeoalleles. We conclude that the observed phylogenetic pattern reflects both vertical inheritance and biased HGT and that the signal caused by common organismal descent is difficult to distinguish from the signal due to biased gene transfer.

  16. Quartet analysis of putative horizontal gene transfer in Crenarchaeota.

    PubMed

    Ching, Travers H; Yoza, Brandon A; Li, Qing X

    2014-02-01

    Horizontal gene transfers (HGT) between four Crenarchaeota species (Metallosphaera cuprina Ar-4T, Acidianus hospitalis W1T, Vulcanisaeta moutnovskia 768-28T, and Pyrobaculum islandicum DSM 4184T) were investigated with quartet analysis. Strong support was found for individual genes that disagree with the phylogeny of the majority, implying genomic mosaicism. One such gene, a ferredoxin-related gene, was investigated further and incorporated into a larger phylogeny, which provided evidence for HGT of this gene from the Vulcanisaeta lineage to the Acidianus lineage. This is the first application of quartet analysis of HGT for the phylum Crenarchaeota. The results have shown that quartet analysis is a powerful technique to screen homologous sequences for putative HGTs and is useful in visually describing genomic mosaicism and HGT within four taxa.

  17. Graphene materials as 2D non-viral gene transfer vector platforms.

    PubMed

    Vincent, M; de Lázaro, I; Kostarelos, K

    2017-01-05

    Advances in genomics and gene therapy could offer solutions to many diseases that remain incurable today, however, one of the critical reasons halting clinical progress is due to the difficulty in designing efficient and safe delivery vectors for the appropriate genetic cargo. Safety and large-scale production concerns counter-balance the high gene transfer efficiency achieved with viral vectors, while non-viral strategies have yet to become sufficiently efficient. The extraordinary physicochemical, optical and photothermal properties of graphene-based materials (GBMs) could offer two-dimensional components for the design of nucleic acid carrier systems. We discuss here such properties and their implications for the optimization of gene delivery. While the design of such vectors is still in its infancy, we provide here an exhaustive and up-to-date analysis of the studies that have explored GBMs as gene transfer vectors, focusing on the functionalization strategies followed to improve vector performance and on the biological effects attained.Gene Therapy advance online publication, 5 January 2017; doi:10.1038/gt.2016.79.

  18. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    PubMed

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation.

  19. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes

    PubMed Central

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F.

    2015-01-01

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners—the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)—and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic—and plant and algal—lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller’s ratchet—the origin of eukaryotic recombination, or sex—might have required surprisingly little evolutionary innovation. PMID:25733873

  20. In vitro study for laser gene transfer in BHK-21 fibroblast cell line

    NASA Astrophysics Data System (ADS)

    Abdel Aziz, M.; Salem, D. S.; Salama, M. S.; Badr, Y.

    2009-02-01

    Modifications to our previously introduced system for laser microbeam cell surgery were carried out in the present work to match animal cells. These modifications included: 1- Using other laser system that used before, Excimer laser with 193 and 308 nm wavelengths. The used laser here, is He-Cd with low power and 441.5 nm wavelength in the visible region. 2- Instead of using pulsed laser, we used here CW He-Cd chopped by electrical chopper, which is synchronized with the mechanical motion of the mobile stage with step 40 microns, according to cell dimensions to avoid puncturing the same cell twice. The advantages of the modified here laser setup for gene transfer is: it is less damaging to the sensitive animal cell which has thin cell membrane. The present work aimed to: 1- Design a modified laser microbeam cell surgery, applicable to animal cells, such as fibroblast cells 2- To examine the efficiency of such system. 3- To assure gene transfer and its expression in the used cells. 4- To evaluate the ultra damages produced from using the laser beam as a modality for gene transfer. On the other wards, to introduce: safe, efficient and less damaging modality for gene transfer in animal cells. To achieve these goals, we applied the introduced here home-made laser setup with its synchronized parameters to introduce pBK-CMV phagemid, containing LacZ and neomycin resistance (neor )genes into BHK-21 fibroblast cell line. The results of the present work showed that: 1- Our modified laser microbeam cell surgery setup proved to be useful and efficient tool for gene transfer into fibroblast cells. 2- The presence and expression of LacZ gene was achieved using histochemical LacZ assay. 3- Selection of G418 antibiotic sensitivity assay confirmed the presence and expression towards stability of neor gene with time. 4- Presence of LacZ and neor genes in the genomic DNA of transfected fibroblast cells was indicated using PCR analysis. 5- Transmission electron microscopy indicated

  1. Lateral gene transfer, bacterial genome evolution, and the Anthropocene.

    PubMed

    Gillings, Michael R

    2017-02-01

    Lateral gene transfer (LGT) has significantly influenced bacterial evolution since the origins of life. It helped bacteria generate flexible, mosaic genomes and enables individual cells to rapidly acquire adaptive phenotypes. In turn, this allowed bacteria to mount strong defenses against human attempts to control their growth. The widespread dissemination of genes conferring resistance to antimicrobial agents has precipitated a crisis for modern medicine. Our actions can promote increased rates of LGT and also provide selective forces to fix such events in bacterial populations. For instance, the use of selective agents induces the bacterial SOS response, which stimulates LGT. We create hotspots for lateral transfer, such as wastewater systems, hospitals, and animal production facilities. Conduits of gene transfer between humans and animals ensure rapid dissemination of recent transfer events, as does modern transport and globalization. As resistance to antibacterial compounds becomes universal, there is likely to be increasing selection pressure for phenotypes with adverse consequences for human welfare, such as enhanced virulence, pathogenicity, and transmission. Improved understanding of the ecology of LGT could help us devise strategies to control this fundamental evolutionary process.

  2. Kidney-specific transposon-mediated gene transfer in vivo.

    PubMed

    Woodard, Lauren E; Cheng, Jizhong; Welch, Richard C; Williams, Felisha M; Luo, Wentian; Gewin, Leslie S; Wilson, Matthew H

    2017-03-20

    Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice.

  3. Kidney-specific transposon-mediated gene transfer in vivo

    PubMed Central

    Woodard, Lauren E.; Cheng, Jizhong; Welch, Richard C.; Williams, Felisha M.; Luo, Wentian; Gewin, Leslie S.; Wilson, Matthew H.

    2017-01-01

    Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice. PMID:28317878

  4. Wolbachia genome integrated in an insect chromosome: Evolution and fate of laterally transferred endosymbiont genes

    PubMed Central

    Nikoh, Naruo; Tanaka, Kohjiro; Shibata, Fukashi; Kondo, Natsuko; Hizume, Masahiro; Shimada, Masakazu; Fukatsu, Takema

    2008-01-01

    Recent accumulation of microbial genome data has demonstrated that lateral gene transfers constitute an important and universal evolutionary process in prokaryotes, while those in multicellular eukaryotes are still regarded as unusual, except for endosymbiotic gene transfers from mitochondria and plastids. Here we thoroughly investigated the bacterial genes derived from a Wolbachia endosymbiont on the nuclear genome of the beetle Callosobruchus chinensis. Exhaustive PCR detection and Southern blot analysis suggested that ∼30% of Wolbachia genes, in terms of the gene repertoire of wMel, are present on the insect nuclear genome. Fluorescent in situ hybridization located the transferred genes on the proximal region of the basal short arm of the X chromosome. Molecular evolutionary and other lines of evidence indicated that the transferred genes are probably derived from a single lateral transfer event. The transferred genes were, for the length examined, structurally disrupted, freed from functional constraints, and transcriptionally inactive. Hence, most, if not all, of the transferred genes have been pseudogenized. Notwithstanding this, the transferred genes were ubiquitously detected from Japanese and Taiwanese populations of C. chinensis, while the number of the transferred genes detected differed between the populations. The transferred genes were not detected from congenic beetle species, indicating that the transfer event occurred after speciation of C. chinensis, which was estimated to be one or several million years ago. These features of the laterally transferred endosymbiont genes are compared with the evolutionary patterns of mitochondrial and plastid genome fragments acquired by nuclear genomes through recent endosymbiotic gene transfers. PMID:18073380

  5. Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer.

    PubMed

    Pan, DengKe; Zhang, Li; Zhou, YanRong; Feng, Chong; Long, Chuan; Liu, Xiao; Wan, Rong; Zhang, Jian; Lin, AiXing; Dong, EnQiu; Wang, ShuChen; Xu, HouGang; Chen, HongXing

    2010-04-01

    Omega-3(omega-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of omega-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.

  6. Horizontal transfer of carbohydrate metabolism genes into ectomycorrhizal Amanita.

    PubMed

    Chaib De Mares, Maryam; Hess, Jaqueline; Floudas, Dimitrios; Lipzen, Anna; Choi, Cindy; Kennedy, Megan; Grigoriev, Igor V; Pringle, Anne

    2015-03-01

    The genus Amanita encompasses both symbiotic, ectomycorrhizal fungi and asymbiotic litter decomposers; all species are derived from asymbiotic ancestors. Symbiotic species are no longer able to degrade plant cell walls. The carbohydrate esterases family 1 (CE1s) is a diverse group of enzymes involved in carbon metabolism, including decomposition and carbon storage. CE1 genes of the ectomycorrhizal A. muscaria appear diverged from all other fungal homologues, and more similar to CE1s of bacteria, suggesting a horizontal gene transfer (HGT) event. In order to test whether AmanitaCE1s were acquired horizontally, we built a phylogeny of CE1s collected from across the tree of life, and describe the evolution of CE1 genes among Amanita and relevant lineages of bacteria. CE1s of symbiotic Amanita were very different from CE1s of asymbiotic Amanita, and are more similar to bacterial CE1s. The protein structure of one CE1 gene of A. muscaria matched a depolymerase that degrades the carbon storage molecule poly((R)-3-hydroxybutyrate) (PHB). Asymbiotic Amanita do not carry sequence or structural homologues of these genes. The CE1s acquired through HGT may enable novel metabolisms, or play roles in signaling or defense. This is the first evidence for the horizontal transfer of carbohydrate metabolism genes into ectomycorrhizal fungi.

  7. Myocardial Gene Transfer: Routes and Devices for Regulation of Transgene Expression by Modulation of Cellular Permeability

    PubMed Central

    Katz, Michael G.; Bridges, Charles R.

    2013-01-01

    Abstract Heart diseases are major causes of morbidity and mortality in Western society. Gene therapy approaches are becoming promising therapeutic modalities to improve underlying molecular processes affecting failing cardiomyocytes. Numerous cardiac clinical gene therapy trials have yet to demonstrate strong positive results and advantages over current pharmacotherapy. The success of gene therapy depends largely on the creation of a reliable and efficient delivery method. The establishment of such a system is determined by its ability to overcome the existing biological barriers, including cellular uptake and intracellular trafficking as well as modulation of cellular permeability. In this article, we describe a variety of physical and mechanical methods, based on the transient disruption of the cell membrane, which are applied in nonviral gene transfer. In addition, we focus on the use of different physiological techniques and devices and pharmacological agents to enhance endothelial permeability. Development of these methods will undoubtedly help solve major problems facing gene therapy. PMID:23427834

  8. Production of human glucocerebrosidase in mice after retroviral gene transfer into multipotential hematopoietic progenitor cells

    SciTech Connect

    Correll, P.H.; Fink, J.K.; Brady, R.O.; Perry, L.K.; Karlsson, S. )

    1989-11-01

    The human glucocerebrosidase (GC) gene has been transferred efficiently into spleen colony-forming unit (CFU-S) multipotential hematopoietic progenitor cells, and production of human GC RNA and protein has been achieved in transduced CFU-S colonies. High-titer retroviral vectors containing the human GC cDNA were constructed. Four vectors were compared with respect to gene-transfer efficiency into CFU-S progenitors. One vector (G vector) required high concentrations of interleukins 3 and 6 during stimulation and coculture for efficient transduction of CFU-S progenitors. The remaining three vectors (NTG, GTN, and GI vectors) transduced these progenitors at infection frequencies approaching 100% using low concentrations of hematopoietic growth factors to simulate cell division prior to and during the infection. Vectors using the viral long terminal repeat enhancer/promoter to drive the human GC cDNA produced high levels of human GC RNA in the progeny of CFU-S progenitors after gene transfer. All three vectors producing human GC RNA in CFU-S colonies can generate human GC as detected by immunochemical analysis of CFU-S colonies. The capacity of the viral long terminal repeat and the internal thymidine kinase promoter to direct synthesis of RNA in transduced bone marrow and spleen cells 5 months after bone marrow transplantation reflected the performance of these promoters in NTG-transduced CFU-S colonies.

  9. Comparison of reprogramming genes in induced pluripotent stem cells and nuclear transfer cloned embryos.

    PubMed

    Duan, Lian; Wang, Zhendong; Shen, Jingling; Shan, Zhiyan; Shen, Xinghui; Wu, Yanshuang; Sun, Ruizhen; Li, Tong; Yuan, Rui; Zhao, Qiaoshi; Bai, Guangyu; Gu, Yanli; Jin, Lianhong; Lei, Lei

    2014-08-01

    The most effective reprogramming methods, somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs), are widely used in biological research and regenerative medicine, yet the mechanism that reprograms somatic cells to totipotency remains unclear and thus reprogramming efficiency is still low. Microarray technology has been employed in analyzing the transcriptomes changes during iPS reprogramming. Unfortunately, it is difficult to obtain enough DNA from SCNT reconstructed embryos to take advantage of this technology. In this study, we aimed to identify critical genes from the transcriptional profile for iPS reprogramming and compared expression levels of these genes in SCNT reprogramming. By integrating gene expression information from microarray databases and published studies comparing somatic cells with either miPSCs or mouse embryonic stem cells (ESCs), we obtained two lists of co-upregulated genes. The gene ontology (GO) enriched analysis of these two lists demonstrated that the reprogramming process is associated with numerous biological processes. Specifically, we selected 32 genes related to heterochromatin, embryonic development, and cell cycle from our co-upregulated gene datasets and examined the gene expression level in iPSCs and SCNT embryos by qPCR. The results revealed that some reprogramming related genes in iPSCs were also expressed in SCNT reprogramming. We established the network of gene interactions that occur with genes differentially expressed in iPS and SCNT reprogramming and then performed GO analysis on the genes in the network. The network genes function in chromatin organization, heterochromatin, transcriptional regulation, and cell cycle. Further researches to improve reprogramming efficiency, especially in SCNT, will focus on functional studies of these selected genes.

  10. Role of Vibrio cholerae exochitinase ChiA2 in horizontal gene transfer.

    PubMed

    Mondal, Moumita; Chatterjee, Nabendu Sekhar

    2016-03-01

    Vibrio cholerae exochitinase ChiA2 plays a key role in acquisition of nutrients by chitin hydrolysis in the natural environment as well as in pathogenesis in the intestinal milieu. In this study we demonstrate the importance of ChiA2 in horizontal gene transfer in the natural environment. We found that the expression of ChiA2 and TfoX, the central regulator of V. cholerae horizontal gene transfer, varied with changes in environmental conditions. The activity of ChiA2 was also dependent on these conditions. In 3 different environmental conditions tested here, we observed that the supporting environmental condition for maximum expression and activity of ChiA2 was 20 °C, pH 5.5, and 100 mmol/L salinity in the presence of chitin. The same condition also induced TfoX expression and was favorable for horizontal gene transfer in V. cholerae. High-performance liquid chromatography analysis showed that ChiA2 released a significant amount of (GlcNAc)2 from chitin hydrolysis under the favorable condition. We hypothesized that under the favorable environmental condition, ChiA2 was upregulated and maximally active to produce a significant amount of (GlcNAc)2 from chitin. The same environmental condition also induced tfoX expression, followed by its translational activation by the (GlcNAc)2 produced, leading to efficient horizontal gene transfer.

  11. In vivo gene transfer targeting in pancreatic adenocarcinoma with cell surface antigens

    PubMed Central

    2012-01-01

    Background Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector. Results Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down. Conclusion This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors

  12. Efficient Heat and Mass Transfer Formulations for Oil Shale Retorting

    NASA Astrophysics Data System (ADS)

    Parker, J. C.; Zhang, F.

    2007-12-01

    A mathematical model for oil shale retorting is described that considers kerogen pyrolysis, oil coking, residual carbon gasification, carbonate mineral decomposition, water-gas shift, and phase equilibria reaction. Reaction rate temperature-dependence is described by Arrhenius kinetics. Fractured rock is modeled as a bi-continuum consisting of fracture porosity in which advective and dispersive gas and heat transport occur, and rock matrix in which diffusive mass transport and thermal conduction occur. Heat transfer between fracture and matrix regions is modeled either by a partial differential equation for spherical conduction or by a linear first-order heat transfer formulation. Mass transfer is modeled in an analogous manner or assuming local equilibrium. First-order mass and heat transfer coefficients are computed by a theoretical model from fundamental rock matrix properties. The governing equations are solved using a 3-D finite element formulation. Simulations of laboratory retort experiments and hypothetical problems indicated thermal disequilibrium to be the dominant factor controlling retort reactions. Simulation accuracy was unaffected by choice of mass transfer formulation. However, computational effort to explicitly simulate diffusive mass transfer in the rock matrix increased computational effort by more than an order of magnitude compared with first-order mass transfer or equilibrium analyses. A first-order heat transfer approximation of thermal conduction can be used without significant loss of accuracy if the block size and/or heating rate are not too large, as quantified by a proposed dimensionless heating rate.

  13. Characterization of an Ancient Lepidopteran Lateral Gene Transfer

    PubMed Central

    Wheeler, David; Redding, Amanda J.; Werren, John H.

    2013-01-01

    Bacteria to eukaryote lateral gene transfers (LGT) are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31) from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65–145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea). Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material. PMID:23533610

  14. Gene transfer from a parasitic flowering plant to a fern.

    PubMed

    Davis, Charles C; Anderson, William R; Wurdack, Kenneth J

    2005-11-07

    The rattlesnake fern (Botrychium virginianum (L.) Sw.) is obligately mycotrophic and widely distributed across the northern hemisphere. Three mitochondrial gene regions place this species with other ferns in Ophioglossaceae, while two regions place it as a member of the largely parasitic angiosperm order Santalales (sandalwoods and mistletoes). These discordant phylogenetic placements suggest that part of the genome in B. virginianum was acquired by horizontal gene transfer (HGT), perhaps from root-parasitic Loranthaceae. These transgenes are restricted to B. virginianum and occur across the range of the species. Molecular and life-history traits indicate that the transfer preceded the global expansion of B. virginianum, and that the latter may have happened very rapidly. This is the first report of HGT from an angiosperm to a fern, through either direct parasitism or the mediation of interconnecting fungal symbionts.

  15. Gene transfer from a parasitic flowering plant to a fern

    PubMed Central

    Davis, Charles C; Anderson, William R; Wurdack, Kenneth J

    2005-01-01

    The rattlesnake fern (Botrychium virginianum (L.) Sw.) is obligately mycotrophic and widely distributed across the northern hemisphere. Three mitochondrial gene regions place this species with other ferns in Ophioglossaceae, while two regions place it as a member of the largely parasitic angiosperm order Santalales (sandalwoods and mistletoes). These discordant phylogenetic placements suggest that part of the genome in B. virginianum was acquired by horizontal gene transfer (HGT), perhaps from root-parasitic Loranthaceae. These transgenes are restricted to B. virginianum and occur across the range of the species. Molecular and life-history traits indicate that the transfer preceded the global expansion of B. virginianum, and that the latter may have happened very rapidly. This is the first report of HGT from an angiosperm to a fern, through either direct parasitism or the mediation of interconnecting fungal symbionts. PMID:16191635

  16. Exploring the limits of polarization transfer efficiency in homonuclear three spin systems.

    PubMed

    Neves, Jorge L; Heitmann, Björn; Reiss, Timo O; Schor, Heloiza H R; Khaneja, Navin; Glaser, Steffen J

    2006-07-01

    The limits of polarization transfer efficiency are explored for systems consisting of three isotropically coupled spins 1/2 in the absence of relaxation. An idealized free evolution and control Hamiltonian is studied, which provides an upper limit of transfer efficiency (in terms of transfer amplitude and transfer time) for realistic homonuclear spin systems with arbitrary Heisenberg-type coupling constants J12, J13, and J23. It is shown that optimal control based pulse sequences have significantly improved transfer efficiencies compared to conventional transfer schemes. An experimental demonstration of optimal polarization transfer is given for the case of the carbon spin system of fully 13C labelled alanine at 62.5 MHz Larmor frequency.

  17. Risks from GMOs due to horizontal gene transfer.

    PubMed

    Keese, Paul

    2008-01-01

    Horizontal gene transfer (HGT) is the stable transfer of genetic material from one organism to another without reproduction or human intervention. Transfer occurs by the passage of donor genetic material across cellular boundaries, followed by heritable incorporation to the genome of the recipient organism. In addition to conjugation, transformation and transduction, other diverse mechanisms of DNA and RNA uptake occur in nature. The genome of almost every organism reveals the footprint of many ancient HGT events. Most commonly, HGT involves the transmission of genes on viruses or mobile genetic elements. HGT first became an issue of public concern in the 1970s through the natural spread of antibiotic resistance genes amongst pathogenic bacteria, and more recently with commercial production of genetically modified (GM) crops. However, the frequency of HGT from plants to other eukaryotes or prokaryotes is extremely low. The frequency of HGT to viruses is potentially greater, but is restricted by stringent selection pressures. In most cases the occurrence of HGT from GM crops to other organisms is expected to be lower than background rates. Therefore, HGT from GM plants poses negligible risks to human health or the environment.

  18. Horizontal Gene Transfer is a Significant Driver of Gene Innovation in Dinoflagellates

    PubMed Central

    Wisecaver, Jennifer H.; Brosnahan, Michael L.; Hackett, Jeremiah D.

    2013-01-01

    The dinoflagellates are an evolutionarily and ecologically important group of microbial eukaryotes. Previous work suggests that horizontal gene transfer (HGT) is an important source of gene innovation in these organisms. However, dinoflagellate genomes are notoriously large and complex, making genomic investigation of this phenomenon impractical with currently available sequencing technology. Fortunately, de novo transcriptome sequencing and assembly provides an alternative approach for investigating HGT. We sequenced the transcriptome of the dinoflagellate Alexandrium tamarense Group IV to investigate how HGT has contributed to gene innovation in this group. Our comprehensive A. tamarense Group IV gene set was compared with those of 16 other eukaryotic genomes. Ancestral gene content reconstruction of ortholog groups shows that A. tamarense Group IV has the largest number of gene families gained (314–1,563 depending on inference method) relative to all other organisms in the analysis (0–782). Phylogenomic analysis indicates that genes horizontally acquired from bacteria are a significant proportion of this gene influx, as are genes transferred from other eukaryotes either through HGT or endosymbiosis. The dinoflagellates also display curious cases of gene loss associated with mitochondrial metabolism including the entire Complex I of oxidative phosphorylation. Some of these missing genes have been functionally replaced by bacterial and eukaryotic xenologs. The transcriptome of A. tamarense Group IV lends strong support to a growing body of evidence that dinoflagellate genomes are extraordinarily impacted by HGT. PMID:24259313

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

    PubMed

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

    2002-11-01

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

  20. Phylogeographic support for horizontal gene transfer involving sympatric bruchid species

    PubMed Central

    Alvarez, Nadir; Benrey, Betty; Hossaert-McKey, Martine; Grill, Andrea; McKey, Doyle; Galtier, Nicolas

    2006-01-01

    Background We report on the probable horizontal transfer of a mitochondrial gene, cytb, between species of Neotropical bruchid beetles, in a zone where these species are sympatric. The bruchid beetles Acanthoscelides obtectus, A. obvelatus, A. argillaceus and Zabrotes subfasciatus develop on various bean species in Mexico. Whereas A. obtectus and A. obvelatus develop on Phaseolus vulgaris in the Mexican Altiplano, A. argillaceus feeds on P. lunatus in the Pacific coast. The generalist Z. subfasciatus feeds on both bean species, and is sympatric with A. obtectus and A. obvelatus in the Mexican Altiplano, and with A. argillaceus in the Pacific coast. In order to assess the phylogenetic position of these four species, we amplified and sequenced one nuclear (28S rRNA) and two mitochondrial (cytb, COI) genes. Results Whereas species were well segregated in topologies obtained for COI and 28S rRNA, an unexpected pattern was obtained in the cytb phylogenetic tree. In this tree, individuals from A. obtectus and A. obvelatus, as well as Z. subfasciatus individuals from the Mexican Altiplano, clustered together in a unique little variable monophyletic unit. In contrast, A. argillaceus and Z. subfasciatus individuals from the Pacific coast clustered in two separated clades, identically to the pattern obtained for COI and 28S rRNA. An additional analysis showed that Z. subfasciatus individuals from the Mexican Altiplano also possessed the cytb gene present in individuals of this species from the Pacific coast. Zabrotes subfasciatus individuals from the Mexican Altiplano thus demonstrated two cytb genes, an "original" one and an "infectious" one, showing 25% of nucleotide divergence. The "infectious" cytb gene seems to be under purifying selection and to be expressed in mitochondria. Conclusion The high degree of incongruence of the cytb tree with patterns for other genes is discussed in the light of three hypotheses: experimental contamination, hybridization, and

  1. CHARACTERIZING RESIDUE TRANSFER EFFICIENCIES USING A FLUORESCENT IMAGING TECHNIQUE

    EPA Science Inventory

    To reduce the uncertainty associated with current estimates of children's exposure to pesticides by dermal contact and indirect ingestion, residue transfer data are required. Prior to conducting exhaustive studies, a screening study to identify the important parameters for chara...

  2. TRANSFER EFFICIENCIES OF PESTICIDES FROM HOUSEHOLD FLOORING SURFACES TO FOODS

    EPA Science Inventory

    The transfer of pesticides from household surfaces to foods was measured to determine if excess dietary exposure potentially occurs when children's foods contact contaminated surfaces prior to being. Three common household surfaces (ceramic tile, hardwood flooring, and carpet) w...

  3. Dynamic monitoring of horizontal gene transfer in soil

    NASA Astrophysics Data System (ADS)

    Cheng, H. Y.; Masiello, C. A.; Silberg, J. J.; Bennett, G. N.

    2015-12-01

    Soil microbial gene expression underlies microbial behaviors (phenotypes) central to many aspects of C, N, and H2O cycling. However, continuous monitoring of microbial gene expression in soils is challenging because genetically-encoded reporter proteins widely used in the lab are difficult to deploy in soil matrices: for example, green fluorescent protein cannot be easily visualized in soils, even in the lab. To address this problem we have developed a reporter protein that releases small volatile gases. Here, we applied this gas reporter in a proof-of-concept soil experiment, monitoring horizontal gene transfer, a microbial activity that alters microbial genotypes and phenotypes. Horizontal gene transfer is central to bacterial evolution and adaptation and is relevant to problems such as the spread of antibiotic resistance, increasing metal tolerance in superfund sites, and bioremediation capability of bacterial consortia. This process is likely to be impacted by a number of matrix properties not well-represented in the petri dish, such as microscale variations in water, nutrients, and O2, making petri-dish experiments a poor proxy for environmental processes. We built a conjugation system using synthetic biology to demonstrate the use of gas-reporting biosensors in safe, lab-based biogeochemistry experiments, and here we report the use of these sensors to monitor horizontal gene transfer in soils. Our system is based on the F-plasmid conjugation in Escherichia coli. We have found that the gas signal reports on the number of cells that acquire F-plasmids (transconjugants) in a loamy Alfisol collected from Kellogg Biological Station. We will report how a gas signal generated by transconjugants varies with the number of F-plasmid donor and acceptor cells seeded in a soil, soil moisture, and soil O2 levels.

  4. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    PubMed

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  5. New Construct Approaches for Efficient Gene Silencing in Plants

    PubMed Central

    Yan, Hua; Chretien, Robert; Ye, Jingsong; Rommens, Caius M.

    2006-01-01

    An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts. PMID:16766670

  6. [Gene transfer system mediated by PEI-cholesterol lipopolymer with lipid microbubbles].

    PubMed

    Jiang, Yong-Nan; Mo, Hong-Ying; Chen, Jian-Hai

    2010-05-01

    The properties of polyethyleneimine-cholesterol cationic lipopolymer (PEI-Chol) as gene carries and its gene transfer efficiency in vitro with lipid microbubbles were presented in this paper. PEI-Chol lipopolymer was synthesized by linking cholesterol chloroformate to the amino groups of branched poly(ethyleneimine) (PEI) of 1 800. The structure and molecular weight of PEI-Chol were confirmed by IR, 1H NMR and MADI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry), respectively. The average molecular weight of PEI-Chol was approximately 2 000. The gene delivery system of bubble/PEI-Chol/DNA was constructed by mixed PEI-Chol/pDNA (N/P 10:1) complexes with lipid microbubbles (2-8 microm) which were prepared by DPPC, DSPE-PEG2000 and perfluoropropane with the reverse phase evaporation technique. pEGFP-Cl (enhanced green fluorescent protein) was used as report gene to investigate the DNA condensing ability of PEI-Chol lipopolymer by agarose gel electrophoresis. And their cytotoxicity and in vitro transfer efficiency of different complexes were compared with each other in A549 and MCF-7. The results indicated PEI-Chol lipopolymer can condense plasmid DNA when N/P ratio upto 4, PEI-Chol complexes and bubble/PEI-Chol/DNA complexes were nontoxic to A549 and MCF-7 when formulated at the N/P ratio of 10/1 as determined by MTT assay. This bubble/PEI-Chol/DNA delivery system provided good transfer efficiency with other desirable characteristics such as against-precipitation of plasma proteins. In conclusion, bubble/PEI-Chol/DNA complex is a novel non-viral gene delivery system.

  7. Investigating rate-limiting barriers to nanoscale nonviral gene transfer with nanobiophotonics

    NASA Astrophysics Data System (ADS)

    Chen, Hunter H.

    Nucleic acids are a novel class of therapeutics poised to address many unmet clinical needs. Safe and efficient delivery remains a significant challenge that has delayed the realization of the full therapeutic potential of nucleic acids. Nanoscale nonviral vectors offer an attractive alternative to viral vectors as natural and synthetic polymers or polypeptides may be rationally designed to meet the unique demands of individual applications. A mechanistic understanding of cellular barriers is necessary to develop guidelines for designing custom gene carriers which are expected to greatly impact this delivery challenge. The work herein focused on the relationships among nanocomplex stability, intracellular trafficking and unpacking kinetics, and DNA degradation. Ultrasensitive nanosensors based on QD-FRET were developed to characterize the biophysical properties of nanocomplexes and study these rate-limiting steps. Quantitative image analysis enabled the distributions of the subpopulation of condensed or released DNA to be determined within the major cellular compartments encountered during gene transfer. The steady state stability and unpacking kinetics within these compartments were found to impact transgene expression, elucidating multiple design strategies to achieve efficient gene transfer. To address enzymatic barriers, a novel two-step QD-FRET nanosensor was developed to analyze unpacking and DNA degradation simultaneously, which has not been accomplished previously. Bioresponsive strategies such as disulfide crosslinking and thermosensitivity were evaluated by QD-FRET and quantitative compartmental analysis as case studies to determine appropriate design specifications for thiolated polymers and thermoresponsive polypeptides. Relevant nanobiophotonic tools were developed as a platform to study major rate-limiting barriers to nanomedicine and demonstrated the feasibility of using mechanistic information gained from these tools to guide the rational design of

  8. Operationally efficient propulsion system study (OEPSS) data book. Volume 6; Space Transfer Propulsion Operational Efficiency Study Task of OEPSS

    NASA Technical Reports Server (NTRS)

    Harmon, Timothy J.

    1992-01-01

    This document is the final report for the Space Transfer Propulsion Operational Efficiency Study Task of the Operationally Efficient Propulsion System Study (OEPSS) conducted by the Rocketdyne Division of Rockwell International. This Study task studied, evaluated and identified design concepts and technologies which minimized launch and in-space operations and optimized in-space vehicle propulsion system operability.

  9. HGTree: database of horizontally transferred genes determined by tree reconciliation.

    PubMed

    Jeong, Hyeonsoo; Sung, Samsun; Kwon, Taehyung; Seo, Minseok; Caetano-Anollés, Kelsey; Choi, Sang Ho; Cho, Seoae; Nasir, Arshan; Kim, Heebal

    2016-01-04

    The HGTree database provides putative genome-wide horizontal gene transfer (HGT) information for 2472 completely sequenced prokaryotic genomes. This task is accomplished by reconstructing approximate maximum likelihood phylogenetic trees for each orthologous gene and corresponding 16S rRNA reference species sets and then reconciling the two trees under parsimony framework. The tree reconciliation method is generally considered to be a reliable way to detect HGT events but its practical use has remained limited because the method is computationally intensive and conceptually challenging. In this regard, HGTree (http://hgtree.snu.ac.kr) represents a useful addition to the biological community and enables quick and easy retrieval of information for HGT-acquired genes to better understand microbial taxonomy and evolution. The database is freely available and can be easily scaled and updated to keep pace with the rapid rise in genomic information.

  10. HGTree: database of horizontally transferred genes determined by tree reconciliation

    PubMed Central

    Jeong, Hyeonsoo; Sung, Samsun; Kwon, Taehyung; Seo, Minseok; Caetano-Anollés, Kelsey; Choi, Sang Ho; Cho, Seoae; Nasir, Arshan; Kim, Heebal

    2016-01-01

    The HGTree database provides putative genome-wide horizontal gene transfer (HGT) information for 2472 completely sequenced prokaryotic genomes. This task is accomplished by reconstructing approximate maximum likelihood phylogenetic trees for each orthologous gene and corresponding 16S rRNA reference species sets and then reconciling the two trees under parsimony framework. The tree reconciliation method is generally considered to be a reliable way to detect HGT events but its practical use has remained limited because the method is computationally intensive and conceptually challenging. In this regard, HGTree (http://hgtree.snu.ac.kr) represents a useful addition to the biological community and enables quick and easy retrieval of information for HGT-acquired genes to better understand microbial taxonomy and evolution. The database is freely available and can be easily scaled and updated to keep pace with the rapid rise in genomic information. PMID:26578597

  11. [Rapid and efficient expression of foreign genes in mammalian cells by baculovirus vectors].

    PubMed

    Cheng, Tong; Xu, Chen-Yu; Wang, Ying-Bin; Chen, Min; Wu, Ting; Xie, Xiao-Yan; Zhang, Jun; Xia, Ning-Shao

    2003-09-01

    The baculovirus insect cell expression system has been used extensively for the expression of recombinant proteins in insect cells. Recently, reports have described that recombinant baculoviruses can transduce a broad spectrum of primary and established mammalian cells, which shows the baculoviruses could serve as a new gene-transfer vehicle for mammalian cells. In this report, we further research the modification of baculovirus vector and the way to deliver exogenous gene into mammalian cells. On the base of Bac-to-Bac baculovirus insect cell expression system, two recombinant baculoviruses (BacV-CMV-EGFPA, BacV-CMV-EGFPB) were constructed containing different direction of CMV promoters which controll the expression of a reporter gene (EGFP). We found that CMV promoter could direct expression of reporter gene in Sf9 cells with relatively low efficiency. The culture supernatant of Sf9 cells which have been infected by the recombinant baculoviruses for four days were collected and the titers of the viruses in culture supernatant were determined by plaque assay on Sf9 cells. The HepG2 cells, an human hepatocellular carcinoma cell line, were directly incubated with the collected culture supernatant which contains the recombinant baculoviruses for 8 hours in 37 degrees C CO2 incubator (moi = 100). Twenty-four hours post transduction the efficiencies of gene-transfer and expression were analyzed by flow cytometry (FCM) which detect the green fluorescence of individual cells. Results show that these two recombinant baculoviruses have similar gene-transfer and expression efficiency in HepG2 cells, which means the direction of CMV promoters has no effects on reporter gene expression. The optimal transduction conditions of incubating the mammalian cells with the culture supernatant of Sf9 cells infected by recombinant baculoviruses for four days were determined by FCM assay in HepG2 cells. The HepG2 cells inoculated in 24-well plate (5 x 10(4)/well) were incubated with the

  12. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

    PubMed Central

    2002-01-01

    Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation). Conclusions Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species. PMID:11964188

  13. Selective gene transfer to endometrial cancer cells by a polymer against matrix metalloproteinase 2 (MMP-2).

    PubMed

    Han, Joo Youn; Choi, Dong Soon; Kim, Changhoon; Joo, Hyun; Min, Churl K

    2008-04-01

    A novel cancer-cell-specific gene delivery vector with high transfection efficiency was designed and tested with an in vitro coculture consisting of the human endometrial adenocarcinoma cell line, HEC-1A cells, and normal endometrial stromal cells. For the cancer-cell targeting, polyethylenimine (PEI), a cationic polymer that can be easily combined with anionic DNA to form a particulate complex, polyplex, being capable of transferring a gene into a variety of cells, was covalently conjugated with antibodies against matrix metalloproteinase 2 (MMP-2), a typical surface-marker protein on cancer cells known for its close correlation with angiogenesis and invasion in many types of cancer, using the heterofunctional cross-linker, n-succinimidyl 3-(2-pyridyldithio)-propionamide. Biophysical properties and transfection efficiencies of anti-MMP-2-conjugated PEI were analyzed by means of dynamic light scattering, laser Doppler anemometry, and flow cytometry. Our results reveal that (1) the PEI-anti-MMP-2 antibody conjugate maintains physical parameters, including sizes and surface charges, which appear to be favorable for gene transfer and (2) when the pEGFP-N3 plasmid complexes of the PEI-anti-MMP-2 antibody conjugate are applied to the coculture consisting of HEC-1A cells and human stromal cells, a high level of green fluorescent protein expression occurs in HEC-1A cells over stromal cells, suggesting a specific gene transfer targeting cancer cells. Therefore, targeting invading cancer cells with the PEI-anti-MMP-2 antibody conjugate could be promising in endometrial cancer treatment, and this gene delivery system deserves further optimization in the context of targeted therapeutic gene delivery.

  14. Automatic graphene transfer system for improved material quality and efficiency.

    PubMed

    Boscá, Alberto; Pedrós, Jorge; Martínez, Javier; Palacios, Tomás; Calle, Fernando

    2016-02-10

    In most applications based on chemical vapor deposition (CVD) graphene, the transfer from the growth to the target substrate is a critical step for the final device performance. Manual procedures are time consuming and depend on handling skills, whereas existing automatic roll-to-roll methods work well for flexible substrates but tend to induce mechanical damage in rigid ones. A new system that automatically transfers CVD graphene to an arbitrary target substrate has been developed. The process is based on the all-fluidic manipulation of the graphene to avoid mechanical damage, strain and contamination, and on the combination of capillary action and electrostatic repulsion between the graphene and its container to ensure a centered sample on top of the target substrate. The improved carrier mobility and yield of the automatically transferred graphene, as compared to that manually transferred, is demonstrated by the optical and electrical characterization of field-effect transistors fabricated on both materials. In particular, 70% higher mobility values, with a 30% decrease in the unintentional doping and a 10% strain reduction are achieved. The system has been developed for lab-scale transfer and proved to be scalable for industrial applications.

  15. Automatic graphene transfer system for improved material quality and efficiency

    PubMed Central

    Boscá, Alberto; Pedrós, Jorge; Martínez, Javier; Palacios, Tomás; Calle, Fernando

    2016-01-01

    In most applications based on chemical vapor deposition (CVD) graphene, the transfer from the growth to the target substrate is a critical step for the final device performance. Manual procedures are time consuming and depend on handling skills, whereas existing automatic roll-to-roll methods work well for flexible substrates but tend to induce mechanical damage in rigid ones. A new system that automatically transfers CVD graphene to an arbitrary target substrate has been developed. The process is based on the all-fluidic manipulation of the graphene to avoid mechanical damage, strain and contamination, and on the combination of capillary action and electrostatic repulsion between the graphene and its container to ensure a centered sample on top of the target substrate. The improved carrier mobility and yield of the automatically transferred graphene, as compared to that manually transferred, is demonstrated by the optical and electrical characterization of field-effect transistors fabricated on both materials. In particular, 70% higher mobility values, with a 30% decrease in the unintentional doping and a 10% strain reduction are achieved. The system has been developed for lab-scale transfer and proved to be scalable for industrial applications. PMID:26860260

  16. Horizontal transfer of expressed genes in a parasitic flowering plant

    PubMed Central

    2012-01-01

    Background Recent studies have shown that plant genomes have potentially undergone rampant horizontal gene transfer (HGT). In plant parasitic systems HGT appears to be facilitated by the intimate physical association between the parasite and its host. HGT in these systems has been invoked when a DNA sequence obtained from a parasite is placed phylogenetically very near to its host rather than with its closest relatives. Studies of HGT in parasitic plants have relied largely on the fortuitous discovery of gene phylogenies that indicate HGT, and no broad systematic search for HGT has been undertaken in parasitic systems where it is most expected to occur. Results We analyzed the transcriptomes of the holoparasite Rafflesia cantleyi Solms-Laubach and its obligate host Tetrastigma rafflesiae Miq. using phylogenomic approaches. Our analyses show that several dozen actively transcribed genes, most of which appear to be encoded in the nuclear genome, are likely of host origin. We also find that hundreds of vertically inherited genes (VGT) in this parasitic plant exhibit codon usage properties that are more similar to its host than to its closest relatives. Conclusions Our results establish for the first time a substantive number of HGTs in a plant host-parasite system. The elevated rate of unidirectional host-to- parasite gene transfer raises the possibility that HGTs may provide a fitness benefit to Rafflesia for maintaining these genes. Finally, a similar convergence in codon usage of VGTs has been shown in microbes with high HGT rates, which may help to explain the increase of HGTs in these parasitic plants. PMID:22681756

  17. Targeted gene transfer to corneal endothelium in vivo by electric pulse.

    PubMed

    Oshima, Y; Sakamoto, T; Yamanaka, I; Nishi, T; Ishibashi, T; Inomata, H

    1998-10-01

    A novel method of in vivo targeted gene transfer to intentionally selected areas of the corneal endothelium was developed. Plasmid DNA with the lacZ gene coding for beta-galactosidase was injected into the anterior chamber of adult Wistar rats, and eight pulses of electricity at intensities ranging from 5 to 40 V/cm were delivered for 50 ms to the cornea with a specially designed electric probe in order to determine the effect of gene transfer on the corneal endothelial cells. Gene expression was visualized by enzymatic color reaction using X-gal in enucleated eyes on days 1, 3, 7, 14 and 21 after gene transfer. The treated eyes were then photographed and the X-gal-positive areas were evaluated by an image analyzer. The ratios of the areas (X-gal-positive area/area of entire corneal endothelium x 100%) were then calculated to determine gene transfection efficiency. The expression of beta-galactosidase was clearly detected in the cytoplasm of the corneal endothelial cells as early as day 1 and lasted until day 21. The most intense gene expression was observed on days 1 and 3 (5.21% on day 1 and 6.45% on day 3). The expression of beta-galactosidase on day 3 was most evident following delivery of 20 V electric pulses (0.09% at 5 V, 0.03% at 10 V, 6.45% at 20 V). beta-Galactosidase expression was limited to the corneal endothelial cells in highly selected areas and no beta-galactosidase expression was detected in any other intra- or axtraocular tissues. In addition, no cell damage was apparent in the cornea and no inflammation was detected in any other intraocular tissues. Thus, low-voltage electric pulses successfully transferred the gene of interest to highly selective areas of the corneal endothelium without inducing any pathological changes. This targeted gene transfer method appears to have great potential for use in gene therapy for ocular diseases.

  18. Immunological ignorance allows long-term gene expression after perinatal recombinant adeno-associated virus-mediated gene transfer to murine airways.

    PubMed

    Carlon, Marianne S; Vidović, Dragana; Dooley, James; da Cunha, Marina Mori; Maris, Michael; Lampi, Youlia; Toelen, Jaan; Van den Haute, Chris; Baekelandt, Veerle; Deprest, Jan; Verbeken, Erik; Liston, Adrian; Gijsbers, Rik; Debyser, Zeger

    2014-06-01

    Gene therapy of the lung has the potential to treat life-threatening diseases such as cystic fibrosis and α(1)-antitrypsin or surfactant deficiencies. A major hurdle for successful gene therapy is the development of an immune response against the transgene and/or viral vector. We hypothesized that by targeting the airways in the perinatal period, induction of an immune response against the vector particle could be prevented because of immaturity of the immune system, in turn allowing repeated gene transfer later in adult life to ensure long-term gene expression. Therefore, we readministered recombinant adeno-associated viral vector serotype 5 (rAAV2/5) to mouse airways 3 and 6 months after initial perinatal gene transfer. Our findings demonstrate that perinatal rAAV2/5-mediated gene transfer to the airways avoids a strong immune response. This immunological ignorance allows the readministration of an autologous vector later in adult life, resulting in efficient and stable gene transfer up to 7 months, without evidence of a decrease in transgene expression. Together, these data provide a basis to further explore perinatal gene therapy for pulmonary conditions with adequate gene expression up to 7 months.

  19. Efficient Radioisotope Energy Transfer by Gold Nanoclusters for Molecular Imaging.

    PubMed

    Volotskova, Olga; Sun, Conroy; Stafford, Jason H; Koh, Ai Leen; Ma, Xiaowei; Cheng, Zhen; Cui, Bianxiao; Pratx, Guillem; Xing, Lei

    2015-08-26

    Beta-emitting isotopes Fluorine-18 and Yttrium-90 are tested for their potential to stimulate gold nanoclusters conjugated with blood serum proteins (AuNCs). AuNCs excited by either medical radioisotope are found to be highly effective ionizing radiation energy transfer mediators, suitable for in vivo optical imaging. AuNCs synthesized with protein templates convert beta-decaying radioisotope energy into tissue-penetrating optical signals between 620 and 800 nm. Optical signals are not detected from AuNCs incubated with Technetium-99m, a pure gamma emitter that is used as a control. Optical emission from AuNCs is not proportional to Cerenkov radiation, indicating that the energy transfer between the radionuclide and AuNC is only partially mediated by Cerenkov photons. A direct Coulombic interaction is proposed as a novel and significant mechanism of energy transfer between decaying radionuclides and AuNCs.

  20. Interferon-β gene transfer induces a strong cytotoxic bystander effect on melanoma cells.

    PubMed

    Rossi, Úrsula A; Gil-Cardeza, María L; Villaverde, Marcela S; Finocchiaro, Liliana M E; Glikin, Gerardo C

    2015-05-01

    A local gene therapy scheme for the delivery of type I interferons could be an alternative for the treatment of melanoma. We evaluated the cytotoxic effects of interferon-β (IFNβ) gene lipofection on tumor cell lines derived from three human cutaneous and four canine mucosal melanomas. The cytotoxicity of human IFNβ gene lipofection resulted higher or equivalent to that of the corresponding addition of the recombinant protein (rhIFNβ) to human cells. IFNβ gene lipofection was not cytotoxic for only one canine melanoma cell line. When cultured as monolayers, three human and three canine IFNβ-lipofected melanoma cell lines displayed a remarkable bystander effect. As spheroids, the same six cell lines were sensitive to IFNβ gene transfer, two displaying a significant multicell resistance phenotype. The effects of conditioned IFNβ-lipofected canine melanoma cell culture media suggested the release of at least one soluble thermolabile cytotoxic factor that could not be detected in human melanoma cells. By using a secretion signal-free truncated human IFNβ, we showed that its intracellular expression was enough to induce cytotoxicity in two human melanoma cell lines. The lower cytoplasmatic levels of reactive oxygen species detected after intracellular IFNβ expression could be related to the resistance displayed by one human melanoma cell line. As IFNβ gene transfer was effective against most of the assayed melanomas in a way not limited by relatively low lipofection efficiencies, the clinical potential of this approach is strongly supported.

  1. Non-Invasive Gene Transfer by Iontophoresis for Therapy of an Inherited Retinal Degeneration

    PubMed Central

    Souied, Eric H.; Reid, Silvia N. M.; Piri, Natik I.; Lerner, Leonid E.; Nusinowitz, Steven; Farber, Debora B.

    2009-01-01

    Despite extensive research on many of the genes responsible for inherited retinal degenerations leading to blindness, no effective treatment is currently available for patients affected with these diseases. Among the therapeutic approaches tested on animal models of human retinal degeneration, gene therapy using different types of viral vectors as delivery agents has yielded promising results. We report here our results on a non-invasive, non-viral delivery approach using transscleral iontophoresis for transfer of plasmid DNA into mouse retina. Proof of principle experiments were carried out using plasmid containing GFP cDNA to demonstrate expression of the transferred gene in the retina after single applications of iontophoresis. Various parameters for multiple applications of iontophoresis were optimized to sustain GFP gene expression in mouse photoreceptors. Subsequently, repeated iontophoresis of plasmid containing normal β-phosphodiesterase (β-PDE) cDNA was performed in the rd1 mouse, an animal model of autosomal recessive retinitis pigmentosa caused by a mutant β-PDE gene. In normal mice, transscleral iontophoresis of the GFP plasmid provided a significant increase in fluorescence of the retina in the treated versus non-treated eyes. In rd1 mice, repeated iontophoresis of β-PDE cDNA plasmid partially rescued photoreceptors morphologically, as observed by microscopy, and functionally, as recorded on ERG measurements, without adverse effects. Therefore, transscleral iontophoresis of plasmid DNA containing therapeutic genes may be an efficient, safe and non-invasive method for the treatment of retinal degenerations. PMID:18653181

  2. An Efficient Method for Transferring Adult Mosquitoes during Field Tests,

    DTIC Science & Technology

    CULICIDAE, *COLLECTING METHODS, REPRINTS, BLOOD SUCKING INSECTS, FIELD TESTS, HAND HELD, EFFICIENCY, LABORATORY EQUIPMENT, MORTALITY RATES , ADULTS, AEDES, ASPIRATORS, CULICIDAE, TEST AND EVALUATION, REPRINTS

  3. Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis

    PubMed Central

    Juhas, Mario; Wong, Christine; Ajioka, James W.

    2016-01-01

    The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis. PMID:27798678

  4. Linear topology confers in vivo gene transfer activity to polyethylenimines.

    PubMed

    Brissault, B; Leborgne, C; Guis, C; Danos, O; Cheradame, H; Kichler, A

    2006-01-01

    Although polyethylenimines (PEIs) are frequently used transfection agents, it is still unclear which of their properties are required for efficient gene delivery. This is even more striking when working in vivo since some PEIs are able to generate significant gene expression, whereas others are not. To facilitate a rational development of compounds with improved transfection activities, studies aimed at identifying the properties involved in the transfection process seem indispensable. In the present work, we investigated how transfection with linear PEI of 22 kDa allows for high reporter gene expression in lungs after intravenous injection, whereas the branched PEI of 25 kDa does not. To this end, we synthesized L-PEI derivatives that are intermediates between linear and branched PEIs. Our results show that the topology plays a crucial role in obtaining in vivo reporter gene expression, whereas the content of primary, secondary, and tertiary amines is only of minor importance.

  5. Correlation of length of linear oligo(ethanamino) amides with gene transfer and cytotoxicity.

    PubMed

    Scholz, Claudia; Kos, Petra; Leclercq, Laurent; Jin, Xiaoyun; Cottet, Hervé; Wagner, Ernst

    2014-09-01

    The optimization of synthetic carriers for gene transfer remains a major challenge. Cationic polymers such as polyethylenimine (PEI) often show increasing gene transfer activity with increasing molecular weight, but this favorable effect is accompanied by an undesired increase in cytotoxicity. Moreover, the polydispersity of polymers prevents accurate determination of optimum size. Herein we describe the step-by-step elongation of precise linear oligo(ethanamino) amides by making use of the artificial amino acid succinoyl-tetraethylene pentamine (Stp) for solid-phase-assisted synthesis. This procedure enabled us to identify the optimal oligomer Stp30-W (8.4 kDa) with a length of 30 Stp units, with which effective gene transfer occurs in the absence of cytotoxicity. The transfection efficiency of Stp30-W exceeded that of standard linear PEI (22 kDa) by sixfold; nevertheless, Stp30-W exhibited tenfold lower cytotoxicity. In addition to the lower molecular weight, the succinate spacer between the oligoamine units may also contribute to the favorable biocompatibility. The cytotoxicity of the cationic polymer PEI is a major concern for use as a carrier for gene delivery, so this comparison between linear PEI and the new Stp oligomers is particularly relevant.

  6. Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro.

    PubMed

    Boussif, O; Delair, T; Brua, C; Veron, L; Pavirani, A; Kolbe, H V

    1999-01-01

    Cationic polymers possessing primary amine groups are inefficient in transferring nucleic acids into eukaryotic cells. With appropriate chemical modification, namely glycolylation of the amine groups of polylysine and polyallylamine, the actual number of free amino groups was decreased, hydrophilic residues were introduced, and the cytotoxicity of both polymers decreased significantly. Furthermore, in the case of polyallylamine, its ability to mediate gene transfer into cells increased by several orders of magnitude. Transfection efficiency was found to be dependent on the substitution level of amino groups and reached highest levels in the presence of lysosomotropic and/or fusogenic agents. At optimal conditions, glycolylated PAM was shown to be as efficient as the linear polyethylenimine of 22 kDa.

  7. Efficiency transfer using the GEANT4 code of CERN for HPGe gamma spectrometry.

    PubMed

    Chagren, S; Ben Tekaya, M; Reguigui, N; Gharbi, F

    2016-01-01

    In this work we apply the GEANT4 code of CERN to calculate the peak efficiency in High Pure Germanium (HPGe) gamma spectrometry using three different procedures. The first is a direct calculation. The second corresponds to the usual case of efficiency transfer between two different configurations at constant emission energy assuming a reference point detection configuration and the third, a new procedure, consists on the transfer of the peak efficiency between two detection configurations emitting the gamma ray in different energies assuming a "virtual" reference point detection configuration. No pre-optimization of the detector geometrical characteristics was performed before the transfer to test the ability of the efficiency transfer to reduce the effect of the ignorance on their real magnitude on the quality of the transferred efficiency. The obtained and measured efficiencies were found in good agreement for the two investigated methods of efficiency transfer. The obtained agreement proves that Monte Carlo method and especially the GEANT4 code constitute an efficient tool to obtain accurate detection efficiency values. The second investigated efficiency transfer procedure is useful to calibrate the HPGe gamma detector for any emission energy value for a voluminous source using one point source detection efficiency emitting in a different energy as a reference efficiency. The calculations preformed in this work were applied to the measurement exercise of the EUROMET428 project. A measurement exercise where an evaluation of the full energy peak efficiencies in the energy range 60-2000 keV for a typical coaxial p-type HpGe detector and several types of source configuration: point sources located at various distances from the detector and a cylindrical box containing three matrices was performed.

  8. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

    PubMed Central

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  9. PESTICIDE TRANSFER EFFICIENCY FROM HOUSEHOLD SURFACES TO FOODS

    EPA Science Inventory

    Application of pesticides around homes presents a potential for exposure to young children. Contaminated surfaces can be contacted by children's hands or foods which could allow transfer of pesticides. The exposures caused by these contacts are uncertain because the amount of pes...

  10. Using business intelligence for efficient inter-facility patient transfer.

    PubMed

    Haque, Waqar; Derksen, Beth Ann; Calado, Devin; Foster, Lee

    2015-01-01

    In the context of inter-facility patient transfer, a transfer operator must be able to objectively identify a destination which meets the needs of a patient, while keeping in mind each facility's limitations. We propose a solution which uses Business Intelligence (BI) techniques to analyze data related to healthcare infrastructure and services, and provides a web based system to identify optimal destination(s). The proposed inter-facility transfer system uses a single data warehouse with an Online Analytical Processing (OLAP) cube built on top that supplies analytical data to multiple reports embedded in web pages. The data visualization tool includes map based navigation of the health authority as well as an interactive filtering mechanism which finds facilities meeting the selected criteria. The data visualization is backed by an intuitive data entry web form which safely constrains the data, ensuring consistency and a single version of truth. The overall time required to identify the destination for inter-facility transfers is reduced from hours to a few minutes with this interactive solution.

  11. CHARACTERIZING PESTICIDE RESIDUE TRANSFER EFFICIENCIES USING FLUORESCENT TRACER IMAGING TECHNIQUES

    EPA Science Inventory

    To reduce the uncertainty associated with current estimates of children's exposure to pesticides by dermal contact and non-dietary ingestion, residue transfer data are required. Prior to conducting exhaustive studies, a screening study was conducted to identify the important pa...

  12. Energy efficient building design. A transfer guide for local governments

    SciTech Connect

    Not Available

    1992-03-01

    The fundamental concepts of the building design process, energy codes and standards, and energy budgets are introduced. These tools were combined into Energy Design Guidelines and design contract requirements. The Guidelines were repackaged for a national audience and a videotape for selling the concept to government executives. An effort to test transfer of the Guidelines to outside agencies is described.

  13. Retroviral-mediated transfer of the human glucocerebrosidase gene into cultured Gaucher bone marrow.

    PubMed Central

    Nolta, J A; Yu, X J; Bahner, I; Kohn, D B

    1992-01-01

    Gaucher disease, a lysosomal glycolipid storage disorder, results from the genetic deficiency of an acidic glucosidase, glucocerebrosidase (GC). The beneficial effects of allogeneic bone marrow transplantation (BMT) for Gaucher disease suggest that GC gene transduction and the transplantation of autologous hematopoietic stem cells (gene therapy) may similarly alleviate symptoms. We have constructed a retroviral vector, L-GC, produced by a clone of the amphotropic packaging cell line PA317, which transduces the normal human GC cDNA with high efficiency. Whole-marrow mononuclear cells and CD34-enriched cells from a 4-yr-old female with type 3 Gaucher disease were transduced by the L-GC vector and studied in long-term bone marrow culture (LTBMC). Prestimulation of marrow with IL-3 and IL-6, followed by co-cultivation with vector-producing fibroblasts, produced gene transfer into 40-45% of the hematopoietic progenitor cells. The levels of GC expression in progeny cells (primarily mature myelomonocytic) produced by the LTBMC were quantitatively analyzed by Northern blot, Western blot, and glucocerebrosidase enzyme assay. Normal levels of GC RNA, immunoreactive protein, and enzymatic activity were detected throughout the duration of culture. These studies demonstrate that retroviral vectors can efficiently transfer the GC gene into long-lived hematopoietic progenitor cells from the bone marrow of patients with Gaucher disease and express physiologically relevant levels of GC enzyme activity. Images PMID:1379609

  14. Site-specific gene transfer into the rat spinal cord by photomechanical waves

    NASA Astrophysics Data System (ADS)

    Ando, Takahiro; Sato, Shunichi; Toyooka, Terushige; Uozumi, Yoichi; Nawashiro, Hiroshi; Ashida, Hiroshi; Obara, Minoru

    2011-10-01

    Nonviral, site-specific gene delivery to deep tissue is required for gene therapy of a spinal cord injury. However, an efficient method satisfying these requirements has not been established. This study demonstrates efficient and targeted gene transfer into the spinal cord by using photomechanical waves (PMWs), which were generated by irradiating a black laser absorbing rubber with 532-nm nanosecond Nd:YAG laser pulses. After a solution of plasmid DNA coding for enhanced green fluorescent protein (EGFP) or luciferase was intraparenchymally injected into the spinal cord, PMWs were applied to the target site. In the PMW application group, we observed significant EGFP gene expression in the white matter and remarkably high luciferase activity only in the spinal cord segment exposed to the PMWs. We also assessed hind limb movements 24 h after the application of PMWs based on the Basso-Beattie-Bresnahan (BBB) score to evaluate the noninvasiveness of this method. Locomotor evaluation showed no significant decrease in BBB score under optimum laser irradiation conditions. These findings demonstrated that exogenous genes can be efficiently and site-selectively delivered into the spinal cord by applying PMWs without significant locomotive damage.

  15. Adaptive Horizontal Gene Transfers between Multiple Cheese-Associated Fungi.

    PubMed

    Ropars, Jeanne; Rodríguez de la Vega, Ricardo C; López-Villavicencio, Manuela; Gouzy, Jérôme; Sallet, Erika; Dumas, Émilie; Lacoste, Sandrine; Debuchy, Robert; Dupont, Joëlle; Branca, Antoine; Giraud, Tatiana

    2015-10-05

    Domestication is an excellent model for studies of adaptation because it involves recent and strong selection on a few, identified traits [1-5]. Few studies have focused on the domestication of fungi, with notable exceptions [6-11], despite their importance to bioindustry [12] and to a general understanding of adaptation in eukaryotes [5]. Penicillium fungi are ubiquitous molds among which two distantly related species have been independently selected for cheese making-P. roqueforti for blue cheeses like Roquefort and P. camemberti for soft cheeses like Camembert. The selected traits include morphology, aromatic profile, lipolytic and proteolytic activities, and ability to grow at low temperatures, in a matrix containing bacterial and fungal competitors [13-15]. By comparing the genomes of ten Penicillium species, we show that adaptation to cheese was associated with multiple recent horizontal transfers of large genomic regions carrying crucial metabolic genes. We identified seven horizontally transferred regions (HTRs) spanning more than 10 kb each, flanked by specific transposable elements, and displaying nearly 100% identity between distant Penicillium species. Two HTRs carried genes with functions involved in the utilization of cheese nutrients or competition and were found nearly identical in multiple strains and species of cheese-associated Penicillium fungi, indicating recent selective sweeps; they were experimentally associated with faster growth and greater competitiveness on cheese and contained genes highly expressed in the early stage of cheese maturation. These findings have industrial and food safety implications and improve our understanding of the processes of adaptation to rapid environmental changes.

  16. Variation in net trophic transfer efficiencies among 21 PCB congeners

    USGS Publications Warehouse

    Madenjian, C.P.; Schmidt, L.J.; Chernyak, S.M.; Elliott, R.F.; Desorcie, T.J.; Quintal, R.T.; Begnoche, L.J.; Hesselberg, R.J.

    1999-01-01

    We tested the hypothesis that the efficiency with which fish retain polychlorinated biphenyl (PCB) congeners from their food strongly depends on Kow and degree of chlorination of the congener. We used diet information, determinations of concentrations of individual PCB congeners in both coho salmon (Oncorhynchus kisutch) and their prey, and bioenergetics modeling to estimate the efficiencies with which Lake Michigan coho salmon retain various PCB congeners from their food. The retention efficiency for the tetrachloro congeners averaged 38%, whereas retention efficiencies for higher chlorinated congeners ranged from 43 to 56%. Not including tetrachloro congeners, we found neither decreasing nor increasing trends in the efficiencies with which the coho salmon retained the PCB congeners from their food with either increasing Kow or increasing degree of chlorination of the PCB congeners. We concluded that (a) for PCB congeners with 5−8 chlorine atoms/molecule, Kow and degree of chlorination had little influence on the efficiency with which coho salmon retained the various PCB congeners in their food, and (b) the efficiency with which coho salmon retained tetrachloro PCB congeners in their food appeared to be slightly lower than that for higher chlorinated PCB congeners.

  17. Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration.

    PubMed

    Nomikou, Nikolitsa; Feichtinger, Georg A; Redl, Heinz; McHale, Anthony P

    2016-01-01

    It has been suggested that gene transfer into donor cells is an efficient and practical means of locally supplying requisite growth factors for applications in tissue regeneration. Here we describe, for the first time, an ultrasound-mediated system that can non-invasively facilitate gene transfer into cells entrapped within fibrin-based matrices. Since ultrasound-mediated gene transfer is enhanced using microbubbles, we compared the efficacy of neutral and cationic forms of these reagents on the ultrasound-stimulated gene transfer process in gel matrices. In doing so we demonstrated the beneficial effects associated with the use of cationic microbubble preparations that interact directly with cells and nucleic acid within matrices. In some cases, gene expression was increased two-fold in gel matrices when cationic microbubbles were compared with neutral microbubbles. In addition, incorporating collagen into fibrin gels yielded a 25-fold increase in gene expression after application of ultrasound to microbubble-containing matrices. We suggest that this novel system may facilitate non-invasive temporal and spatial control of gene transfer in gel-based matrices for the purposes of tissue regeneration.

  18. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.

    PubMed

    Deverman, Benjamin E; Pravdo, Piers L; Simpson, Bryan P; Kumar, Sripriya Ravindra; Chan, Ken Y; Banerjee, Abhik; Wu, Wei-Li; Yang, Bin; Huber, Nina; Pasca, Sergiu P; Gradinaru, Viviana

    2016-02-01

    Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.

  19. Gene therapy of the central nervous system: general considerations on viral vectors for gene transfer into the brain.

    PubMed

    Serguera, C; Bemelmans, A-P

    2014-12-01

    The last decade has nourished strong doubts on the beneficial prospects of gene therapy for curing fatal diseases. However, this climate of reservation is currently being transcended by the publication of several successful clinical protocols, restoring confidence in the appropriateness of therapeutic gene transfer. A strong sign of this present enthusiasm for gene therapy by clinicians and industrials is the market approval of the therapeutic viral vector Glybera, the first commercial product in Europe of this class of drug. This new field of medicine is particularly attractive when considering therapies for a number of neurological disorders, most of which are desperately waiting for a satisfactory treatment. The central nervous system is indeed a very compliant organ where gene transfer can be stable and successful if provided through an appropriate strategy. The purpose of this review is to present the characteristics of the most efficient virus-derived vectors used by researchers and clinicians to genetically modify particular cell types or whole regions of the brain. In addition, we discuss major issues regarding side effects, such as genotoxicity and immune response associated to the use of these vectors.

  20. Efficiency of pulse high-current generator energy transfer into plasma liner energy

    NASA Astrophysics Data System (ADS)

    Oreshkin, V. I.

    2013-08-01

    The efficiency of capacitor-bank energy transfer from a high-current pulse generator into kinetic energy of a plasma liner has been analyzed. The analysis was performed using a model including the circuit equations and equations of the cylindrical shell motion. High efficiency of the energy transfer into kinetic energy of the liner is shown to be achieved only by a low-inductance generator. We considered an "ideal" liner load in which the load current is close to zero in the final of the shell compression. This load provides a high (up to 80%) efficiency of energy transfer and higher stability when compressing the liner.

  1. Massive Mitochondrial Gene Transfer in a Parasitic Flowering Plant Clade

    PubMed Central

    Bradley, Robert K.; Sugumaran, M.; Marx, Christopher J.; Rest, Joshua S.; Davis, Charles C.

    2013-01-01

    Recent studies have suggested that plant genomes have undergone potentially rampant horizontal gene transfer (HGT), especially in the mitochondrial genome. Parasitic plants have provided the strongest evidence of HGT, which appears to be facilitated by the intimate physical association between the parasites and their hosts. A recent phylogenomic study demonstrated that in the holoparasite Rafflesia cantleyi (Rafflesiaceae), whose close relatives possess the world's largest flowers, about 2.1% of nuclear gene transcripts were likely acquired from its obligate host. Here, we used next-generation sequencing to obtain the 38 protein-coding and ribosomal RNA genes common to the mitochondrial genomes of angiosperms from R. cantleyi and five additional species, including two of its closest relatives and two host species. Strikingly, our phylogenetic analyses conservatively indicate that 24%–41% of these gene sequences show evidence of HGT in Rafflesiaceae, depending on the species. Most of these transgenic sequences possess intact reading frames and are actively transcribed, indicating that they are potentially functional. Additionally, some of these transgenes maintain synteny with their donor and recipient lineages, suggesting that native genes have likely been displaced via homologous recombination. Our study is the first to comprehensively assess the magnitude of HGT in plants involving a genome (i.e., mitochondria) and a species interaction (i.e., parasitism) where it has been hypothesized to be potentially rampant. Our results establish for the first time that, although the magnitude of HGT involving nuclear genes is appreciable in these parasitic plants, HGT involving mitochondrial genes is substantially higher. This may represent a more general pattern for other parasitic plant clades and perhaps more broadly for angiosperms. PMID:23459037

  2. Massive mitochondrial gene transfer in a parasitic flowering plant clade.

    PubMed

    Xi, Zhenxiang; Wang, Yuguo; Bradley, Robert K; Sugumaran, M; Marx, Christopher J; Rest, Joshua S; Davis, Charles C

    2013-01-01

    Recent studies have suggested that plant genomes have undergone potentially rampant horizontal gene transfer (HGT), especially in the mitochondrial genome. Parasitic plants have provided the strongest evidence of HGT, which appears to be facilitated by the intimate physical association between the parasites and their hosts. A recent phylogenomic study demonstrated that in the holoparasite Rafflesia cantleyi (Rafflesiaceae), whose close relatives possess the world's largest flowers, about 2.1% of nuclear gene transcripts were likely acquired from its obligate host. Here, we used next-generation sequencing to obtain the 38 protein-coding and ribosomal RNA genes common to the mitochondrial genomes of angiosperms from R. cantleyi and five additional species, including two of its closest relatives and two host species. Strikingly, our phylogenetic analyses conservatively indicate that 24%-41% of these gene sequences show evidence of HGT in Rafflesiaceae, depending on the species. Most of these transgenic sequences possess intact reading frames and are actively transcribed, indicating that they are potentially functional. Additionally, some of these transgenes maintain synteny with their donor and recipient lineages, suggesting that native genes have likely been displaced via homologous recombination. Our study is the first to comprehensively assess the magnitude of HGT in plants involving a genome (i.e., mitochondria) and a species interaction (i.e., parasitism) where it has been hypothesized to be potentially rampant. Our results establish for the first time that, although the magnitude of HGT involving nuclear genes is appreciable in these parasitic plants, HGT involving mitochondrial genes is substantially higher. This may represent a more general pattern for other parasitic plant clades and perhaps more broadly for angiosperms.

  3. In vivo Cytokine Gene Transfer by Gene Gun Reduces Tumor Growth in Mice

    NASA Astrophysics Data System (ADS)

    Sun, Wenn H.; Burkholder, Joseph K.; Sun, Jian; Culp, Jerilyn; Turner, Joel; Lu, Xing G.; Pugh, Thomas D.; Ershler, William B.; Yang, Ning-Sun

    1995-03-01

    Implantation of tumor cells modified by in vitro cytokine gene transfer has been shown by many investigators to result in potent in vivo antitumor activities in mice. Here we describe an approach to tumor immunotherapy utilizing direct transfection of cytokine genes into tumorbearing animals by particle-mediated gene transfer. In vivo transfection of the human interleukin 6 gene into the tumor site reduced methylcholanthrene-induced fibrosarcoma growth, and a combination of murine tumor necrosis factor α and interferon γ genes inhibited growth of a renal carcinoma tumor model (Renca). In addition, treatment with murine interleukin 2 and interferon γ genes prolonged the survival of Renca tumor-bearing mice and resulted in tumor eradication in 25% of the test animals. Transgene expression was demonstrated in treated tissues by ELISA and immunohistochemical analysis. Significant serum levels of interleukin 6 and interferon γ were detected, demonstrating effective secretion of transgenic proteins from treated skin into the bloodstream. This in vivo cytokine gene therapy approach provides a system for evaluating the antitumor properties of various cytokines in different tumor models and has potential utility for human cancer gene therapy.

  4. Horizontal gene transfer and recombination in Streptococcus dysgalactiae subsp. equisimilis

    PubMed Central

    McNeilly, Celia L.; McMillan, David J.

    2014-01-01

    Streptococcus dysgalactiae subsp. equisimilis (SDSE) is a human pathogen that colonizes the skin or throat, and causes a range of diseases from relatively benign pharyngitis to potentially fatal invasive diseases. While not as virulent as the close relative Streptococcus pyogenes the two share a number of virulence factors and are known to coexist in a human host. Both pre- and post-genomic studies have revealed that horizontal gene transfer (HGT) and recombination occurs between these two organisms and plays a major role in shaping the population structure of SDSE. This review summarizes our current knowledge of HGT and recombination in the evolution of SDSE. PMID:25566202

  5. Elongation and gene expression in bovine cloned embryos transferred to temporary recipients.

    PubMed

    Rodríguez-Alvarez, Lleretny; Cox, José; Navarrete, Felipe; Valdés, Cristián; Zamorano, Teresa; Einspanier, Ralf; Castro, Fidel Ovidio

    2009-11-01

    SummaryElongated embryos provide a unique source of information about trophoblastic differentiation, gene expression and maternal-embryonic interactions; however they are difficult and costly to obtain, especially elongated cloned embryos. One alternative is their production in heterologous temporary recipients such as sheep and goats. We aimed to produce elongated bovine cloned embryos using heterologous transfer to temporary recipients. Day-7 cloned cattle blastocysts were transferred to the uteri of ewes and goats and recovered as elongated structures at day 17. We evaluated elongation, length, presence of embryonic disc and expression of several important genes for embryonic development. We also produced homologous (cloned cattle embryos transferred into cattle uteri). Cloned bovine blastocysts were able to proceed with preimplantation development through elongation with high efficiency despite the species to which they were transferred. In qualitative and quantitative RT-PCR experiments we found differences in the pattern of gene expression among embryos recovered from different species. Sox2, Nanog and FGF-4 were markedly deregulated. No previous reports about the expression pattern of the studied genes had been published for elongated bovine cloned embryos produced in intermediate recipients, furthermore, the pattern of expression of Nanog, Oct4, Eomes, Cdx2, IFN-tau, Dicer, FGF-4 and Sox2 shown here are novel for elongated cloned bovine embryos created by hand-made cloning. Our data confirmed that sheep and goats can be used as temporary recipients. This model could serve as a basis for further research on gene expression and cellular changes during bovine peri-implantation development.

  6. Passive immunization against HIV/AIDS by antibody gene transfer.

    PubMed

    Yang, Lili; Wang, Pin

    2014-01-27

    Despite tremendous efforts over the course of many years, the quest for an effective HIV vaccine by the classical method of active immunization remains largely elusive. However, two recent studies in mice and macaques have now demonstrated a new strategy designated as Vectored ImmunoProphylaxis (VIP), which involves passive immunization by viral vector-mediated delivery of genes encoding broadly neutralizing antibodies (bnAbs) for in vivo expression. Robust protection against virus infection was observed in preclinical settings when animals were given VIP to express monoclonal neutralizing antibodies. This unorthodox approach raises new promise for combating the ongoing global HIV pandemic. In this article, we survey the status of antibody gene transfer, review the revolutionary progress on isolation of extremely bnAbs, detail VIP experiments against HIV and its related virus conduced in humanized mice and macaque monkeys, and discuss the pros and cons of VIP and its opportunities and challenges towards clinical applications to control HIV/AIDS endemics.

  7. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes.

    PubMed Central

    Peng, H; Armentano, D; MacKenzie-Graham, L; Shen, R F; Darlington, G; Ledley, F D; Woo, S L

    1988-01-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. We report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human alpha 1-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating form the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the alpha 1-antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes. Images PMID:3186716

  8. Laterally Transferred Gene Recruited as a Venom in Parasitoid Wasps.

    PubMed

    Martinson, Ellen O; Martinson, Vincent G; Edwards, Rachel; Mrinalini; Werren, John H

    2016-04-01

    Parasitoid wasps use venom to manipulate the immunity and metabolism of their host insects in a variety of ways to provide resources for their offspring. Yet, how genes are recruited and evolve to perform venom functions remain open questions. A recently recognized source of eukaryotic genome innovation is lateral gene transfer (LGT). Glycoside hydrolase family 19 (GH19) chitinases are widespread in bacteria, microsporidia, and plants where they are used in nutrient acquisition or defense, but have previously not been known in metazoans. In this study, a GH19 chitinase LGT is described from the unicellular microsporidia/Rozella clade into parasitoid wasps of the superfamily Chalcidoidea, where it has become recruited as a venom protein. The GH19 chitinase is present in 15 species of chalcidoid wasps representing four families, and phylogenetic analysis indicates that it was laterally transferred near or before the origin of Chalcidoidea (∼95 Ma). The GH19 chitinase gene is highly expressed in the venom gland of at least seven species, indicating a role in the complex host manipulations performed by parasitoid wasp venom. RNAi knockdown in the model parasitoid Nasonia vitripennis reveals that-following envenomation-the GH19 chitinase induces fly hosts to upregulate genes involved in an immune response to fungi. A second, independent LGT of GH19 chitinase from microsporidia into mosquitoes was also found, also supported by phylogenetic reconstructions. Besides these two LGT events, GH19 chitinase is not found in any other sequenced animal genome, or in any fungi outside the microsporidia/Rozella clade.

  9. Efficiently finding regulatory elements using correlation with gene expression.

    PubMed

    Bannai, Hideo; Inenaga, Shunsuke; Shinohara, Ayumi; Takeda, Masayuki; Miyano, Satoru

    2004-06-01

    We present an efficient algorithm for detecting putative regulatory elements in the upstream DNA sequences of genes, using gene expression information obtained from microarray experiments. Based on a generalized suffix tree, our algorithm looks for motif patterns whose appearance in the upstream region is most correlated with the expression levels of the genes. We are able to find the optimal pattern, in time linear in the total length of the upstream sequences. We implement and apply our algorithm to publicly available microarray gene expression data, and show that our method is able to discover biologically significant motifs, including various motifs which have been reported previously using the same data set. We further discuss applications for which the efficiency of the method is essential, as well as possible extensions to our algorithm.

  10. Genome-wide experimental determination of barriers to horizontal gene transfer

    SciTech Connect

    Rubin, Edward; Sorek, Rotem; Zhu, Yiwen; Creevey, Christopher J.; Francino, M. Pilar; Bork, Peer; Rubin, Edward M.

    2007-09-24

    Horizontal gene transfer, in which genetic material is transferred from the genome of one organism to another, has been investigated in microbial species mainly through computational sequence analyses. To address the lack of experimental data, we studied the attempted movement of 246,045 genes from 79 prokaryotic genomes into E. coli and identified genes that consistently fail to transfer. We studied the mechanisms underlying transfer inhibition by placing coding regions from different species under the control of inducible promoters. Their toxicity to the host inhibited transfer regardless of the species of origin and our data suggest that increased gene dosage and associated increased expression is a predominant cause for transfer failure. While these experimental studies examined transfer solely into E. coli, a computational analysis of gene transfer rates across available bacterial and archaeal genomes indicates that the barriers observed in our study are general across the tree of life.

  11. Interspecific evolution: microbial symbiosis, endosymbiosis and gene transfer.

    PubMed

    Hoffmeister, Meike; Martin, William

    2003-08-01

    Microbial symbioses are interesting in their own right and also serve as exemplary models to help biologists to understand two important symbioses in the evolutionary past of eukaryotic cells: the origins of chloroplasts and mitochondria. Most, if not all, microbial symbioses have a chemical basis: compounds produced by one partner are useful for the other. But symbioses can also entail the transfer of genes from one partner to the other, which in some cases cements two cells into a bipartite, co-evolving unit. Here, we discuss some microbial symbioses in which progress is being made in uncovering the nature of symbiotic interactions: anaerobic methane-oxidizing consortia, marine worms that possess endosymbionts instead of a digestive tract, amino acid-producing endosymbionts of aphids, prokaryotic endosymbionts living within a prokaryotic host within mealybugs, endosymbionts of an insect vector of human disease and a photosynthetic sea slug that steals chloroplasts from algae. In the case of chloroplasts and mitochondria, examples of recent and ancient gene transfer to the chromosomes of their host cell illustrate the process of genetic merger in the wake of organelle origins.

  12. Resistance Gene Transfer during Treatments for Experimental Avian Colibacillosis

    PubMed Central

    Dheilly, Alexandra; Le Devendec, Laëtitia; Mourand, Gwenaëlle; Bouder, Axelle; Jouy, Eric

    2012-01-01

    An experiment was conducted in animal facilities to compare the impacts of four avian colibacillosis treatments—oxytetracycline (OTC), trimethoprim-sulfadimethoxine (SXT), amoxicillin (AMX), or enrofloxacin (ENR)—on the susceptibility of Escherichia coli in broiler intestinal tracts. Birds were first orally inoculated with rifampin-resistant E. coli strains bearing plasmid genes conferring resistance to fluoroquinolones (qnr), cephalosporins (blaCTX-M or blaFOX), trimethoprim-sulfonamides, aminoglycosides, or tetracyclines. Feces samples were collected before, during, and after antimicrobial treatments. The susceptibilities of E. coli strains were studied, and resistance gene transfer was analyzed. An increase in the tetracycline-resistant E. coli population was observed only in OTC-treated birds, whereas multiresistant E. coli was detected in the dominant E. coli populations of SXT-, AMX-, or ENR-treated birds. Most multiresistant E. coli strains were susceptible to rifampin and exhibited various pulsed-field gel electrophoresis profiles, suggesting the transfer of one of the multiresistance plasmids from the inoculated strains to other E. coli strains in the intestinal tract. In conclusion, this study clearly illustrates how, in E. coli, “old” antimicrobials may coselect antimicrobial resistance to recent and critical molecules. PMID:21986830

  13. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

    PubMed Central

    Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

    2015-01-01

    ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

  14. High-efficiency quantum state transfer and quantum memory using a mechanical oscillator

    NASA Astrophysics Data System (ADS)

    Sete, Eyob A.; Eleuch, H.

    2015-03-01

    We analyze an optomechanical system that can be used to efficiently transfer a quantum state between an optical cavity and a distant mechanical oscillator coupled to a second optical cavity. We show that for a moderate mechanical Q factor it is possible to achieve a transfer efficiency of 99.4 % by using adjustable cavity damping rates and destructive interference. We also show that the quantum mechanical oscillator can be used as a quantum memory device with an efficiency of 96 % employing a pulsed optomechanical coupling. Although the mechanical dissipation slightly decreases the efficiency, its effect can be significantly reduced by designing a high-Q mechanical oscillator.

  15. Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells.

    PubMed

    Sapinoro, Ramil; Volcy, Ketna; Rodrigo, W W Shanaka I; Schlesinger, Jacob J; Dewhurst, Stephen

    2008-04-10

    Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

  16. Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages.

    PubMed

    Chen, John; Carpena, Nuria; Quiles-Puchalt, Nuria; Ram, Geeta; Novick, Richard P; Penadés, José R

    2015-05-01

    Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.

  17. Effect of dowel bar looseness on measured load transfer efficiency using FWD load

    NASA Astrophysics Data System (ADS)

    Shoukry, Samir N.; William, Gergis W.; Riad, Mourad Y.

    2001-07-01

    The effect of dowel bar looseness on the joint load transfer efficiency using Falling Weight Deflectometer is the subject of this paper. The mechanism of dynamic load transfer at transverse joints of Jointed Plain Concrete Pavement is examined using nonlinear 3D finite element analysis.

  18. A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in vivo: Polyethylenimine

    NASA Astrophysics Data System (ADS)

    Boussif, Otmane; Lezoualc'h, Frank; Zanta, Maria Antonietta; Djavaheri Mergny, Mojgan; Scherman, Daniel; Demeneix, Barbara; Behr, Jean-Paul

    1995-08-01

    Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se-i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its genedelivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

  19. Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts.

    PubMed

    Smith, Craig; Berg, Debbie; Beaumont, Sue; Standley, Neil T; Wells, David N; Pfeffer, Peter L

    2007-01-01

    During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4, Otx2, Ifitm3, GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (beta-actin, beta-tubulin and GAPDH) in 21 NT blastocysts with that in genetically half-identical in vitro produced (IVP, n=19) and in vivo (n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts. Ifitm3 expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NT and IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP or in vivo embryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer. Oct4 levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed that in

  20. Adaptive Horizontal Gene Transfers between Multiple Cheese-Associated Fungi

    PubMed Central

    Ropars, Jeanne; Rodríguez de la Vega, Ricardo C.; López-Villavicencio, Manuela; Gouzy, Jérôme; Sallet, Erika; Dumas, Émilie; Lacoste, Sandrine; Debuchy, Robert; Dupont, Joëlle; Branca, Antoine; Giraud, Tatiana

    2015-01-01

    Summary Domestication is an excellent model for studies of adaptation because it involves recent and strong selection on a few, identified traits [1–5]. Few studies have focused on the domestication of fungi, with notable exceptions [6–11], despite their importance to bioindustry [12] and to a general understanding of adaptation in eukaryotes [5]. Penicillium fungi are ubiquitous molds among which two distantly related species have been independently selected for cheese making—P. roqueforti for blue cheeses like Roquefort and P. camemberti for soft cheeses like Camembert. The selected traits include morphology, aromatic profile, lipolytic and proteolytic activities, and ability to grow at low temperatures, in a matrix containing bacterial and fungal competitors [13–15]. By comparing the genomes of ten Penicillium species, we show that adaptation to cheese was associated with multiple recent horizontal transfers of large genomic regions carrying crucial metabolic genes. We identified seven horizontally transferred regions (HTRs) spanning more than 10 kb each, flanked by specific transposable elements, and displaying nearly 100% identity between distant Penicillium species. Two HTRs carried genes with functions involved in the utilization of cheese nutrients or competition and were found nearly identical in multiple strains and species of cheese-associated Penicillium fungi, indicating recent selective sweeps; they were experimentally associated with faster growth and greater competitiveness on cheese and contained genes highly expressed in the early stage of cheese maturation. These findings have industrial and food safety implications and improve our understanding of the processes of adaptation to rapid environmental changes. PMID:26412136

  1. Gene transfer into older chicken embryos by ex ovo electroporation.

    PubMed

    Luo, Jiankai; Yan, Xin; Lin, Juntang; Rolfs, Arndt

    2012-07-27

    The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos(1). Different structures such as DNA plasmids encoding genes(2-4), small interfering RNA (siRNA) plasmids(5), small synthetic RNA oligos(6), and morpholino antisense oligonucleotides(7) can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20--according to Hamburg and Hamilton)(8) and there are some disadvantages for its application in embryos at later stages (older than stage HH22--approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell. In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes(9) and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos(10-12).

  2. Comparative analysis of magnetosome gene clusters in magnetotactic bacteria provides further evidence for horizontal gene transfer.

    PubMed

    Jogler, Christian; Kube, Michael; Schübbe, Sabrina; Ullrich, Susanne; Teeling, Hanno; Bazylinski, Dennis A; Reinhardt, Richard; Schüler, Dirk

    2009-05-01

    The organization of magnetosome genes was analysed in all available complete or partial genomic sequences of magnetotactic bacteria (MTB), including the magnetosome island (MAI) of the magnetotactic marine vibrio strain MV-1 determined in this study. The MAI was found to differ in gene content and organization between Magnetospirillum species and strains MV-1 or MC-1. Although a similar organization of magnetosome genes was found in all MTB, distinct variations in gene order and sequence similarity were uncovered that may account for the observed diversity of biomineralization, cell biology and magnetotaxis found in various MTB. While several magnetosome genes were present in all MTB, others were confined to Magnetospirillum species, indicating that the minimal set of genes required for magnetosome biomineralization might be smaller than previously suggested. A number of novel candidate genes were implicated in magnetosome formation by gene cluster comparison. Based on phylogenetic and compositional evidence we present a model for the evolution of magnetotaxis within the Alphaproteobacteria, which suggests the independent horizontal transfer of magnetosome genes from an unknown ancestor of magnetospirilla into strains MC-1 and MV-1.

  3. Gene transfer properties and structural modeling of human stem cell-derived AAV.

    PubMed

    Smith, Laura J; Ul-Hasan, Taihra; Carvaines, Sarah K; Van Vliet, Kim; Yang, Ethel; Wong, Kamehameha K; Agbandje-McKenna, Mavis; Chatterjee, Saswati

    2014-09-01

    Adeno-associated virus (AAV) vectors are proving to be remarkably successful for in vivo gene delivery. Based upon reports of abundant AAV in the human marrow, we tested CD34(+) hematopoietic stem cells for the presence of natural AAV. Here, we report for the first time, the presence of novel AAV variants in healthy CD34(+) human peripheral blood stem cells. The majority of healthy peripheral blood stem cell donors were found to harbor AAV in their CD34(+) cells. Every AAV isolated from CD34(+) cells mapped to AAV Clade F. Gene transfer vectors derived from these novel AAVs efficiently underwent entry and postentry processing in human cord blood stem cells and supported stable gene transfer into long-term, in vivo engrafting human HSCs significantly better than other serotypes. AAVHSC-transduced human CD34(+) cells engrafted in vivo and gave rise to differentiated transgene-expressing progeny. Importantly, gene-marked CD34(+) stem cells persisted long term in xenograft recipients, indicating transduction of primitive progenitors. Notably, correlation of structure with function permitted identification of potential capsid components important for HSC transduction. Thus, AAVHSCs represent a new class of genetic vectors for the manipulation of HSC genomes.

  4. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer.

    PubMed

    Zhou, Xiaoqing; Xin, Jige; Fan, Nana; Zou, Qingjian; Huang, Jiao; Ouyang, Zhen; Zhao, Yu; Zhao, Bentian; Liu, Zhaoming; Lai, Sisi; Yi, Xiaoling; Guo, Lin; Esteban, Miguel A; Zeng, Yangzhi; Yang, Huaqiang; Lai, Liangxue

    2015-03-01

    The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR (-/-) pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 (-/-)/PINK1 (-/-) pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.

  5. Phylogenetic analyses of cyanobacterial genomes: Quantification of horizontal gene transfer events

    PubMed Central

    Zhaxybayeva, Olga; Gogarten, J. Peter; Charlebois, Robert L.; Doolittle, W. Ford; Papke, R. Thane

    2006-01-01

    Using 1128 protein-coding gene families from 11 completely sequenced cyanobacterial genomes, we attempt to quantify horizontal gene transfer events within cyanobacteria, as well as between cyanobacteria and other phyla. A novel method of detecting and enumerating potential horizontal gene transfer events within a group of organisms based on analyses of “embedded quartets” allows us to identify phylogenetic signal consistent with a plurality of gene families, as well as to delineate cases of conflict to the plurality signal, which include horizontally transferred genes. To infer horizontal gene transfer events between cyanobacteria and other phyla, we added homologs from 168 available genomes. We screened phylogenetic trees reconstructed for each of these extended gene families for highly supported monophyly of cyanobacteria (or lack of it). Cyanobacterial genomes reveal a complex evolutionary history, which cannot be represented by a single strictly bifurcating tree for all genes or even most genes, although a single completely resolved phylogeny was recovered from the quartets’ plurality signals. We find more conflicts within cyanobacteria than between cyanobacteria and other phyla. We also find that genes from all functional categories are subject to transfer. However, in interphylum as compared to intraphylum transfers, the proportion of metabolic (operational) gene transfers increases, while the proportion of informational gene transfers decreases. PMID:16899658

  6. Creating Efficient Instrumentation Networks to Support Parametric Risk Transfer

    NASA Astrophysics Data System (ADS)

    Rockett, P.

    2009-04-01

    The development and institutionalisation of Catastrophe modelling during the 1990s opened the way for Catastrophe risk securitization transactions in which catastrophe risk held by insurers is transferred to the capital markets in the form of a bond. Cat Bonds have been one of the few areas of the capital markets in which the risk modelling has remained secure and the returns on the bonds have held up well through the 2008 Credit Crunch. There are three ways of structuring the loss triggers on bonds: ‘indemnity triggers' - reflecting the actual losses to the issuers; ‘index triggers' reflecting the losses to some index such as reported insurance industry loss and ‘parametric triggers' reflecting the parameters of the underlying catastrophe event itself. Indemnity triggers require that the investors trust that the insurer is reporting all their underlying exposures, while both indemnity and index losses may take 1-2 years to settle before all the claims are reported and resolved. Therefore parametric structures have many advantages, in particular in that the bond can be settled rapidly after an event. The challenge is to create parametric indices that closely reflect the actual losses to the insurer - ie that minimise ‘basis risk'. First generation parametric indices had high basis risk as they were crudely based on the magnitude of an earthquake occurring within some defined geographical box, or the intensity of a hurricane relative to the distance of the storm from some location. Second generation triggers involve taking measurements of ground motion or windspeed or flood depths at many locations and weighting each value so that the overall index closely mimics insurance loss. Cat bonds with second generation parametric triggers have been successfully issued for European Windstorm, UK Flood and California and Japan Earthquake. However the spread of second generation parametric structures is limited by the availability of suitable networks of

  7. Research of Efficient C/S Model Based File Transfer Method

    NASA Astrophysics Data System (ADS)

    Li, Xubin; Zou, Quan; Jiang, Yi

    The one-to-multiple file transfer problem has not been solved under the Linux system. In this paper, we take socket programming to accomplish Client/Server model based file transfer system and develop efficient software which not only support multiple clients and files transferring simultaneously, but also support transmission resuming from breakpoint. Our research focuses on solving the multi-thread concurrency, transmission resuming from breakpoint, thread pool, buffer queue and other aspects of problem. Our work is the basic foundation of FTP servers, file transfer software in local area network and large-scale examination system, etc.

  8. In Vivo Gene Transfer Strategies to Achieve Partial Correction of von Willebrand Disease

    PubMed Central

    Wang, Lan; Rosenberg, Jonathan B.; De, Bishnu P.; Ferris, Barbara; Wang, Rui; Rivella, Stefano; Kaminsky, Stephen M.

    2012-01-01

    Abstract von Willebrand disease (VWD), the most common hereditary coagulation disorder, results from mutations in the 52-exon gene for von Willebrand factor (VWF), which encodes an 8.4-kB cDNA. Studies with VWF cDNA plasmids have demonstrated that in vivo gene transfer to the liver will correct the coagulation dysfunction in VWF−/− mice, but the correction is transient. To develop gene therapy for VWF that would mediate long-term expression of the VWF cDNA in liver, we first evaluated segmental pre-mRNA trans-splicing (SPTS) with two adeno-associated virus (AAV) serotype 8 vectors, each delivering one-half of the VWF cDNA. However, although the two vectors functioned well to generate VWF multimers after infection of cells in vitro, the efficiency of SPTS was insufficient to correct the VWF−/− mouse in vivo. As an alternative, we assessed the ability of a lentiviral vector to transfer the intact murine VWF cDNA in vivo directly to the neonatal liver of VWF−/− mice, using generation of VWF multimers, bleeding time, and bleeding volume as efficacy parameters. The VWF lentivirus generated VWF multimers and partially or completely corrected the coagulation defect on a persistent basis in 33% of the treated VWF-deficient mice. On the basis of the concept that partial persistent correction with gene transfer could be beneficial in VWD patients, these observations suggest that lentiviral delivery of VWF cDNA should be explored as a candidate for gene therapy in patients with a severe form of VWD. PMID:22482515

  9. EXCAVATOR: a computer program for efficiently mining gene expression data.

    PubMed

    Xu, Dong; Olman, Victor; Wang, Li; Xu, Ying

    2003-10-01

    Massive amounts of gene expression data are generated using microarrays for functional studies of genes and gene expression data clustering is a useful tool for studying the functional relationship among genes in a biological process. We have developed a computer package EXCAVATOR for clustering gene expression profiles based on our new framework for representing gene expression data as a minimum spanning tree. EXCAVATOR uses a number of rigorous and efficient clustering algorithms. This program has a number of unique features, including capabilities for: (i) data- constrained clustering; (ii) identification of genes with similar expression profiles to pre-specified seed genes; (iii) cluster identification from a noisy background; (iv) computational comparison between different clustering results of the same data set. EXCAVATOR can be run from a Unix/Linux/DOS shell, from a Java interface or from a Web server. The clustering results can be visualized as colored figures and 2-dimensional plots. Moreover, EXCAVATOR provides a wide range of options for data formats, distance measures, objective functions, clustering algorithms, methods to choose number of clusters, etc. The effectiveness of EXCAVATOR has been demonstrated on several experimental data sets. Its performance compares favorably against the popular K-means clustering method in terms of clustering quality and computing time.

  10. Supra-operonic clusters of functionally related genes (SOCs) are a source of horizontal gene co-transfers

    PubMed Central

    Pang, Tin Yau; Lercher, Martin J.

    2017-01-01

    Adaptation of bacteria occurs predominantly via horizontal gene transfer (HGT). While it is widely recognized that horizontal acquisitions frequently encompass multiple genes, it is unclear what the size distribution of successfully transferred DNA segments looks like and what evolutionary forces shape this distribution. Here, we identified 1790 gene family pairs that were consistently co-gained on the same branches across a phylogeny of 53 E. coli strains. We estimated a lower limit of their genomic distances at the time they were transferred to their host genomes; this distribution shows a sharp upper bound at 30 kb. The same gene-pairs can have larger distances (up to 70 kb) in other genomes. These more distant pairs likely represent recent acquisitions via transduction that involve the co-transfer of excised prophage genes, as they are almost always associated with intervening phage-associated genes. The observed distribution of genomic distances of co-transferred genes is much broader than expected from a model based on the co-transfer of genes within operons; instead, this distribution is highly consistent with the size distribution of supra-operonic clusters (SOCs), groups of co-occurring and co-functioning genes that extend beyond operons. Thus, we propose that SOCs form a basic unit of horizontal gene transfer. PMID:28067311

  11. Integrating network and transfer metrics to optimize transfer efficiency and experiment workflows

    NASA Astrophysics Data System (ADS)

    McKee, S.; Babik, M.; Campana, S.; Di Girolamo, A.; Wildish, T.; Closier, J.; Roiser, S.; Grigoras, C.; Vukotic, I.; Salichos, M.; De, Kaushik; Garonne, V.; Cruz, J. A. D.; Forti, A.; Walker, C. J.; Rand, D.; de Salvo, A.; Mazzoni, E.; Gable, I.; Chollet, F.; Caillat, L.; Schaer, F.; Chen, Hsin-Yen; Tigerstedt, U.; Duckeck, G.; Hoeft, B.; Petzold, A.; Lopez, F.; Flix, J.; Stancu, S.; Shade, J.; O'Connor, M.; Kotlyar, V.; Zurawski, J.

    2015-12-01

    The Worldwide LHC Computing Grid relies on the network as a critical part of its infrastructure and therefore needs to guarantee effective network usage and prompt detection and resolution of any network issues, including connection failures, congestion, traffic routing, etc. The WLCG Network and Transfer Metrics project aims to integrate and combine all network-related monitoring data collected by the WLCG infrastructure. This includes FTS monitoring information, monitoring data from the XRootD federation, as well as results of the perfSONAR tests. The main challenge consists of further integrating and analyzing this information in order to allow the optimizing of data transfers and workload management systems of the LHC experiments. In this contribution, we present our activity in commissioning WLCG perfSONAR network and integrating network and transfer metrics: We motivate the need for the network performance monitoring, describe the main use cases of the LHC experiments as well as status and evolution in the areas of configuration and capacity management, datastore and analytics, including integration of transfer and network metrics and operations and support.

  12. Evaluation of ADA gene expression and transduction efficiency in ADA/SCID patients undergoing gene therapy.

    PubMed

    Carlucci, F; Tabucchi, A; Aiuti, A; Rosi, F; Floccari, F; Pagani, R; Marinello, E

    2004-10-01

    A capillary electrophoresis (CE) method was developed for ADA/SCID diagnosis and monitoring of enzyme replacement therapy, as well as for exploring the transfection efficiency for different retroviral vectors in gene therapy.

  13. Personalised Adaptive Task Selection in Air Traffic Control: Effects on Training Efficiency and Transfer

    ERIC Educational Resources Information Center

    Salden, Ron J. C. M.; Paas, Fred; van Merrienboer, Jeroen J. G.

    2006-01-01

    The differential effects of four task selection methods on training efficiency and transfer in a computer-based training for Air Traffic Control were investigated. Two personalised conditions were compared with two corresponding yoked control conditions. The hypothesis that personalised adaptive task selection leads to more efficient training than…

  14. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

    DOE PAGES

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.; ...

    2015-03-02

    Some of the most damaging tree diseases are caused by pathogens that induce cankers, a stem deformation often lethal. To investigate the cause of this adaptation, we sequenced the genomes of poplar pathogens that do and do not cause cankers. We found a unique cluster of genes that produce secondary metabolites and are co-activated when the canker pathogen is grown on poplar wood and leaves. The gene genealogy is discordant with the species phylogeny, showing a signature of horizontal transfer from fungi associated with wood decay. Furthermore, genes encoding hemicellulose-degrading enzymes are up-regulated on poplar wood chips, with some havingmore » been acquired horizontally. In conclusion, we propose that adaptation to colonize poplar woody stems is the result of acquisition of these genes.« less

  15. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

    SciTech Connect

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M.; Levasseur, Anthony; Lindquist, Erika A.; Majoor, Eline; Ohm, Robin A.; Pangilinan, Jasmyn L.; Pribowo, Amadeus; Saddler, John N.; Sakalidis, Monique L.; de Vries, Ronald P.; Grigoriev, Igor V.; Goodwin, Stephen B.; Tanguay, Philippe; Hamelin, Richard C.

    2015-03-02

    Some of the most damaging tree diseases are caused by pathogens that induce cankers, a stem deformation often lethal. To investigate the cause of this adaptation, we sequenced the genomes of poplar pathogens that do and do not cause cankers. We found a unique cluster of genes that produce secondary metabolites and are co-activated when the canker pathogen is grown on poplar wood and leaves. The gene genealogy is discordant with the species phylogeny, showing a signature of horizontal transfer from fungi associated with wood decay. Furthermore, genes encoding hemicellulose-degrading enzymes are up-regulated on poplar wood chips, with some having been acquired horizontally. In conclusion, we propose that adaptation to colonize poplar woody stems is the result of acquisition of these genes.

  16. Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile

    PubMed Central

    2013-01-01

    Background Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression. Methods Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3). Results Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies. Conclusions We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer. PMID:23937994

  17. Energy Efficient Cryogenic Transfer Line with Magnetic Suspension

    NASA Astrophysics Data System (ADS)

    Shu, Quan-Sheng; Cheng, Guangfeng; Yu, Kun; Hull, John R.; Demko, Jonathan A.; Britcher, Colin P.; Fesmire, James E.; Augustynowicz, Stan D.

    2003-07-01

    An energy efficient, cost effective cryogenic distribution system (up to several miles) has been identified as important for spaceport and in-space cryogenic systems. The conduction heat loss from the supports that connect the lines cold mass to the warm support structure is ultimately the most serious heat leak after thermal radiation has been minimized. The use of magnetic levitation by permanent magnets and high temperature superconductors provides support without mechanical contact and thus, the conduction part of the heat leak can be reduced to zero. A stop structure is carefully designed to hold the center tube when the system is warm. The novel design will provide the potential of extending many missions by saving cryogens, or reducing the overall launch mass to accomplish a given mission.

  18. Efficient transfer of weather information to the pilot in flight

    NASA Technical Reports Server (NTRS)

    Mcfarland, R. H.

    1982-01-01

    Efficient methods for providing weather information to the pilot in flight are summarized. Use of discrete communications channels in the aeronautical, VHF band or subcarriers in the VOR navigation band are considered the best possibilities. Data rates can be provided such that inputs to the ground based transmitters from 2400 band telephone lines are easily accommodated together with additional data. The crucial weather data considered for uplinking are identified as radar reflectivity patterns relating to precipitation, spherics data, hourly sequences, nowcasts, forecasts, cloud top heights with freezing and icing conditions, the critical weather map and satellite maps. NEXRAD, the ground based, Doppler weather radar which will produce an improved weather product also encourages use of an uplink to fully utilize its capability to improve air safety.

  19. Immunomodulation by mucosal gene transfer using TGF-beta DNA.

    PubMed Central

    Kuklin, N A; Daheshia, M; Chun, S; Rouse, B T

    1998-01-01

    This report evaluates the efficacy of DNA encoding TGF-beta administered mucosally to suppress immunity and modulate the immunoinflammatory response to herpes simplex virus (HSV) infection. A single intranasal administration of an eukaryotic expression vector encoding TGF-beta1 led to expression in the lung and lymphoid tissue. T cell-mediated immune responses to HSV infection were suppressed with this effect persisting as measured by the delayed-type hypersensitivity reaction for at least 7 wk. Treated animals were more susceptible to systemic infection with HSV. Multiple prophylactic mucosal administrations of TGF-beta DNA also suppressed the severity of ocular lesions caused by HSV infection, although no effects on this immunoinflammatory response were evident after therapeutic treatment with TGF-beta DNA. Our results demonstrate that the direct mucosal gene transfer of immunomodulatory cytokines provides a convenient means of modulating immunity and influencing the expression of inflammatory disorders. PMID:9664086

  20. Horizontal gene transfer: building the web of life.

    PubMed

    Soucy, Shannon M; Huang, Jinling; Gogarten, Johann Peter

    2015-08-01

    Horizontal gene transfer (HGT) is the sharing of genetic material between organisms that are not in a parent-offspring relationship. HGT is a widely recognized mechanism for adaptation in bacteria and archaea. Microbial antibiotic resistance and pathogenicity are often associated with HGT, but the scope of HGT extends far beyond disease-causing organisms. In this Review, we describe how HGT has shaped the web of life using examples of HGT among prokaryotes, between prokaryotes and eukaryotes, and even between multicellular eukaryotes. We discuss replacement and additive HGT, the proposed mechanisms of HGT, selective forces that influence HGT, and the evolutionary impact of HGT on ancestral populations and existing populations such as the human microbiome.

  1. Statistical Mechanics of Horizontal Gene Transfer in Evolutionary Ecology

    NASA Astrophysics Data System (ADS)

    Chia, Nicholas; Goldenfeld, Nigel

    2011-04-01

    The biological world, especially its majority microbial component, is strongly interacting and may be dominated by collective effects. In this review, we provide a brief introduction for statistical physicists of the way in which living cells communicate genetically through transferred genes, as well as the ways in which they can reorganize their genomes in response to environmental pressure. We discuss how genome evolution can be thought of as related to the physical phenomenon of annealing, and describe the sense in which genomes can be said to exhibit an analogue of information entropy. As a direct application of these ideas, we analyze the variation with ocean depth of transposons in marine microbial genomes, predicting trends that are consistent with recent observations using metagenomic surveys.

  2. Extensive Intra-Kingdom Horizontal Gene Transfer Converging on a Fungal Fructose Transporter Gene

    PubMed Central

    Coelho, Marco A.; Gonçalves, Carla; Sampaio, José Paulo; Gonçalves, Paula

    2013-01-01

    Comparative genomics revealed in the last decade a scenario of rampant horizontal gene transfer (HGT) among prokaryotes, but for fungi a clearly dominant pattern of vertical inheritance still stands, punctuated however by an increasing number of exceptions. In the present work, we studied the phylogenetic distribution and pattern of inheritance of a fungal gene encoding a fructose transporter (FSY1) with unique substrate selectivity. 109 FSY1 homologues were identified in two sub-phyla of the Ascomycota, in a survey that included 241 available fungal genomes. At least 10 independent inter-species instances of horizontal gene transfer (HGT) involving FSY1 were identified, supported by strong phylogenetic evidence and synteny analyses. The acquisition of FSY1 through HGT was sometimes suggestive of xenolog gene displacement, but several cases of pseudoparalogy were also uncovered. Moreover, evidence was found for successive HGT events, possibly including those responsible for transmission of the gene among yeast lineages. These occurrences do not seem to be driven by functional diversification of the Fsy1 proteins because Fsy1 homologues from widely distant lineages, including at least one acquired by HGT, appear to have similar biochemical properties. In summary, retracing the evolutionary path of the FSY1 gene brought to light an unparalleled number of independent HGT events involving a single fungal gene. We propose that the turbulent evolutionary history of the gene may be linked to the unique biochemical properties of the encoded transporter, whose predictable effect on fitness may be highly variable. In general, our results support the most recent views suggesting that inter-species HGT may have contributed much more substantially to shape fungal genomes than heretofore assumed. PMID:23818872

  3. Applying horizontal gene transfer phenomena to enhance non-viral gene therapy

    PubMed Central

    Elmer, Jacob J.; Christensen, Matthew D.; Rege, Kaushal

    2014-01-01

    Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases. PMID:23994344

  4. Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector.

    PubMed

    Choudhury, Sourav R; Harris, Anne F; Cabral, Damien J; Keeler, Allison M; Sapp, Ellen; Ferreira, Jennifer S; Gray-Edwards, Heather L; Johnson, Jacob A; Johnson, Aime K; Su, Qin; Stoica, Lorelei; DiFiglia, Marian; Aronin, Neil; Martin, Douglas R; Gao, Guangping; Sena-Esteves, Miguel

    2016-04-01

    Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.

  5. Efficient decoding algorithms for generalized hidden Markov model gene finders

    PubMed Central

    Majoros, William H; Pertea, Mihaela; Delcher, Arthur L; Salzberg, Steven L

    2005-01-01

    Background The Generalized Hidden Markov Model (GHMM) has proven a useful framework for the task of computational gene prediction in eukaryotic genomes, due to its flexibility and probabilistic underpinnings. As the focus of the gene finding community shifts toward the use of homology information to improve prediction accuracy, extensions to the basic GHMM model are being explored as possible ways to integrate this homology information into the prediction process. Particularly prominent among these extensions are those techniques which call for the simultaneous prediction of genes in two or more genomes at once, thereby increasing significantly the computational cost of prediction and highlighting the importance of speed and memory efficiency in the implementation of the underlying GHMM algorithms. Unfortunately, the task of implementing an efficient GHMM-based gene finder is already a nontrivial one, and it can be expected that this task will only grow more onerous as our models increase in complexity. Results As a first step toward addressing the implementation challenges of these next-generation systems, we describe in detail two software architectures for GHMM-based gene finders, one comprising the common array-based approach, and the other a highly optimized algorithm which requires significantly less memory while achieving virtually identical speed. We then show how both of these architectures can be accelerated by a factor of two by optimizing their content sensors. We finish with a brief illustration of the impact these optimizations have had on the feasibility of our new homology-based gene finder, TWAIN. Conclusions In describing a number of optimizations for GHMM-based gene finders and making available two complete open-source software systems embodying these methods, it is our hope that others will be more enabled to explore promising extensions to the GHMM framework, thereby improving the state-of-the-art in gene prediction techniques. PMID:15667658

  6. Widespread impact of horizontal gene transfer on plant colonization of land

    PubMed Central

    Yue, Jipei; Hu, Xiangyang; Sun, Hang; Yang, Yongping; Huang, Jinling

    2012-01-01

    In complex multicellular eukaryotes such as animals and plants, horizontal gene transfer is commonly considered rare with very limited evolutionary significance. Here we show that horizontal gene transfer is a dynamic process occurring frequently in the early evolution of land plants. Our genome analyses of the moss Physcomitrella patens identified 57 families of nuclear genes that were acquired from prokaryotes, fungi or viruses. Many of these gene families were transferred to the ancestors of green or land plants. Available experimental evidence shows that these anciently acquired genes are involved in some essential or plant-specific activities such as xylem formation, plant defence, nitrogen recycling as well as the biosynthesis of starch, polyamines, hormones and glutathione. These findings suggest that horizontal gene transfer had a critical role in the transition of plants from aquatic to terrestrial environments. On the basis of these findings, we propose a model of horizontal gene transfer mechanism in nonvascular and seedless vascular plants. PMID:23093189

  7. Widespread impact of horizontal gene transfer on plant colonization of land.

    PubMed

    Yue, Jipei; Hu, Xiangyang; Sun, Hang; Yang, Yongping; Huang, Jinling

    2012-01-01

    In complex multicellular eukaryotes such as animals and plants, horizontal gene transfer is commonly considered rare with very limited evolutionary significance. Here we show that horizontal gene transfer is a dynamic process occurring frequently in the early evolution of land plants. Our genome analyses of the moss Physcomitrella patens identified 57 families of nuclear genes that were acquired from prokaryotes, fungi or viruses. Many of these gene families were transferred to the ancestors of green or land plants. Available experimental evidence shows that these anciently acquired genes are involved in some essential or plant-specific activities such as xylem formation, plant defence, nitrogen recycling as well as the biosynthesis of starch, polyamines, hormones and glutathione. These findings suggest that horizontal gene transfer had a critical role in the transition of plants from aquatic to terrestrial environments. On the basis of these findings, we propose a model of horizontal gene transfer mechanism in nonvascular and seedless vascular plants.

  8. Cyclen-Based Cationic Lipids for Highly Efficient Gene Delivery towards Tumor Cells

    PubMed Central

    Zhang, Yang; Ren, Jiang; Fu, Yun; Zhang, Ji; Zhu, Wen; Yu, Xiao-Qi

    2011-01-01

    Background Gene therapy has tremendous potential for both inherited and acquired diseases. However, delivery problems limited their clinical application, and new gene delivery vehicles with low cytotoxicity and high transfection efficiency are greatly required. Methods In this report, we designed and synthesized three amphiphilic molecules (L1–L3) with the structures involving 1, 4, 7, 10-tetraazacyclododecane (cyclen), imidazolium and a hydrophobic dodecyl chain. Their interactions with plasmid DNA were studied via electrophoretic gel retardation assays, fluorescent quenching experiments, dynamic light scattering and transmission electron microscopy. The in vitro gene transfection assay and cytotoxicity assay were conducted in four cell lines. Results Results indicated that L1 and L3-formed liposomes could effectively bind to DNA to form well-shaped nanoparticles. Combining with neutral lipid DOPE, L3 was found with high efficiency in gene transfer in three tumor cell lines including A549, HepG2 and H460. The optimized gene transfection efficacy of L3 was nearly 5.5 times more efficient than that of the popular commercially available gene delivery agent Lipofectamine 2000™ in human lung carcinoma cells A549. In addition, since L1 and L3 had nearly no gene transfection performance in normal cells HEK293, these cationic lipids showed tumor cell-targeting property to a certain extent. No significant cytotoxicity was found for the lipoplexes formed by L1–L3, and their cytotoxicities were similar to or slightly lower than the lipoplexes prepared from Lipofectamine 2000™. Conclusion Novel cyclen-based cationic lipids for effective in vitro gene transfection were founded, and these studies here may extend the application areas of macrocyclic polyamines, especially for cyclen. PMID:21887233

  9. Gut inflammation can boost horizontal gene transfer between pathogenic and commensal Enterobacteriaceae

    PubMed Central

    Stecher, Bärbel; Denzler, Rémy; Maier, Lisa; Bernet, Florian; Sanders, Mandy J.; Pickard, Derek J.; Barthel, Manja; Westendorf, Astrid M.; Krogfelt, Karen A.; Walker, Alan W.; Ackermann, Martin; Dobrindt, Ulrich; Thomson, Nicholas R.; Hardt, Wolf-Dietrich

    2012-01-01

    The mammalian gut harbors a dense microbial community interacting in multiple ways, including horizontal gene transfer (HGT). Pangenome analyses established particularly high levels of genetic flux between Gram-negative Enterobacteriaceae. However, the mechanisms fostering intraenterobacterial HGT are incompletely understood. Using a mouse colitis model, we found that Salmonella-inflicted enteropathy elicits parallel blooms of the pathogen and of resident commensal Escherichia coli. These blooms boosted conjugative HGT of the colicin-plasmid p2 from Salmonella enterica serovar Typhimurium to E. coli. Transconjugation efficiencies of ∼100% in vivo were attributable to high intrinsic p2-transfer rates. Plasmid-encoded fitness benefits contributed little. Under normal conditions, HGT was blocked by the commensal microbiota inhibiting contact-dependent conjugation between Enterobacteriaceae. Our data show that pathogen-driven inflammatory responses in the gut can generate transient enterobacterial blooms in which conjugative transfer occurs at unprecedented rates. These blooms may favor reassortment of plasmid-encoded genes between pathogens and commensals fostering the spread of fitness-, virulence-, and antibiotic-resistance determinants. PMID:22232693

  10. Successful gene transfer into dendritic cells with cationized gelatin and plasmid DNA complexes via a phagocytosis-dependent mechanism.

    PubMed

    Inada, Satoshi; Fujiwara, Hitoshi; Atsuji, Kiyoto; Takashima, Kazuhiro; Araki, Yasunobu; Kubota, Takeshi; Tabata, Yasuhiko; Yamagishi, Hisakazu

    2006-01-01

    The use of gene-modified dendritic cells (DC) is a powerful tool to enhance antitumor immune responses stimulated by these cells in cancer immunotherapy. Cationized gelatin is preferably incorporated via phagocytosis and is gradually degraded by proteolysis while buffering lysosomal activity. This may be appropriate for gene transfer into phagocytic cells, such as immature DC. In the present study, successful transfection into monocyte-derived immature DC was demonstrated using cationized gelatin and plasmid DNA complexes. A high transfection efficiency, approaching 16%, was obtained upon transfection of the enhanced green fluorescent protein (EGFP) gene as evaluated by flow cytometry. Transgene expression of EGFP and murine interleukin 12 were also detected by RT-PCR. The antigen-presenting capacity of the transfected DC was equal to that of untransfected DC as evaluated by the allogeneic mixed lymphocyte reaction. Cationized gelatin has the potential to be a unique non-viral vector for gene transfer into DC.

  11. Technology transfer of energy efficient technologies in industry: A review of trends and policy issues

    SciTech Connect

    Worrell, Ernst; van Berkel, Rene; Fengqi, Zhou; Menke, Christoph; Schaeffer, Roberto; Williams, Robert O.

    2000-03-01

    In 1995, industry accounted for 41 percent of global energy use. Although the efficiency of industrial processes has increased greatly during the past decades, energy efficiency improvements remain the major opportunity to reduce CO2 emissions. Industrialization may affect the environment adversely, stressing the need for transfer of cleaner technologies to developing countries. A review of trends, barriers and opportunities for technology transfer is presented. Technology transfer is a process involving assessment, agreement, implementation, evaluation and adaptation, and repetition. Institutional barriers and policies influence the transaction process. Investments in industrial technology are dominated by the private sector. In industry, energy efficiency is often the result of investments in modern equipment, stressing the importance and need for environmentally sound and long-term investment policies. The interactive and dynamic character of technology transfer stresses the need for innovative and flexible approaches, through partnerships between various stakeholders. Adaptation of technology to local conditions is essential, but practices vary widely. Countries that spend on average more on adaptation, seem to be more successful in technology transfer, hence successful technology transfer depends on transfer of technological capabilities.

  12. Gene Transfer Potential of Outer Membrane Vesicles of Acinetobacter baylyi and Effects of Stress on Vesiculation

    PubMed Central

    Fulsundar, Shweta; Harms, Klaus; Flaten, Gøril E.; Johnsen, Pål J.; Chopade, Balu Ananda

    2014-01-01

    Outer membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negative Acinetobacter baylyi as a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations of Escherichia coli and A. baylyi, including naturally transformation-deficient mutants of A. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using anti-fluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT to A. baylyi and E. coli at transfer frequencies ranging from 10−6 to 10−8, with transfer efficiencies of approximately 103 and 102 per μg of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs from A. baylyi interact with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released from A. baylyi. PMID:24657872

  13. Efficient Virus-Induced Gene Silencing in Solanum rostratum

    PubMed Central

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  14. Triticale powdery mildew: population characterization and wheat gene efficiency.

    PubMed

    Bouguennec, Annaig; Trottet, Maxime; du Cheyron, Philippe; Lonnet, Philippe

    2014-01-01

    Powdery mildew has emerged on triticale in the early 2000s in many locations, probably due to a host range expansion of the wheat formae speciales, Blumeria graminis f.sp. tritici. Many triticale cultivars are highly susceptible to powdery mildew, mainly in seedling stage, revealing a probably narrow genetic basis for powdery mildew resistance genes (Pm). Moreover, as Blumeria graminis is an obligate biotrophic fungus, it is very time consuming and difficult to maintain powdery mildew isolates for a non-specialized laboratory and populations can evolve. In order to identify wheat Pm genes efficient against natural populations of powdery mildew, wheat differential hosts and triticale seedlings were inoculated below susceptible triticale crop naturally contaminated by mildew, in several locations and several years. Symptoms on seedlings were measured after approximately two weeks of incubation in favorable fungus growth conditions. According to these data, we classified the Pm genes presents in our wheat differential hosts set in 3 classes: Pm already overcame by triticale powdery mildew, Pm having variable effects and Pm still efficient against triticale mildew. Data on triticale seedlings allowed us to identify some few triticale cultivars resistant to Blumeria graminis in seedling stage. We will try to identify Pm genes present in those cultivars next year by testing them with the characterized isolates of powdery mildew from Gent University. Nevertheless, interspecific crossing of wheat, resistant to powdery mildew in seedling stage, and rye have been initiated to introduce potentially interesting genes for resistance in triticale.

  15. Enhancement of DNA-mediated gene transfer by inhibitors of autophagic-lysosomal function.

    PubMed

    Ege, T; Reisbig, R R; Rogne, S

    1984-11-01

    A variety of compounds, known to influence the intravesicular transport and degradation of macromolecules, was studied for their effect on the efficiency of DNA-mediated gene transfer (transfection). The efficiency of transfection was measured by transformation of rat 2 thymidine kinase-deficient (tk-) cells by the cloned herpes simplex I thymidine kinase gene (pAGO). When salmon sperm DNA (average molecular weight, 6 X 10(6) D) was used as a carrier, the presence of either 20 mM NH4Cl, 1 microM carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), or 5 mM 3-methyl adenine (3-MA) in the medium during incubation of the cells with the DNA-calcium-phosphate (DNA-Ca-Pi) precipitate, enhanced the efficiency of transfection by a factor of 10. If rat thymus DNA (greater than 30 X 10(6) D) was used as a carrier, the transformation efficiency was much higher than with salmon sperm DNA. However, in this case treatment with 3-MA, NH4Cl and FCCP enhanced the transformation frequency by slightly less than a factor of two. 3-MA further increased the transfection frequency if the cells were incubated with the compound after removal of the DNA-Ca-Pi coprecipitate, whereas NH4Cl and FCCP had no such effect. Our results strongly suggest that these inhibitors of intracellular degradation can increase the frequency of transformation by increasing the cytoplasmic levels of exogenous DNA.

  16. Gene-transfer study approval awaits more data

    SciTech Connect

    Marwick, C.

    1988-11-18

    Approval of the gene-transfer study in cancer patients has been delayed. The proposal was recommended for approval by a National Institutes of Health (NIH) advisory committee, but has been put on hold by James B. Wyngaarden, MD, NIH director, pending submission in writing of further information. Some of this information, now forthcoming, had been withheld because data on preliminary studies had been submitted to peer-reviewed journals. The study involves placing the gene for neomycin-resistance to tumor-infiltrating lymphocytes as a marker. When these cells are injected into the patient, the presence of the marker should enable their fate to be studied over a prolonged period and an improved antitumor regimen could result. The use of tumor-infiltrating lymphocytes as immunotherapy has been studied for two years at the NIH's National Cancer Institute. The patients' tumors are removed and the tumor-infiltrating lymphocytes are cultivated to obtain several billion cells. These cells are then injected back into the patient. Early clinical experience has shown a substantial decease in tumor size in some patients, but not in all, an no one knows why.

  17. Heterologous gene expression in Hansenula polymorpha: Efficient secretion of glucoamylase

    SciTech Connect

    Gellissen, G.; Janowicz, Z.A.; Merckelbach, A.; Keup, P.; Weydemann, U.; Strasser, A.W.M. ); Piontek, M.; Hollenberg, C.P. )

    1991-03-01

    The authors have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein. Transformants of H. polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified. Yields of up to 1.4 g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter.

  18. Influence of embryo handling and transfer method on pig cloning efficiency.

    PubMed

    Shi, Junsong; Zhou, Rong; Luo, Lvhua; Mai, Ranbiao; Zeng, Haiyu; He, Xiaoyan; Liu, Dewu; Zeng, Fang; Cai, Gengyuan; Ji, Hongmei; Tang, Fei; Wang, Qinglai; Wu, Zhenfang; Li, Zicong

    2015-03-01

    The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs.

  19. Multiple Inter-Kingdom Horizontal Gene Transfers in the Evolution of the Phosphoenolpyruvate Carboxylase Gene Family

    PubMed Central

    Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought. PMID:23251445

  20. Frequent, independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes

    PubMed Central

    Bruto, Maxime; Prigent-Combaret, Claire; Luis, Patricia; Moënne-Loccoz, Yvan; Muller, Daniel

    2014-01-01

    Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes—as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude. PMID:24990676

  1. Frequent, independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes.

    PubMed

    Bruto, Maxime; Prigent-Combaret, Claire; Luis, Patricia; Moënne-Loccoz, Yvan; Muller, Daniel

    2014-08-22

    Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes-as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude.

  2. Gene Transfer into Rat Brain Using Adenoviral Vectors

    PubMed Central

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

    2010-01-01

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

  3. The elucidation of gene transferring mechanism by ultrasound-responsive unmodified and mannose-modified lipoplexes.

    PubMed

    Un, Keita; Kawakami, Shigeru; Yoshida, Mitsuru; Higuchi, Yuriko; Suzuki, Ryo; Maruyama, Kazuo; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2011-07-01

    The development of gene transfection methods enhancing the level of gene expression under simple and low-toxic condition is required for gene therapy in clinical. Our group has developed the ultrasound (US)-mediated gene transfection method using Man-PEG(2000) bubble lipoplexes, which are US-responsive and mannose-modified gene carriers, and succeeded in obtaining the enhanced gene expression in mannose receptor-expressing cells selectively by the gene transfer using Man-PEG(2000) bubble lipoplexes with US exposure in vitro and in vivo. Here, we investigated pDNA transferring mechanism followed by US exposure to unmodified and Man-PEG(2000) bubble lipoplexes, in particular, focused on US exposure timing. Following investigation of intracellular transferring characteristics, a large amount of pDNA was transferred into the cytoplasm followed by US-mediated destruction of bubble lipoplexes in the gene transfer using both bubble lipoplexes with US exposure. Moreover, the effective gene expression was obtained without TNF-α production when US was exposed until 5 min after the addition of bubble lipoplexes. These findings suggest that the gene transfer using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure enables to transfer pDNA into the cytoplasm, and optimized US exposure timing is important to achieve the high level of gene expression and the low level of pro-inflammatory cytokine production.

  4. Bacterial Transport and Fate and Its Effect on Horizontal Gene Transfer in Soil

    NASA Astrophysics Data System (ADS)

    Lv, N.; Massoudieh, A.; Nguyen, T. H.; Kamai, T.; Zilles, J. L.; Ginn, T. R.; Liang, X.

    2013-12-01

    Biogeochemical cycling in ecosystems relies heavily on soil bacterial communities. Bacterial communities adapt to natural or anthropogenic disruptions through mutation and horizontal gene transfer. Horizontal gene transfer alters bacterial communities rapidly by transferring DNA across species. A systematic understanding of bacterial transport and fate and its effects on horizontal gene transfer is critical for predicting and harnessing bacterial adaption and evolution in soil. In this work, a multi-scale approach was applied to study the effects of both flagella and motility on transport and fate of the soil bacterium Azotobacter vinelandii in porous media. Both micromodel and column experiments showed decreasing deposition over time, suggesting that both flagellated and non-flagellated cells were blocked from deposition by previously deposited cells. In later stages, ripening effects were also observed, and they appeared earlier for the non-flagellated strain. Based on the overall clean collector removal efficiencies determined from micromodel and column experiments, the non-motile and non-flagellated strain DJNM deposited the most, while the motile, wild-type strain DJ showed the least deposition. The overall clean collector removal efficiencies was due to decreased deposition of motile cells on the front sides of the collectors (relative to the flow direction). The horizontal gene transfer of extracellular DNA, known as natural transformation, was evaluated with both dissolved and adsorbed extracellular DNA and with motile and non-motile but flagellated strains (DJ and DJ77, respectively). The distinct transport mechanisms of these strains resulted in different natural transformation rates and relationships to the concentration of cells and dissolved extracellular DNA. A modified mass action type relationship with power relationships was established to model the differences in natural transformation between DJ and DJ77. A cell-DNA pairing hypothesis was

  5. Horizontal gene transfer of chlamydial-like tRNA genes into early vascular plant mitochondria.

    PubMed

    Knie, Nils; Polsakiewicz, Monika; Knoop, Volker

    2015-03-01

    Mitochondrial genomes of lycophytes are surprisingly diverse, including strikingly different transfer RNA (tRNA) gene complements: No mitochondrial tRNA genes are present in the spikemoss Selaginella moellendorffii, whereas 26 tRNAs are encoded in the chondrome of the clubmoss Huperzia squarrosa. Reinvestigating the latter we found that trnL(gag) and trnS(gga) had never before been identified in any other land plant mitochondrial DNA. Sensitive sequence comparisons showed these two tRNAs as well as trnN(guu) and trnS(gcu) to be very similar to their respective counterparts in chlamydial bacteria. We identified homologs of these chlamydial-type tRNAs also in other lycophyte, fern, and gymnosperm DNAs, suggesting horizontal gene transfer (HGT) into mitochondria in the early vascular plant stem lineages. These findings extend plant mitochondrial HGT to affect individual tRNA genes, to include bacterial donors, and suggest that Chlamydiae on top of their recently proposed key role in primary chloroplast establishment may also have participated in early tracheophyte genome evolution.

  6. GENE TRANSFER FOR THE TREATMENT OF NEOVASCULAR OCULAR DISEASE (AN AMERICAN OPHTHALMOLOGICAL SOCIETY THESIS)

    PubMed Central

    Stout, John Timothy

    2006-01-01

    Purpose As vasoproliferative diseases account for a substantial fraction of worldwide blindness and share the activation of the angiogenic pathway as a common etiology, the expression of antiangiogenic proteins offers a promising means of treatment. This study was designed to develop viral vectors, harboring angiostatic genes, for the study and treatment of experimental proliferative ocular disease. A variety of methods (in vitro, ex vivo tissue, and in vivo) were employed to model the process of proliferation and test the effectiveness of these reagents. Methods Antiangiogenic genes included single genes as well as hybrid genes that fused the active elements of different genes. Genes studied included the soluble vascular endothelial growth factor receptor (sKDR), soluble neuropilin (sNRP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1), plasminogen gene fragments (Kringle 1-3, 1-4, and 1-5), and soluble receptors for advanced glycosylation end products (sRAGE) genes, as well as the Endo:Ang, MIG:IP10, and Endo:Kringle5 fusion genes. All genes were cloned into a lentiviral vector system and were used to produce replication deficient lentiviral particles. These viral particles were used to transduce a variety of ocular cells and tissues to test viral transfer efficiency and transgene expression. In vivo systems were employed to explore the potential of these genes as antiangiogenic agents in models of corneal and retinal neovascular disease. Results Recombinant lentiviral particles, capable of transducing cell lines germane to eye disease (ocular endothelial, epithelial, and fibroblast cells), were successfully produced. These vectors were demonstrated to be effective in long-term transformation of cells and tissues. In vivo experiments confirmed that at least three different potentially angiostatic genes were successful in aborting the angiogenic process in the ocular models tested. Conclusions Lentiviral vectors are a viable means to deliver angiostatic genes

  7. In utero lung gene transfer using adeno-associated viral and lentiviral vectors in mice.

    PubMed

    Joyeux, Luc; Danzer, Enrico; Limberis, Maria P; Zoltick, Philip W; Radu, Antoneta; Flake, Alan W; Davey, Marcus G

    2014-06-01

    Virus-mediated gene transfer to the fetal lung epithelium holds considerable promise for the therapeutic management of prenatally diagnosed, potentially life-threatening inherited lung diseases. In this study we hypothesized that efficient and life-long lung transduction can be achieved by in utero gene therapy, using viral vectors. To facilitate diffuse entry into the lung, viral vector was injected into the amniotic sac of C57BL/6 mice on embryonic day 16 (term, ∼ 20 days) in a volume of 10 μl. Vectors investigated included those based on adeno-associated virus (AAV) (serotypes 5, 6.2, 9, rh.64R1) and vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1-based lentivirus (LV). All vectors expressed green fluorescent protein (GFP) under the transcriptional control of various promoters including chicken β-actin (CB) or cytomegalovirus (CMV) for AAV and CMV or MND (myeloproliferative sarcoma virus enhancer, negative control region deleted) for LV. Pulmonary GFP gene expression was detected by fluorescence stereoscopic microscopy and immunohistochemistry for up to 9 months after birth. At equivalent vector doses (mean, 12 × 10(10) genome copies per fetus) three AAV vectors resulted in long-term (up to 9 months) pulmonary epithelium transduction. AAV2/6.2 transduced predominantly cells of the conducting airway epithelium, although transduction decreased 2 months after vector delivery. AAV2/9-transduced cells of the alveolar epithelium with a type 1 pneumocyte phenotype for up to 6 months. Although minimal levels of GFP expression were observed with AAV2/5 up to 9 months, the transduced cells immunostained positive for F480 and were retrievable by bronchoalveolar lavage, confirming an alveolar macrophage phenotype. No GFP expression was observed in lung epithelial cells after AAV2/rh.64R1 and VSV-G-LV vector-mediated gene transfer. We conclude that these experiments demonstrate that prenatal lung gene transfer with AAV vectors engineered to target

  8. Efficient femtosecond energy transfer from carotenoid to retinal in gloeobacter rhodopsin-salinixanthin complex.

    PubMed

    Iyer, E Siva Subramaniam; Gdor, Itay; Eliash, Tamar; Sheves, Mordechai; Ruhman, Sanford

    2015-02-12

    The retinal proton pump xanthorhodopsin (XR) was recently found to function with an attached carotenoid light harvesting antenna, salinixanthin (SX). It is intriguing to discover if this departure from single chromophore architecture is singular or if it has been adopted by other microbial rhodopsins. In search of other cases, retinal protein encoding genes in numerous bacteria have been identified containing sequences corresponding to carotenoid binding sites like that in XR. Gloeobacter rhodopsin (GR), exhibiting particularly close homology to XR, has been shown to attach SX, and fluorescence measurements suggest SX can function as a light harvesting (LH) antenna in GR as well. In this study, we test this suggestion in real time using ultrafast transient absorption. Results show that energy transfer indeed occurs from S2 of SX to retinal in the GR-SX composite with an efficiency of ∼40%, even higher than that in XR. This validates the earlier fluorescence study, and supports the notion that many microbial retinal proteins use carotenoid antennae to harvest light.

  9. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.

    PubMed

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M; Levasseur, Anthony; Lindquist, Erika A; Majoor, Eline; Ohm, Robin A; Pangilinan, Jasmyn L; Pribowo, Amadeus; Saddler, John N; Sakalidis, Monique L; de Vries, Ronald P; Grigoriev, Igor V; Goodwin, Stephen B; Tanguay, Philippe; Hamelin, Richard C

    2015-03-17

    Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems.

  10. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

    PubMed Central

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M.; Levasseur, Anthony; Lindquist, Erika A.; Majoor, Eline; Ohm, Robin A.; Pangilinan, Jasmyn L.; Pribowo, Amadeus; Saddler, John N.; Sakalidis, Monique L.; de Vries, Ronald P.; Grigoriev, Igor V.; Goodwin, Stephen B.; Tanguay, Philippe; Hamelin, Richard C.

    2015-01-01

    Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems. PMID:25733908

  11. Sensitivity control through attenuation of signal transfer efficiency by negative regulation of cellular signalling.

    PubMed

    Toyoshima, Yu; Kakuda, Hiroaki; Fujita, Kazuhiro A; Uda, Shinsuke; Kuroda, Shinya

    2012-03-13

    Sensitivity is one of the hallmarks of biological and pharmacological responses. However, the principle of controlling sensitivity remains unclear. Here we theoretically analyse a simple biochemical reaction and find that the signal transfer efficiency of the transient peak amplitude attenuates depending on the strength of negative regulation. We experimentally find that many signalling pathways in various cell lines, including the Akt and ERK pathways, can be approximated by simple biochemical reactions and that the same property of the attenuation of signal transfer efficiency was observed for such pathways. Because of this property, a downstream molecule should show higher sensitivity to an activator and lower sensitivity to an inhibitor than an upstream molecule. Indeed, we experimentally verify that S6, which lies downstream of Akt, shows lower sensitivity to an epidermal growth factor receptor inhibitor than Akt. Thus, cells can control downstream sensitivity through the attenuation of signal transfer efficiency by changing the expression level of negative regulators.

  12. [Post-translational ligation and function of dual-vector transferred split CFTR gene].

    PubMed

    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2010-01-01

    The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

  13. Modification of nanostructured calcium carbonate for efficient gene delivery.

    PubMed

    Zhao, Dong; Wang, Chao-Qun; Zhuo, Ren-Xi; Cheng, Si-Xue

    2014-06-01

    In this study, a facile method to modify nanostructured calcium carbonate (CaCO3) gene delivery systems by adding calcium phosphate (CaP) component was developed. CaCO3/CaP/DNA nanoparticles were prepared by the co-precipitation of Ca(2+) ions with plasmid DNA in the presence of carbonate and phosphate ions. For comparison, CaCO3/DNA nanoparticles and CaP/DNA co-precipitates were also prepared. The effects of carbonate ion/phosphate ion (CO3(2-)/PO4(3-)) ratio on the particle size and gene delivery efficiency were investigated. With an appropriate CO3(2-)/PO4(3-) ratio, the co-existence of carbonate and phosphate ions could control the size of co-precipitates effectively, and CaCO3/CaP/DNA nanoparticles with a decreased size and improved stability could be obtained. The in vitro gene transfections mediated by different nanoparticles in 293T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. The gene transfection efficiency of CaCO3/CaP/DNA nanoparticles could be significantly improved as compared with CaCO3/DNA nanoparticles and CaP/DNA co-precipitates. The confocal microscopy study indicated that the cellular uptake and nuclear localization of CaCO3/CaP/DNA nanoparticles were significantly enhanced as compared with unmodified CaCO3/DNA nanoparticles.

  14. Quantum dot solar cells: hole transfer as a limiting factor in boosting the photoconversion efficiency.

    PubMed

    Kamat, Prashant V; Christians, Jeffrey A; Radich, James G

    2014-05-27

    Semiconductor nanostructures are attractive for designing low-cost solar cells with tunable photoresponse. The recent advances in size- and shape-selective synthesis have enabled the design of quantum dot solar cells with photoconversion efficiencies greater than 5%. To make them competitive with other existing thin film or polycrystalline photovoltaic technologies, it is important to overcome kinetic barriers for charge transfer at semiconductor interfaces. This feature article focuses on the limitations imposed by slow hole transfer in improving solar cell performance and its role in the stability of metal chalcogenide solar cells. Strategies to improve the rate of hole transfer through surface-modified redox relays offer new opportunities to overcome the hole-transfer limitation. The mechanistic and kinetic aspects of hole transfer in quantum dot solar cells (QDSCs), nanowire solar cells (NWSCs), and extremely thin absorber (ETA) solar cells are discussed.

  15. Transduction-Like Gene Transfer in the Methanogen Methanococcus voltae

    PubMed Central

    Bertani, Giuseppe

    1999-01-01

    Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 × 10−5 (BES resistance) to a maximum of 10−3 (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae. PMID:10321998

  16. Parvalbumin gene transfer impairs skeletal muscle contractility in old mice.

    PubMed

    Murphy, Kate T; Ham, Daniel J; Church, Jarrod E; Naim, Timur; Trieu, Jennifer; Williams, David A; Lynch, Gordon S

    2012-08-01

    Sarcopenia is the progressive age-related loss of skeletal muscle mass associated with functional impairments that reduce mobility and quality of life. Overt muscle wasting with sarcopenia is usually preceded by a slowing of the rate of relaxation and a reduction in maximum force production. Parvalbumin (PV) is a cytosolic Ca(2+) buffer thought to facilitate relaxation in muscle. We tested the hypothesis that restoration of PV levels in muscles of old mice would increase the magnitude and hasten relaxation of submaximal and maximal force responses. The tibialis anterior (TA) muscles of young (6 month), adult (13 month), and old (26 month) C57BL/6 mice received electroporation-assisted gene transfer of plasmid encoding PV or empty plasmid (pcDNA3.1). Contractile properties of TA muscles were assessed in situ 14 days after transfer. In old mice, muscles with increased PV expression had a 40% slower rate of tetanic force development (p<0.01), and maximum twitch and tetanic force were 22% and 16% lower than control values, respectively (p<0.05). Muscles with increased PV expression from old mice had an 18% lower maximum specific (normalized) force than controls, and absolute force was `26% lower at higher stimulation frequencies (150-300 Hz, p<0.05). In contrast, there was no effect of increased PV expression on TA muscle contractile properties in young and adult mice. The impairments in skeletal muscle function in old mice argue against PV overexpression as a therapeutic strategy for ameliorating aspects of contractile dysfunction with sarcopenia and help clarify directions for therapeutic interventions for age-related changes in skeletal muscle structure and function.

  17. Transduction-like gene transfer in the methanogen Methanococcus voltae

    NASA Technical Reports Server (NTRS)

    Bertani, G.

    1999-01-01

    Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.

  18. Effects of oxidants and reductants on the efficiency of excitation transfer in green photosynthetic bacteria

    NASA Technical Reports Server (NTRS)

    Wang, J.; Brune, D. C.; Blankenship, R. E.

    1990-01-01

    The efficiency of energy transfer in chlorosome antennas in the green sulfur bacteria Chlorobium vibrioforme and Chlorobium limicola was found to be highly sensitive to the redox potential of the suspension. Energy transfer efficiencies were measured by comparing the absorption spectrum of the bacteriochlorophyll c or d pigments in the chlorosome to the excitation spectrum for fluorescence arising from the chlorosome baseplate and membrane-bound antenna complexes. The efficiency of energy transfer approaches 100% at low redox potentials induced by addition of sodium dithionite or other strong reductants, and is lowered to 10-20% under aerobic conditions or after addition of a variety of membrane-permeable oxidizing agents. The redox effect on energy transfer is observed in whole cells, isolated membranes and purified chlorosomes, indicating that the modulation of energy transfer efficiency arises within the antenna complexes and is not directly mediated by the redox state of the reaction center. It is proposed that chlorosomes contain a component that acts as a highly quenching center in its oxidized state, but is an inefficient quencher when reduced by endogenous or exogenous reductants. This effect may be a control mechanism that prevents cellular damage resulting from reaction of oxygen with reduced low-potential electron acceptors found in the green sulfur bacteria. The redox modulation effect is not observed in the green gliding bacterium Chloroflexus aurantiacus, which contains chlorosomes but does not contain low-potential electron acceptors.

  19. Feline immunodeficiency virus and retrovirus-mediated adventitial ex vivo gene transfer to rabbit carotid artery using autologous vascular smooth muscle cells.

    PubMed

    Kankkonen, Hanna M; Turunen, Mikko P; Hiltunen, Mikko O; Lehtolainen, Pauliina; Koponen, Jonna; Leppänen, Pia; Turunen, Anna-Mari; Ylä-Herttuala, Seppo

    2004-03-01

    We have developed an ex vivo gene transfer technique to rabbit arterial wall using autologous smooth muscle cells (SMCs). SMCs were harvested from rabbit ear artery, transduced in vitro with vesicular stomatitis virus G-glycoprotein pseudotyped retrovirus or feline immunodeficiency virus (FIV) and returned to the adventitial surface of the carotid artery using a periadventitial silicone collar or collagen sheet placed around the artery. Beta-galactosidase (lacZ) and human apolipoprotein E3 (apoE3) cDNAs were used as transgenes. After retrovirus-mediated gene transfer of lacZ the selected cells implanted with high efficiency and expressed lacZ marker gene at a very high level 7 and 14 days after the operation. The level of lacZ expression decreased thereafter but was still detectable 12 weeks after the gene transfer, and was exclusively localized to the site of cell implantation inside the collar. Utilizing FIV vector expressing apoE3, low levels of apoE were measured from serum collected from a low-density lipoprotein receptor deficient Watanabe heritable hyperlipidemic rabbits 1 month after the gene transfer. The physiological effect of apoE expression was detected as transiently elevated serum cholesterol levels. The results indicate that the model can be used for high efficiency local gene transfer in arteries, e.g. during vascular surgery. The model is also valuable for studying expression, stability and safety of new gene transfer vectors and their expression products in vivo.

  20. Highly Efficient and Anomalous Charge Transfer in van der Waals Trilayer Semiconductors.

    PubMed

    Ceballos, Frank; Ju, Ming-Gang; Lane, Samuel D; Zeng, Xiao Cheng; Zhao, Hui

    2017-03-08

    Two-dimensional materials, such as graphene and monolayer transition metal dichalcogenides, allow the fabrication of multilayer structures without lattice matching restriction. A central issue in developing such artificial materials is to understand and control the interlayer electron transfer process, which plays a key role in harnessing their emergent properties. Recent photoluminescence and transient absorption measurements revealed that the electron transfer in heterobilayers occurs on ultrafast time scales. However, there is still a lack of fundamental understanding on how this process can be so efficient at van der Waals interfaces. Here we show evidence suggesting the coherent nature of such interlayer electron transfer. In a trilayer of MoS2-WS2-MoSe2, electrons excited in MoSe2 transfer to MoS2 in about one picosecond. Surprisingly, these electrons do not populate the middle WS2 layer during this process. Calculations showed the coherent nature of the charge transfer and reproduced the measured electron transfer time. The hole transfer from MoS2 to MoSe2 is also found to be efficient and ultrafast. The separation of electrons and holes extends their lifetimes to more than one nanosecond, suggesting potential applications of such multilayer structures in optoelectronics.

  1. DNA-water interactions distinguish messenger RNA genes from transfer RNA genes.

    PubMed

    Khandelwal, Garima; Jayaram, B

    2012-05-30

    Physicochemical properties of DNA sequences as a guide to developing insights into genome organization has received little attention. Here, we utilize the energetics of DNA to further advance the knowledge on its language at a molecular level. Specifically, we ask the question whether physicochemical properties of different functional units on genomes differ. We extract intramolecular and solvation energies of different DNA base pair steps from a comprehensive set of molecular dynamics simulations. We then investigate the solvation behavior of DNA sequences coding for mRNAs and tRNAs. Distinguishing mRNA genes from tRNA genes is a tricky problem in genome annotation without assumptions on length of DNA and secondary structure of the product of transcription. We find that solvation energetics of DNA behaves as an extremely efficient property in discriminating 2,063,537 genes coding for mRNAs from 56,251 genes coding for tRNAs in all (~1500) completely sequenced prokaryotic genomes.

  2. Detection of Neutralizing Antibodies against Human Papillomaviruses (HPV) by Inhibition of Gene Transfer Mediated by HPV Pseudovirions

    PubMed Central

    Bousarghin, Latifa; Combita-Rojas, Alba-Lucia; Touzé, Antoine; Mehdaoui, Slimane El; Sizaret, Pierre-Yves; Bravo, Maria-Mercedes; Coursaget, Pierre

    2002-01-01

    The goal of this study was to develop a human papillomavirus (HPV) neutralization assay using HPV pseudovirions generated in vitro. For this purpose, gene transfer efficiency of HPV virus-like particles (VLPs) was improved by using direct interaction between a reporter plasmid and the VLPs. Electron microscopic observation of the interaction between DNA molecules and VLPs revealed that VLPs always interact with a single DNA molecule and that VLPs bind to the end of linearized DNA molecules. An 100-fold improvement in the gene transfer was obtained by simple interaction between a linearized DNA molecule and VLPs. Moreover, direct interaction methods offer the possibility of transferring plasmids a size higher than that of the papillomavirus genome. The approach that we developed to generate HPV-16 and HPV-31 pseudovirions proved to be suitable for testing neutralizing antibodies in human sera both after immunization and after natural infection. PMID:11880418

  3. Multimodality Imaging of Gene Transfer with a Receptor-Based Reporter Gene

    PubMed Central

    Chen, Ron; Parry, Jesse J.; Akers, Walter J.; Berezin, Mikhail Y.; El Naqa, Issam M.; Achilefu, Samuel; Edwards, W. Barry; Rogers, Buck E.

    2010-01-01

    Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Non-invasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor–enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging. Methods An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of 111In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice. Results The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of 111In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2

  4. Microbial Evolution Is in the Cards: Horizontal Gene Transfer in the Classroom

    ERIC Educational Resources Information Center

    Kagle, Jeanne; Hay, Anthony G.

    2007-01-01

    Horizontal gene transfer, the exchange of genetic material between bacteria, is a potentially important factor in the degradation of synthetic compounds introduced to the environment and in the acquisition of other characteristics including antibiotic resistance. This game-based activity illustrates the role of horizontal gene transfer in the…

  5. Trophic transfer efficiency of methylmercury and inorganic mercury to lake trout Salvelinus namaycush from its prey

    USGS Publications Warehouse

    Madenijian, C.P.; David, S.R.; Krabbenhoft, D.P.

    2012-01-01

    Based on a laboratory experiment, we estimated the net trophic transfer efficiency of methylmercury to lake trout Salvelinus namaycush from its prey to be equal to 76.6 %. Under the assumption that gross trophic transfer efficiency of methylmercury to lake trout from its prey was equal to 80 %, we estimated that the rate at which lake trout eliminated methylmercury was 0.000244 day−1. Our laboratory estimate of methylmercury elimination rate was 5.5 times lower than the value predicted by a published regression equation developed from estimates of methylmercury elimination rates for fish available from the literature. Thus, our results, in conjunction with other recent findings, suggested that methylmercury elimination rates for fish have been overestimated in previous studies. In addition, based on our laboratory experiment, we estimated that the net trophic transfer efficiency of inorganic mercury to lake trout from its prey was 63.5 %. The lower net trophic transfer efficiency for inorganic mercury compared with that for methylmercury was partly attributable to the greater elimination rate for inorganic mercury. We also found that the efficiency with which lake trout retained either methylmercury or inorganic mercury from their food did not appear to be significantly affected by the degree of their swimming activity.

  6. Targeted gene transfer of different genes to presynaptic and postsynaptic neocortical neurons connected by a glutamatergic synapse.

    PubMed

    Zhang, Guo-rong; Zhao, Hua; Cao, Haiyan; Li, Xu; Geller, Alfred I

    2012-09-14

    Genetic approaches to analyzing neuronal circuits and learning would benefit from a technology to first deliver a specific gene into presynaptic neurons, and then deliver a different gene into an identified subset of their postsynaptic neurons, connected by a specific synapse type. Here, we describe targeted gene transfer across a neocortical glutamatergic synapse, using as the model the projection from rat postrhinal to perirhinal cortex. The first gene transfer, into the presynaptic neurons in postrhinal cortex, used a virus vector and standard gene transfer procedures. The vector expresses an artificial peptide neurotransmitter containing a dense core vesicle targeting domain, a NMDA NR1 subunit binding domain (from a monoclonal antibody), and the His tag. Upon release, this peptide neurotransmitter binds to NMDA receptors on the postsynaptic neurons. Antibody-mediated targeted gene transfer to these postsynaptic neurons in perirhinal cortex used a His tag antibody, as the peptide neurotransmitter contains the His tag. Confocal microscopy showed that with untargeted gene transfer, ~3% of the transduced presynaptic axons were proximal to a transduced postsynaptic dendrite. In contrast, with targeted gene transfer, ≥ 20% of the presynaptic axons were proximal to a transduced postsynaptic dendrite. Targeting across other types of synapses might be obtained by modifying the artificial peptide neurotransmitter to contain a binding domain for a different neurotransmitter receptor. This technology may benefit elucidating how specific neurons and subcircuits contribute to circuit physiology, behavior, and learning.

  7. Use of HIV as a gene transfer vector.

    PubMed

    Pluta, Krzysztof; Kacprzak, Magdalena Marta

    2009-01-01

    Despite the extensive research efforts over the past 25 years that have focused on HIV, there is still no cure for AIDS. However, tremendous progress in the understanding of the structure and biology of the HIV virus led to the development of safe and potent HIV-based transgene delivery vectors. These genetic vehicles are referred to as lentiviral vectors. They appear to be better suited for particular applications, such as transgene delivery into stem cells, compared to other viral- and non-viral vectors. This is because Lentivirus-based vectors can efficiently infect nondividing and slowly dividing cells. In the present review article, the current state of understanding of HIV-1 is discussed and the main characteristics that had an impact on vector design are outlined. A historical view on the vector concept is presented to facilitate discussion of recent results in vector engineering in a broader context. Subsequently, a state of the art overview concerning vector construction and vector production is given. This review also touches upon the subject of lentiviral vector safety and related topics that can be helpful in addressing this issue are discussed. Finally, examples of Lentivirus-based gene delivery systems and their applications are presented, with emphasis on animal transgenesis and human gene therapy.

  8. Efficient light harvesting and energy transfer in a red phosphorescent iridium dendrimer.

    PubMed

    Cho, Yang-Jin; Hong, Seong Ahn; Son, Ho-Jin; Han, Won-Sik; Cho, Dae Won; Kang, Sang Ook

    2014-12-15

    A series of red phosphorescent iridium dendrimers of the type [Ir(btp)2(pic-PCn)] (Ir-Gn; n = 0, 1, 2, and 3) with two 2-(benzo[b]thiophen-2-yl)pyridines (btp) and 3-hydroxypicolinate (pic) as the cyclometalating and ancillary ligands were prepared in good yields. Dendritic generation was grown at the 3 position of the pic ligand with 4-(9H-carbazolyl)phenyl dendrons connected to 3,5-bis(methyleneoxy)benzyloxy branches (PCn; n = 0, 2, 4, and 8). The harvesting photons on the PCn dendrons followed by efficient energy transfer to the iridium center resulted in high red emissions at ∼600 nm by metal-to-ligand charge transfer. The intensity of the phosphorescence gradually increased with increasing dendrimer generation. Steady-state and time-resolved spectroscopy were used to investigate the energy-transfer mechanism. On the basis of the fluorescence quenching rate constants of the PCn dendrons, the energy-transfer efficiencies for Ir-G1, Ir-G2, and Ir-G3 were 99, 98, and 96%, respectively. The energy-transfer efficiency for higher-generation dendrimers decreased slightly because of the longer distance between the PC dendrons and the core iridium(III) complex, indicating that energy transfer in Ir-Gn is a Förster-type energy transfer. Finally, the light-harvesting efficiencies for Ir-G1, Ir-G2, and Ir-G3 were determined to be 162, 223, and 334%, respectively.

  9. Simultaneous Enhancement of Efficiency and Stability of Phosphorescent OLEDs Based on Efficient Förster Energy Transfer from Interface Exciplex.

    PubMed

    Zhang, Dongdong; Cai, Minghan; Zhang, Yunge; Bin, Zhengyang; Zhang, Deqiang; Duan, Lian

    2016-02-17

    Exciplex forming cohosts have been widely adopted in phosphorescent organic light-emitting diodes (PHOLEDs), achieving high efficiency with low roll-off and low driving voltage. However, the influence of the exciplex-forming hosts on the lifetimes of the devices, which is one of the essential characteristics, remains unclear. Here, we compare the influence of the bulk exciplex and interface exciplex on the performances of the devices, demonstrating highly efficient orange PHOLEDs with long lifetime at low dopant concentration by efficient Förster energy transfer from the interface exciplex. A bipolar host, (3'-(4,6-diphenyl-1,3,5-triazin-2-yl)-(1,1'-biphenyl)-3-yl)-9-carbazole (CzTrz), was adopted to combine with a donor molecule, tris(4-(9H-carbazol-9-yl)phenyl)amine (TCTA), to form exciplex. Devices with energy transfer from the interface exciplex achieve lifetime almost 2 orders of magnitude higher than the ones based on bulk exciplex as the host by avoiding the formation of the donor excited states. Moreover, a highest EQE of 27% was obtained at the dopant concentration as low as 3 wt % for a device with interface exciplex, which is favorable for reducing the cost of fabrication. We believe that our work may shed light on future development of ideal OLEDs with high efficiency, long-lifetime, low roll-off and low cost simultaneously.

  10. Horizontal gene transfer in the acquisition of novel traits by metazoans

    PubMed Central

    Boto, Luis

    2014-01-01

    Horizontal gene transfer is accepted as an important evolutionary force modulating the evolution of prokaryote genomes. However, it is thought that horizontal gene transfer plays only a minor role in metazoan evolution. In this paper, I critically review the rising evidence on horizontally transferred genes and on the acquisition of novel traits in metazoans. In particular, I discuss suspected examples in sponges, cnidarians, rotifers, nematodes, molluscs and arthropods which suggest that horizontal gene transfer in metazoans is not simply a curiosity. In addition, I stress the scarcity of studies in vertebrates and other animal groups and the importance of forthcoming studies to understand the importance and extent of horizontal gene transfer in animals. PMID:24403327

  11. Gene transfer for neovascular age-related macular degeneration.

    PubMed

    Campochiaro, Peter A

    2011-05-01

    Age-related macular degeneration (AMD) is a complex disease that has two phases: a degenerative phase often referred to as nonneovascular AMD (non-NVAMD) or dry AMD and a phase dominated by growth of new blood vessels in the subretinal space, referred to as NVAMD or wet AMD. Advances in the understanding of the molecular pathogenesis of NVAMD have led to new drug therapies that have provided major benefits to patients. However, those treatments require frequent intraocular injections that in many patients must be continued indefinitely to maintain visual benefits. Gene transfer to augment expression of endogenous antiangiogenic proteins is an alternative approach that has the potential to provide long-term stability in patients with NVAMD. Studies in animal models that mimic aspects of NVAMD have identified several possible transgenes, and a clinical trial in patients with advanced NVAMD has suggested that the approach may be feasible. Many important questions remain, but the rationale and preliminary data are compelling. The results of two ongoing clinical trials may answer several of the questions and help direct future research.

  12. Gene recruitment--a common mechanism in the evolution of transfer RNA gene families.

    PubMed

    Wang, Xiujuan; Lavrov, Dennis V

    2011-04-01

    The evolution of alloacceptor transfer RNAs (tRNAs) has been traditionally thought to occur vertically and reflect the evolution of the genetic code. Yet there have been several indications that a tRNA gene could evolve horizontally, from a copy of an alloacceptor tRNA gene in the same genome. Earlier, we provided the first unambiguous evidence for the occurrence of such "tRNA gene recruitment" in nature--in the mitochondrial (mt) genome of the demosponge Axinella corrugata. Yet the extent and the pattern of this process in the evolution of tRNA gene families remained unclear. Here we analyzed tRNA genes from 21 mt genomes of demosponges as well as nuclear genomes of rhesus macaque, chimpanzee and human. We found four new cases of alloacceptor tRNA gene recruitment in mt genomes and eleven cases in the nuclear genomes. In most of these cases we observed a single nucleotide substitution at the middle position of the anticodon, which resulted in the change of not only the tRNA's amino-acid identity but also the class of the amino-acyl tRNA synthetases (aaRSs) involved in amino-acylation. We hypothesize that the switch to a different class of aaRSs may have prevented the conflict between anticodon and amino-acid identities of recruited tRNAs. Overall our results suggest that gene recruitment is a common phenomenon in tRNA multigene family evolution and should be taken into consideration when tRNA evolutionary history is reconstructed.

  13. Genomic Data Quality Impacts Automated Detection of Lateral Gene Transfer in Fungi

    PubMed Central

    Dupont, Pierre-Yves; Cox, Murray P.

    2017-01-01

    Lateral gene transfer (LGT, also known as horizontal gene transfer), an atypical mechanism of transferring genes between species, has almost become the default explanation for genes that display an unexpected composition or phylogeny. Numerous methods of detecting LGT events all rely on two fundamental strategies: primary structure composition or gene tree/species tree comparisons. Discouragingly, the results of these different approaches rarely coincide. With the wealth of genome data now available, detection of laterally transferred genes is increasingly being attempted in large uncurated eukaryotic datasets. However, detection methods depend greatly on the quality of the underlying genomic data, which are typically complex for eukaryotes. Furthermore, given the automated nature of genomic data collection, it is typically impractical to manually verify all protein or gene models, orthology predictions, and multiple sequence alignments, requiring researchers to accept a substantial margin of error in their datasets. Using a test case comprising plant-associated genomes across the fungal kingdom, this study reveals that composition- and phylogeny-based methods have little statistical power to detect laterally transferred genes. In particular, phylogenetic methods reveal extreme levels of topological variation in fungal gene trees, the vast majority of which show departures from the canonical species tree. Therefore, it is inherently challenging to detect LGT events in typical eukaryotic genomes. This finding is in striking contrast to the large number of claims for laterally transferred genes in eukaryotic species that routinely appear in the literature, and questions how many of these proposed examples are statistically well supported. PMID:28235827

  14. Identification of multiple independent horizontal gene transfers into poxviruses using a comparative genomics approach

    PubMed Central

    2008-01-01

    Background Poxviruses are important pathogens of humans, livestock and wild animals. These large dsDNA viruses have a set of core orthologs whose gene order is extremely well conserved throughout poxvirus genera. They also contain many genes with sequence and functional similarity to host genes which were probably acquired by horizontal gene transfer. Although phylogenetic trees can indicate the occurrence of horizontal gene transfer and even uncover multiple events, their use may be hampered by uncertainties in both the topology and the rooting of the tree. We propose to use synteny conservation around the horizontally transferred gene (HTgene) to distinguish between single and multiple events. Results Here we devise a method that incorporates comparative genomic information into the investigation of horizontal gene transfer, and we apply this method to poxvirus genomes. We examined the synteny conservation around twenty four pox genes that we identified, or which were reported in the literature, as candidate HTgenes. We found support for multiple independent transfers into poxviruses for five HTgenes. Three of these genes are known to be important for the survival of the virus in or out of the host cell and one of them increases susceptibility to some antiviral drugs. Conclusion In related genomes conserved synteny information can provide convincing evidence for multiple independent horizontal gene transfer events even in the absence of a robust phylogenetic tree for the HTgene. PMID:18304319

  15. Energy transfer efficiency in the chromophore network strongly coupled to a vibrational mode

    NASA Astrophysics Data System (ADS)

    Mourokh, Lev G.; Nori, Franco

    2015-11-01

    Using methods from condensed matter and statistical physics, we examine the transport of excitons through the photosynthetic complex from a receiving antenna to a reaction center. Writing the equations of motion for the exciton creation-annihilation operators, we are able to describe the exciton dynamics, even in the regime when the reorganization energy is of the order of the intrasystem couplings. We determine the exciton transfer efficiency in the presence of a quenching field and protein environment. While the majority of the protein vibrational modes are treated as a heat bath, we address the situation when specific modes are strongly coupled to excitons and examine the effects of these modes on the energy transfer efficiency in the steady-state regime. Using the structural parameters of the Fenna-Matthews-Olson complex, we find that, for vibrational frequencies below 16 meV, the exciton transfer is drastically suppressed. We attribute this effect to the formation of a "mixed exciton-vibrational mode" where the exciton is transferred back and forth between the two pigments with the absorption or emission of vibrational quanta, instead of proceeding to the reaction center. The same effect suppresses the quantum beating at the vibrational frequency of 25 meV. We also show that the efficiency of the energy transfer can be enhanced when the vibrational mode strongly couples to the third pigment only, instead of coupling to the entire system.

  16. Sequences in the U3 region of Human Immunodeficiency Virus 1 improve efficiency of minus strand transfer in infected cells

    PubMed Central

    Piekna-Przybylska, Dorota; Dykes, Carrie; Demeter, Lisa M.; Bambara, Robert A.

    2010-01-01

    A tRNA gene-like sequence has been identified near the 3’ end of HIV-1. Two segments of this sequence (motif 9 and segment 1) promoted minus strand transfer in vitro. The segments are complementary to the tRNA3Lys primer, and apparently act by binding the tRNA, thereby bringing the 3’ and 5’ ends of viral RNA into proximity for strand transfer. In this report, we used full-length HIV-1 to demonstrate biological relevance of these segments. We constructed HIV-1 genomes capable of single cycle infection and altered in one or both of two segments. We devised a real time PCR method for quantifying the amount of (-)ssDNA that completes transfer. Results showed that depending on the mutation the efficiency of transfer decreased 9% to 26%. Alteration of segment 1 had the greatest effect. Alteration of motif 9 or both sequences also caused a reduction, but smaller than alteration of segment 1 alone. PMID:21193211

  17. Recovery efficiency of aquifer storage and recovery (ASR) with mass transfer limitation

    NASA Astrophysics Data System (ADS)

    Lu, Chunhui; Du, Pengfei; Chen, Yiming; Luo, Jian

    2011-08-01

    Aquifer storage and recovery (ASR) is an effective strategy for water resources management and has been widely used in many contaminated and saline aquifers. However, its recovery efficiency (RE) may be significantly affected by mass transfer limitations. A numerical model is developed to simulate ASR performance by combining the convergent and divergent dispersion models with a first-order mass transfer model. By analyzing the concentration history at the pumping well, we obtain simple and effective relationships for investigating ASR efficiency under various mass transfer parameters, including capacity ratio and mass transfer timescale, and operational parameters, including injection durations and well-pumping rates. On the basis of such relationships, one can conveniently determine whether a site with mass transfer limitations is appropriate or not for ASR and how many ASR cycles are required for achieving a positive RE. Results indicate that the immobile domain may function as a contaminant source or sink or both during the recovery phase and RE usually improves with well-flow rate, the decrease of capacity ratio, and the ASR cycles. However, RE is a nonmonotonic function of the mass transfer timescale and the injection duration. A critical timescale is given for quantifying this nonmonotonic behavior. When the injection period is greater than such a critical value, increasing injection period results in a higher RE. Contrarily, when the injection period is less than the critical value, increasing the injection period may even yield a lower RE.

  18. Efficient transfer of information from hexitol nucleic acids to RNA during nonenzymatic oligomerization

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; De Bouvere, B.; Van Aerschot, A.; Herdewijn, P.; Orgel, L. E.

    1999-01-01

    Hexitol nucleic acids (HNAs) are DNA analogues that contain the standard nucleoside bases attached to a phosphorylated 1,5-anhydrohexitol backbone. We find that HNAs support efficient information transfer in nonensymatic template-directed reactions. HNA heterosequences appeared to be superior to the corresponding DNA heterosequences in facilitating synthesis of complementary oligonucleotides from nucleoside-5'-phosphoro-2-methyl imidazolides.

  19. Multiple LHCII antennae can transfer energy efficiently to a single Photosystem I.

    PubMed

    Bos, Inge; Bland, Kaitlyn M; Tian, Lijin; Croce, Roberta; Frankel, Laurie K; van Amerongen, Herbert; Bricker, Terry M; Wientjes, Emilie

    2017-02-22

    Photosystems I and II (PSI and PSII) work in series to drive oxygenic photosynthesis. The two photosystems have different absorption spectra, therefore changes in light quality can lead to imbalanced excitation of the photosystems and a loss in photosynthetic efficiency. In a short-term adaptation response termed state transitions, excitation energy is directed to the light-limited photosystem. In higher plants a special pool of LHCII antennae, which can be associated with either PSI or PSII, participates in these state transitions. It is known that one LHCII antenna can associate with the PsaH site of PSI. However, membrane fractions were recently isolated in which multiple LHCII antennae appear to transfer energy to PSI. We have used time-resolved fluorescence-streak camera measurements to investigate the energy transfer rates and efficiency in these membrane fractions. Our data show that energy transfer from LHCII to PSI is relatively slow. Nevertheless, the trapping efficiency in supercomplexes of PSI with ~2.4 LHCIIs attached is 94%. The absorption cross section of PSI can thus be increased with ~65% without having significant loss in quantum efficiency. Comparison of the fluorescence dynamics of PSI-LHCII complexes, isolated in a detergent or located in their native membrane environment, indicates that the environment influences the excitation energy transfer rates in these complexes. This demonstrates the importance of studying membrane protein complexes in their natural environment.

  20. Efficient Synchronization of Dipolarly Coupled Vortex-Based Spin Transfer Nano-Oscillators

    PubMed Central

    Locatelli, Nicolas; Hamadeh, Abbass; Abreu Araujo, Flavio; Belanovsky, Anatoly D.; Skirdkov, Petr N.; Lebrun, Romain; Naletov, Vladimir V.; Zvezdin, Konstantin A.; Muñoz, Manuel; Grollier, Julie; Klein, Olivier; Cros, Vincent; de Loubens, Grégoire

    2015-01-01

    Due to their nonlinear properties, spin transfer nano-oscillators can easily adapt their frequency to external stimuli. This makes them interesting model systems to study the effects of synchronization and brings some opportunities to improve their microwave characteristics in view of their applications in information and communication technologies and/or to design innovative computing architectures. So far, mutual synchronization of spin transfer nano-oscillators through propagating spinwaves and exchange coupling in a common magnetic layer has been demonstrated. Here we show that the dipolar interaction is also an efficient mechanism to synchronize neighbouring oscillators. We experimentally study a pair of vortex-based spin transfer nano-oscillators, in which mutual synchronization can be achieved despite a significant frequency mismatch between oscillators. Importantly, the coupling efficiency is controlled by the magnetic configuration of the vortices, as confirmed by an analytical model and micromagnetic simulations highlighting the physics at play in the synchronization process. PMID:26608230

  1. Efficient Synchronization of Dipolarly Coupled Vortex-Based Spin Transfer Nano-Oscillators

    NASA Astrophysics Data System (ADS)

    Locatelli, Nicolas; Hamadeh, Abbass; Abreu Araujo, Flavio; Belanovsky, Anatoly D.; Skirdkov, Petr N.; Lebrun, Romain; Naletov, Vladimir V.; Zvezdin, Konstantin A.; Muñoz, Manuel; Grollier, Julie; Klein, Olivier; Cros, Vincent; de Loubens, Grégoire

    2015-11-01

    Due to their nonlinear properties, spin transfer nano-oscillators can easily adapt their frequency to external stimuli. This makes them interesting model systems to study the effects of synchronization and brings some opportunities to improve their microwave characteristics in view of their applications in information and communication technologies and/or to design innovative computing architectures. So far, mutual synchronization of spin transfer nano-oscillators through propagating spinwaves and exchange coupling in a common magnetic layer has been demonstrated. Here we show that the dipolar interaction is also an efficient mechanism to synchronize neighbouring oscillators. We experimentally study a pair of vortex-based spin transfer nano-oscillators, in which mutual synchronization can be achieved despite a significant frequency mismatch between oscillators. Importantly, the coupling efficiency is controlled by the magnetic configuration of the vortices, as confirmed by an analytical model and micromagnetic simulations highlighting the physics at play in the synchronization process.

  2. High-Speed OQPSK and Efficient Power Transfer Through Inductive Link for Biomedical Implants.

    PubMed

    Simard, Guillaume; Sawan, Mohamad; Massicotte, Daniel

    2010-06-01

    Biomedical implants require wireless power and bidirectional data transfer. We pursue our previous work on a novel topology for a multiple carrier inductive link by presenting the fabricated coils. We show that the coplanar geometry approach is better suited for displacement tolerance. We provide a theoretical analysis of the efficiency of power transfer and phase-shift-keying communications through an inductive link. An efficiency of up to 61% has been achieved experimentally for power transfer and a data rate of 4.16 Mb/s with a bit-error rate of less than 2 × 10(-6) has been obtained with our fabricated offset quadrature phase-shift keying modules due to the inductive link optimization presented in this paper.

  3. In vivo repopulation of cytoplasmically gene transferred hematopoietic cells by temperature-sensitive mutant of recombinant Sendai viral vector.

    PubMed

    Yoshida, Kumi; Yonemitsu, Yoshikazu; Tanaka, Sakura; Yoshida, Shuro; Shibata, Satoko; Kondo, Haruhiko; Okano, Shinji; Ishikawa, Fumihiko; Akashi, Koichi; Inoue, Makoto; Hasegawa, Mamoru; Sueishi, Katsuo

    2007-09-28

    Recent clinical studies revealed 'proof of concept' of gene therapy targeting hematopoietic stem cells (HSCs) to treat hematopoietic disorders. However, vector integration-related adverse events of retroviral vectors have slowed progress in this field. As an initial step to overcoming this hurdle, we examined the potential of an improved cytoplasmic RNA vector, temperature-sensitive mutant non-transmissible recombinant Sendai virus (ts-rSeV/dF), for gene transfer to murine HSCs and progenitors. Both conventional vector and ts-rSeV/dF-GFP showed efficient gene transfer to T-lymphocyte-depleted syngeneic bone marrow cells (BMCs) (>85%), but only BMCs treated with ts-rSeV/dF-GFP but not with conventional vector efficiently repopulated in the recipient mice, associated with multilineage differentiation in vitro and in vivo. To our knowledge, this is the first demonstration of the in vivo reconstruction of hematopoietic series by cytoplasmically gene transferred BMCs, that warrants further investigation to realize this strategy in clinical settings.

  4. Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells.

    PubMed Central

    Golzio, M; Mora, M P; Raynaud, C; Delteil, C; Teissié, J; Rols, M P

    1998-01-01

    Cells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10 min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with beta-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for beta-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer. PMID:9635756

  5. The effect of synthetic surfactant Exosurf on gene transfer in mouse lung in vivo.

    PubMed

    Raczka, E; Kukowska-Latallo, J F; Rymaszewski, M; Chen, C; Baker, J R

    1998-10-01

    Gene transfer in the lung holds promise for the treatment of diseases such as pulmonary fibrosis, cystic fibrosis and asthma. Pulmonary surfactant has been reported to enhance expression from endobronchial, adenovirus-mediated gene transfer in experimental animals. This study examines the effect of exogenous synthetic surfactant (Exosurf) on gene expression from naked plasmid DNA administered endobronchially to adult mice. Transfection efficiency was evaluated by quantifying the expression of chloramphenicol acetyltransferase (CAT) and luciferase (Luc) genes in the lung. Endobronchial administration of either CAT or Luc expression plasmid DNA resulted in detectable concentrations of each reporter protein. CAT expression from plasmid DNA was monitored after endobronchial administration with the maximal expression observed at 3-5 days after administration and decreasing for 5 days thereafter. When DNA was delivered in a 50% suspension of Exosurf, the expression of either CAT or Luc was significantly reduced by 89.6 +/- 1.4% and 82.7 +/- 10.5%, respectively. The decrease in Luc expression was closely correlated (r = 0.99, P < 0.001) to log concentration of surfactant in the plasmid buffer solution (IC50 = 8.6%). CAT expression was not altered when surfactant was administered either 2 h before or after plasmid DNA instillation. Examination of the components of Exosurf revealed that two compounds, DPPC and tyloxapol, showed inhibitory effects on CAT expression. However, the inhibition caused by Exosurf appeared greater than that of either component. Our results suggest that the lung surfactant is a barrier to transfection of the endobronchial airway and may be partly responsible for the low expression of exogenous DNA in vivo in the bronchial tree.

  6. What Controls the Rate of Ultrafast Charge Transfer and Charge Separation Efficiency in Organic Photovoltaic Blends.

    PubMed

    Jakowetz, Andreas C; Böhm, Marcus L; Zhang, Jiangbin; Sadhanala, Aditya; Huettner, Sven; Bakulin, Artem A; Rao, Akshay; Friend, Richard H

    2016-09-14

    In solar energy harvesting devices based on molecular semiconductors, such as organic photovoltaics (OPVs) and artificial photosynthetic systems, Frenkel excitons must be dissociated via charge transfer at heterojunctions to yield free charges. What controls the rate and efficiency of charge transfer and charge separation is an important question, as it determines the overall power conversion efficiency (PCE) of these systems. In bulk heterojunctions between polymer donor and fullerene acceptors, which provide a model system to understand the fundamental dynamics of electron transfer in molecular systems, it has been established that the first step of photoinduced electron transfer can be fast, of order 100 fs. But here we report the first study which correlates differences in the electron transfer rate with electronic structure and morphology, achieved with sub-20 fs time resolution pump-probe spectroscopy. We vary both the fullerene substitution and donor/fullerene ratio which allow us to control both aggregate size and the energetic driving force for charge transfer. We observe a range of electron transfer times from polymer to fullerene, from 240 fs to as short as 37 fs. Using ultrafast electro-optical pump-push-photocurrent spectroscopy, we find the yield of free versus bound charges to be weakly dependent on the energetic driving force, but to be very strongly dependent on fullerene aggregate size and packing. Our results point toward the importance of state accessibility and charge delocalization and suggest that energetic offsets between donor and acceptor levels are not an important criterion for efficient charge generation. This provides design rules for next-generation materials to minimize losses related to driving energy and boost PCE.

  7. RE 3+ emission in garnets: multisites, energy transfer and quantum efficiency

    NASA Astrophysics Data System (ADS)

    Lupei, V.

    2002-02-01

    The connection between the high-resolution spectral disordering of the rare earth-doped garnets and the manifestation of the energy transfer in the global or selective emission decay is discussed. It is shown that the identification of the pair satellites and the investigation of their decay properties are important for determination or verification of the mechanisms of interaction that determine the energy transfer in simply activated or in sensitized crystals. The competition of various energy transfer processes in de-excitation of the metastable levels such as down-conversion and up-conversion in Nd: YAG under high intensity pump is discussed. It is shown that the energy transfer parameters determined from the emission decay could give a consistent description of data on quantum efficiency or heating effects.

  8. Dendrimeric siRNA for Efficient Gene Silencing.

    PubMed

    Hong, Cheol Am; Eltoukhy, Ahmed A; Lee, Hyukjin; Langer, Robert; Anderson, Daniel G; Nam, Yoon Sung

    2015-06-01

    Programmable molecular self-assembly of siRNA molecules provides precisely controlled generation of dendrimeric siRNA nanostructures. The second-generation dendrimers of siRNA can be effectively complexed with a low-molecular-weight, cationic polymer (poly(β-amino ester), PBAE) to generate stable nanostructures about 160 nm in diameter via strong electrostatic interactions. Condensation and gene silencing efficiencies increase with the increased generation of siRNA dendrimers due to a high charge density and structural flexibility.

  9. Improvement of oxygen transfer efficiency in aerated ponds using liquid-film-assisted approach.

    PubMed

    Zhu, H; Imai, T; Tani, K; Ukita, M; Sekine, M; Higuchi, T; Zhang, Z

    2007-01-01

    In aerated ponds, oxygen is generally supplied through either diffused or mechanical aeration means. Surface transfer and bubble transfer both contribute significantly to oxygen transfer in a diffused aeration system. In the present study, a liquid-film-forming apparatus (LFFA) is successfully developed on a laboratory scale to improve considerably the surface transfer via the unique liquid film transfer technique. The experimental results show that the volumetric mass transfer coefficient for LFFA alone is found to be as much as 5.3 times higher than that for water surface and that the total volumetric mass transfer coefficient for the liquid film aeration system increases by 37% in comparison with a conventional aeration system. Additionally, by tuning finely the structural parameters of the LFFA, it can also lead to high dissolved oxygen (DO) water with the DO percent saturation greater than 90%. More importantly, this result is accomplished by simply offering a single-pass aeration at a depth as shallow as 26 cm. As a result, the objective of economical energy consumption in aerated ponds can be realized by lowering the aeration depth without sacrificing the aeration efficiency. It is noteworthy that the data presented in this study are acquired either numerically or experimentally.

  10. Enhancement of oxygen transfer efficiency in diffused aeration systems using liquid-film-forming apparatus.

    PubMed

    Zhu, H; Imai, T; Tani, K; Ukita, M; Sekine, M; Higuchi, T; Zhang, Z J

    2007-05-01

    Surface transfer and bubble transfer both contribute significantly to oxygen transfer in a diffused aeration system. In the present study, liquid-film-forming apparatus is successfully developed on a laboratory scale to improve considerably the surface transfer via the unique liquid film transfer technique. The experimental results show that the volumetric mass transfer coefficient for liquid-film-forming apparatus alone is found to be as much as 5.3 times higher than that for water surface and that the total volumetric mass transfer coefficient for liquid film aeration system increases by 37 % in comparison with conventional aeration system. Additionally, by tuning finely the structural parameters of the liquid-film-forming apparatus, it can also lead to high dissolved oxygen water with the dissolved oxygen percent saturation greater than 90 %. More importantly, this result is accomplished by simply offering a single-pass aeration at the depth as shallow as 26 cm. As a result, the objective of economical energy consumption in diffused aeration systems can be realized by lowering the aeration depth without sacrificing the aeration efficiency.

  11. Environmental factors influencing gene transfer agent (GTA) mediated transduction in the subtropical ocean.

    PubMed

    McDaniel, Lauren D; Young, Elizabeth C; Ritchie, Kimberly B; Paul, John H

    2012-01-01

    Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the

  12. Efficient phosphorescent polymer light-emitting diodes by suppressing triplet energy back transfer.

    PubMed

    Gong, Shaolong; Yang, Chuluo; Qin, Jingui

    2012-07-21

    Phosphorescent polymer light-emitting diodes (PhPLEDs) are promising devices in flat panel displays and solid state lighting sources since they can combine the advantages of the high efficiency of electrophosphorescence and low-cost, large-scale manufacture by using a solution process. However, their efficiencies are generally much lower than those of small-molecule-based devices fabricated by using a thermal deposition approach. One of the major reasons for their low efficiency is that energy is lost by back transfer to a polymer host. This tutorial review gives a brief introduction to the fundamentals of PhPLEDs, and then highlights recent progress in the main approaches to suppress triplet energy back transfer from the phosphor to the polymer host towards realizing highly efficient PhPLEDs. The suppressing mechanisms are discussed, and the achievement of high device efficiencies are demonstrated. Emphasis is placed on the relationships between molecular structure, the extent of suppressing triplet energy back transfer, and device performance.

  13. Gene transfer into Solanum tuberosum via Rhizobium spp.

    PubMed

    Wendt, Toni; Doohan, Fiona; Winckelmann, Dominik; Mullins, Ewen

    2011-04-01

    Agrobacterium tumefaciens-mediated transformation (ATMT) is the preferred technique for gene transfer into crops. A major disadvantage of the technology remains the complexity of the patent landscape that surrounds ATMT which restricts its use for commercial applications. An alternative system has been described (Broothaerts et al. in Nature 433:629-633, 2005) detailing the propensity of three rhizobia to transform the model crop Arabidopsis thaliana, the non-food crop Nicotiana tabacum and, at a very low frequency, the monocotyledonous crop Oryza sativa. In this report we describe for the first time the genetic transformation of Solanum tuberosum using the non-Agrobacterium species Sinorhizobium meliloti, Rhizobium sp. NGR234 and Mesorhizobium loti. This was achieved by combining an optimal bacterium and host co-cultivation period with a low antibiotic regime during the callus and shoot induction stages. Using this optimized protocol the transformation frequency (calculated as % of shoots equipped with root systems with the ability to grow in rooting media supplemented with 25 μg/ml hygromycin) of the rhizobia strains was calculated at 4.72, 5.85 and 1.86% for S. meliloti, R. sp. NGR234 and M. loti respectively, compared to 47.6% for the A. tumefaciens control. Stable transgene integration and expression was confirmed via southern hybridisation, quantitative PCR analysis and histochemical screening of both leaf and/or tuber tissue. In light of the rapid advances in potato genomics, combined with the sequencing of the potato genome, the ability of alternative bacteria species to genetically transform this major food crop will provide a novel resource to the Solanaceae community as it continues to develop potato as both a food and non-food crop.

  14. Octaarginine-modified chitosan as a nonviral gene delivery vector: properties and in vitro transfection efficiency

    NASA Astrophysics Data System (ADS)

    Zhao, Xiaoli; Li, Zhaoyang; Liu, Wenguang; Lam, Wingmoon; Sun, Peng; Kao, Richard Y. T.; Luk, Keith D. K.; Lu, William W.

    2011-02-01

    Protein transduction domains (PTD) have been identified to have the capacity to facilitate molecular cargo to translocate through cell membrane. This study aims to utilize the cell membrane penetrating ability of octaarginine oligopeptide, a simplified prototype of the PTD, to enhance the transfection efficiency of chitosan. Octaarginine-modified chitosan (R8-CS) was synthesized as a gene transfer carrier by carbodiimide chemistry. The structure and composition of R8-CSs were characterized using FTIR and 1H NMR. Agarose gel electrophoresis assay showed that R8-CS could efficiently condense the DNA. The particle size of R8-CS/DNA complexes were determined to be around 100-200 nm. The nanoparticle complexes exhibited a spherical and compact morphology. R8-CS demonstrated higher transfection activity and lower cytotoxicity as compared to the unmodified chitosan and also showed good serum resistance.

  15. Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors

    PubMed Central

    Merienne, Nicolas; Douce, Juliette Le; Faivre, Emilie; Déglon, Nicole; Bonvento, Gilles

    2013-01-01

    Astrocytes are now considered as key players in brain information processing because of their newly discovered roles in synapse formation and plasticity, energy metabolism and blood flow regulation. However, our understanding of astrocyte function is still fragmented compared to other brain cell types. A better appreciation of the biology of astrocytes requires the development of tools to generate animal models in which astrocyte-specific proteins and pathways can be manipulated. In addition, it is becoming increasingly evident that astrocytes are also important players in many neurological disorders. Targeted modulation of protein expression in astrocytes would be critical for the development of new therapeutic strategies. Gene transfer is valuable to target a subpopulation of cells and explore their function in experimental models. In particular, viral-mediated gene transfer provides a rapid, highly flexible and cost-effective, in vivo paradigm to study the impact of genes of interest during central nervous system development or in adult animals. We will review the different strategies that led to the recent development of efficient viral vectors that can be successfully used to selectively transduce astrocytes in the mammalian brain. PMID:23847471

  16. Apramycin resistance as a selective marker for gene transfer in mycobacteria.

    PubMed Central

    Paget, E; Davies, J

    1996-01-01

    We have explored the potential of using the apramycin resistance gene as a marker in mycobacterial gene transfer studies. Shuttle plasmids available for both electroporation and conjugation studies have been constructed, and we have successfully validated the use of the apramycin resistance gene as a component of cloning vectors for Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis. PMID:8892841

  17. uv excision-repair gene transfer in Chinese hamster ovary (CHO) cells

    SciTech Connect

    MacInnes, M.A.; Bingham, J.M.; Strniste, G.F.; Thompson, L.H.

    1983-01-01

    uvc-sensitive mutants of CHO cells provide a model system for molecular studies of DNA repair. We present our recent results which show that these mutants are competent recipients for plasmid marker gene transfer and incorporation of a putative CHO repair gene. The applicability and advantages of this system for interspecies human repair gene identification are discussed.

  18. Recent tissue engineering-based advances for effective rAAV-mediated gene transfer in the musculoskeletal system.

    PubMed

    Rey-Rico, Ana; Cucchiarini, Magali

    2016-04-01

    Musculoskeletal tissues are diverse and significantly different in their ability to repair upon injury. Current treatments often fail to reproduce the natural functions of the native tissue, leading to an imperfect healing. Gene therapy might improve the repair of tissues by providing a temporarily and spatially defined expression of the therapeutic gene(s) at the site of the injury. Several gene transfer vehicles have been developed to modify various human cells and tissues from musculoskeletal system among which the non-pathogenic, effective, and relatively safe recombinant adeno-associated viral (rAAV) vectors that have emerged as the preferred gene delivery system to treat human disorders. Adapting tissue engineering platforms to gene transfer approaches mediated by rAAV vectors is an attractive tool to circumvent both the limitations of the current therapeutic options to promote an effective healing of the tissue and the natural obstacles from these clinically adapted vectors to achieve an efficient and durable gene expression of the therapeutic sequences within the lesions.

  19. Evaluation of Vascular Delivery Methodologies to Enhance rAAV6-mediated Gene Transfer to Canine Striated Musculature

    PubMed Central

    Gregorevic, Paul; Schultz, Brian R; Allen, James M; Halldorson, Jeffrey B; Blankinship, Michael J; Meznarich, Norman A; Kuhr, Christian S; Doremus, Caitlin; Finn, Eric; Liggitt, Denny; Chamberlain, Jeffrey S

    2009-01-01

    A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow, (ii) exsanguination via compression bandage, and (iii) vector “dwell” time of <20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans. PMID:19471246

  20. Efficient Transfer Entropy Analysis of Non-Stationary Neural Time Series

    PubMed Central

    Vicente, Raul; Díaz-Pernas, Francisco J.; Wibral, Michael

    2014-01-01

    Information theory allows us to investigate information processing in neural systems in terms of information transfer, storage and modification. Especially the measure of information transfer, transfer entropy, has seen a dramatic surge of interest in neuroscience. Estimating transfer entropy from two processes requires the observation of multiple realizations of these processes to estimate associated probability density functions. To obtain these necessary observations, available estimators typically assume stationarity of processes to allow pooling of observations over time. This assumption however, is a major obstacle to the application of these estimators in neuroscience as observed processes are often non-stationary. As a solution, Gomez-Herrero and colleagues theoretically showed that the stationarity assumption may be avoided by estimating transfer entropy from an ensemble of realizations. Such an ensemble of realizations is often readily available in neuroscience experiments in the form of experimental trials. Thus, in this work we combine the ensemble method with a recently proposed transfer entropy estimator to make transfer entropy estimation applicable to non-stationary time series. We present an efficient implementation of the approach that is suitable for the increased computational demand of the ensemble method's practical application. In particular, we use a massively parallel implementation for a graphics processing unit to handle the computationally most heavy aspects of the ensemble method for transfer entropy estimation. We test the performance and robustness of our implementation on data from numerical simulations of stochastic processes. We also demonstrate the applicability of the ensemble method to magnetoencephalographic data. While we mainly evaluate the proposed method for neuroscience data, we expect it to be applicable in a variety of fields that are concerned with the analysis of information transfer in complex biological, social, and

  1. Evolution of the cutinase gene family: evidence for lateral gene transfer of a candidate Phytophthora virulence factor.

    PubMed

    Belbahri, Lassaad; Calmin, Gautier; Mauch, Felix; Andersson, Jan O

    2008-01-31

    Lateral gene transfer (LGT) can facilitate the acquisition of new functions in recipient lineages, which may enable them to colonize new environments. Several recent publications have shown that gene transfer between prokaryotes and eukaryotes occurs with appreciable frequency. Here we present a study of interdomain gene transfer of cutinases -- well documented virulence factors in fungi -- between eukaryotic plant pathogens Phytophthora species and prokaryotic bacterial lineages. Two putative cutinase genes were cloned from Phytophthora brassicae and Northern blotting experiments showed that these genes are expressed early during the infection of the host Arabidopsis thaliana and induced during cyst germination of the pathogen. Analysis of the gene organisation of this gene family in Phytophthora ramorum and P. sojae showed three and ten copies in tight succession within a region of 5 and 25 kb, respectively, probably indicating a recent expansion in Phytophthora lineages by gene duplications. Bioinformatic analyses identified orthologues only in three genera of Actinobacteria, and in two distantly related eukaryotic groups: oomycetes and fungi. Together with phylogenetic analyses this limited distribution of the gene in the tree of life strongly support a scenario where cutinase genes originated after the origin of land plants in a microbial lineage living in proximity of plants and subsequently were transferred between distantly related plant-degrading microbes. More precisely, a cutinase gene was likely acquired by an ancestor of P. brassicae, P. sojae, P. infestans and P. ramorum, possibly from an actinobacterial source, suggesting that gene transfer might be an important mechanism in the evolution of their virulence. These findings could indeed provide an interesting model system to study acquisition of virulence factors in these important plant pathogens.

  2. IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

    NASA Astrophysics Data System (ADS)

    Awazu, Kunio; Kinpara, Takeshi; Tamiya, Eiichi

    2002-05-01

    A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm 2. FEL irradiation at a wavelength of 5.75 and 6.1 μm, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 μm. The maximum transfer efficiency was about 0.5%.

  3. Eyeglasses-powered, contact lens-like platform with high power transfer efficiency.

    PubMed

    Kim, Young-Joon; Maeng, Jimin; Irazoqui, Pedro P

    2015-08-01

    We present a contact lens-like platform that is wirelessly powered by an external coil embedded in eyeglasses via magnetic resonance coupling at 13.56 MHz. The platform is composed of a transparent parylene film as a host substrate, an embedded spiral inductor as a power receiving coil, and metal interconnects for additional electronics. A multilayer thin-film parylene packaging process is used to meet the form factor of a contact lens. A 36 μm-thick metal plating technique is employed on a parylene film to enhance the quality factor (Q) of the receiving coil (Q = 27.3 at 13.56 MHz). The power transfer method and techniques to compensate for coil misalignment are demonstrated on a pig eye, achieving a power transfer efficiency of 17.5 % at a 20-mm powering distance. The effect of tissue on the coil and the power transfer efficiency is examined. The high power transfer efficiency along with the wearable prototype demonstrated herein make promising progress toward smart contact lens in ocular diagnostics.

  4. Gene Transfer from Targeted Liposomes to Specific Lymphoid Cells by Electroporation

    NASA Astrophysics Data System (ADS)

    Machy, Patrick; Lewis, Florence; McMillan, Lynette; Jonak, Zdenka L.

    1988-11-01

    Large unilamellar liposomes, coated with protein A and encapsulating the gene that confers resistance to mycophenolic acid, were used as a model system to demonstrate gene transfer into specific lymphoid cells. Protein A, which selectively recognizes mouse IgG2a antibodies, was coupled to liposomes to target them specifically to defined cell types coated with IgG2a antibody. Protein A-coated liposomes bound human B lymphoblastoid cells preincubated with a mouse IgG2a anti-HLA monoclonal antibody but failed to adhere to cells challenged with an irrelevant (anti-H-2) antibody of the same isotype or to cells incubated in the absence of antibody. Transfection of target cells bound to protein A-coated liposomes was achieved by electroporation. This step was essential since only electroporated cells survived in a selective medium containing mycophenolic acid. Transfection efficiency with electroporation and targeted liposomes was as efficient as conventional procedures that used unencapsulated plasmids free in solution but, in the latter case, cell selectivity is not possible. This technique provides a methodology for introducing defined biological macromolecules into specific cell types.

  5. Information Transfer Efficiency of X-Ray Image Intensifier-Based Imaging Systems.

    NASA Astrophysics Data System (ADS)

    Fu, Tao-Yi.

    The information transfer efficiency of any quantum detection imaging system can be described by a unique measure: the detective quantum efficiency {DQE(f)}, which is a function of the statistically independent frequency channels. The DQE(f) is a combined descriptor which takes into account the signal transfer as well as noise transfer properties of a complete system. For a complicated multistage imaging system, each system component contributes noise. In this dissertation, physical and mathematical models for noise analysis are developed and verified experimentally with an x-ray image intensifier (XRII)-based imaging system. In such a system, the DQE at low frequency range is primarily determined by the x-ray detection and scintillation processes at the CsI layer of the XRII. The effects of x-ray photon energy and sensor layer thickness on DQE are measured in detail. Numerical calculations based on a physical model of x-ray interactions show a general agreement with the experimental data. At higher frequencies, the DQE behavior becomes more complicated. A mathematical model which combines the micro-image properties and noise statistics is formulated to analyze the noise power spectrum (NPS) of a linear n-stage imaging system. Measurement of NPS components of an XRII system verifies the validity of this analytical prediction. The associated image transfer properties are also measured with emphasis on the effect of signal-induced background on the image information transfer. The low frequency data derived from these image property measurements show further agreement with the numerical calculations based on the physical model. As a result of this predictability of information transfer efficiency, system gain and recording capacity are emphasized in the design consideration of a projected high performance XRII radiographic system.

  6. Cyclic RGD-poly(ethylene glycol)-polyethyleneimine is more suitable for glioblastoma targeting gene transfer in vivo.

    PubMed

    Zhan, Changyou; Qian, Jun; Feng, Linglin; Zhong, Gaoren; Zhu, Jianhua; Lu, Weiyue

    2011-08-01

    Arginine-glycine-aspartic acid (RGD) is a widely chosen ligand to improve the specific gene targeting transfection efficiency of polyethyleneimine (PEI) in vivo. However, the optimal RGD conjugating mode, RGD-poly(ethylene glycol)-PEI (RGD-PEG-PEI) or RGD-PEI-methoxyl poly(ethylene glycol) (RGD-PEI-mPEG) still remains controversial. In this study, RGD-PEG-PEI and RGD-PEI-mPEG were synthesized and compared with respects to their glioblastoma cell-binding capability and tumor-targeting ability of their complexes with plasmid DNA. These results demonstrated that RGD-PEG-PEI/plasmid enhanced green fluorescent protein (pEGFP)-N2 complexes had higher binding affinities with U87 cells than RGD-PEI-mPEG/pEGFP-N2 complexes. The gene transfection was also performed on U87 cells in vitro and in vivo. In vitro, both of the RGD-modified PEI derivatives enhanced the gene transfection efficiency to some extent. However, all of the complexes (with or without RGD modification) had high transfection efficiency. The biodistribution of RGD-PEG-PEI/pEGFP-N2 complexes in mice bearing subcutaneous glioblastomas were significantly greater than that of RGD-PEI-mPEG/pEGFP-N2 complexes, suggesting a more efficient gene transfection in vivo. In the RGD-PEG-PEI, the use of a PEG spacer was particularly important. These results indicated that RGD-PEG-PEI was more suitable for targeted gene transfer in vivo.

  7. Novel and effective gene transfer technique for study of vascular renin angiotensin system.

    PubMed Central

    Morishita, R; Gibbons, G H; Kaneda, Y; Ogihara, T; Dzau, V J

    1993-01-01

    Vascular renin angiotensin system (RAS) has been reported to exist in vascular wall. However, there is no direct evidence whether the vascular RAS per se can modulate growth of vascular smooth muscle cells (VSMC), because there is no suitable method to investigate the effect of endogenously produced vasoactive substances on growth of these cells. In this study, we transferred angiotensin-converting enzyme (ACE) and/or renin cDNAs into cultured VSMC using the efficient Sendai virus (hemagglutinating virus of Japan) liposome-mediated gene transfer method, to examine their relative roles in VSMC growth in vitro. Within 35 min or 6 h, the transfection of ACE cDNA into VSMC by hemagglutinating virus of Japan method resulted in a twofold higher ACE activity than control vector, whereas a cationic liposome (Lipofectin)-mediated method failed to show any effect. This in vitro system provided us with the opportunity to investigate the influence of endogenous vascular RAS on VSMC growth. Transfection of ACE or renin cDNA resulted in increased DNA and RNA synthesis, which was inhibited with the specific angiotensin II receptor antagonist (DuP 753: 10(-6) M). Angiotensin I added to ACE-transfected VSMC increased RNA synthesis in a dose-dependent manner. Cotransfection of renin and ACE cDNAs stimulated further RNA synthesis as compared to ACE or renin cDNA alone. These results showed that transfected components of RAS can modulate VSMC growth through the endogenous production of vascular angiotensin II, and that ACE as well as renin are rate limiting in determining the VSMC RAS activity. We conclude that the hemagglutinating virus of Japan liposome-mediated gene transfer technique provides a new and useful tool for study of endogenous vascular modulators such as vascular RAS. Images PMID:8390484

  8. Horizontal gene transfer and the evolution of transcriptionalregulation in Escherichia coli

    SciTech Connect

    Price, Morgan N.; Dehal, Paramvir S.; Arkin, Adam P.

    2007-12-20

    Background: Most bacterial genes were acquired by horizontalgene transfer from other bacteria instead of being inherited bycontinuous vertical descent from an ancient ancestor}. To understand howthe regulation of these {acquired} genes evolved, we examined theevolutionary histories of transcription factors and of regulatoryinteractions from the model bacterium Escherichia coli K12. Results:Although most transcription factors have paralogs, these usually arose byhorizontal gene transfer rather than by duplication within the E. colilineage, as previously believed. In general, most neighbor regulators --regulators that are adjacent to genes that they regulate -- were acquiredby horizontal gene transfer, while most global regulators evolvedvertically within the gamma-Proteobacteria. Neighbor regulators wereoften acquired together with the adjacent operon that they regulate, sothe proximity might be maintained by repeated transfers (like "selfishoperons"). Many of the as-yet-uncharacterized (putative) regulators havealso been acquired together with adjacent genes, so we predict that theseare neighbor regulators as well. When we analyzed the histories ofregulatory interactions, we found that the evolution of regulation byduplication was rare, and surprisingly, many of the regulatoryinteractions that are shared between paralogs result from convergentevolution. Another surprise was that horizontally transferred genes aremore likely than other genes to be regulated by multiple regulators, andmost of this complex regulation probably evolved after the transfer.Conclusions: Our results highlight the rapid evolution of niche-specificgene regulation in bacteria.

  9. Near-infrared fluorescent single walled carbon nanotube-chitosan composite: Interfacial strain transfer efficiency assessment

    NASA Astrophysics Data System (ADS)

    Mol Menamparambath, Mini; Arabale, Girish; Nikolaev, Pavel; Baik, Seunghyun; Arepalli, Sivaram

    2013-04-01

    Effective load transfer at the single walled carbon nanotube (SWCNT)-polymer interface is most desirable for mechanically reinforced polymer composites. Versatile layer-by-layer assembly technique achieved dispersion and uniform distribution of sodium carboxymethylcellulose (CMC)-solubilized SWCNTs within the polymer matrix. Electrostatic interaction between positively charged chitosan and negatively charged CMC facilitates design of an optically active biocompatible nanocomposite. Interfacial strain transfer efficiency of SWCNT-chitosan nanocomposite was assessed via SWCNT Raman and photoluminescence band shifts under uniaxial strain. Photoluminescence peak shift rates of individual semiconducting SWCNTs were investigated and compared with tight binding model calculations.

  10. Plasmid transfer by conjugation as a possible route of horizontal gene transfer and recombination in Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Horizontal gene transfer is an important component of evolution and adaptation of bacterial species. Xylella fastidiosa has the ability to incorporate exogenous DNA into its genome by homologous recombination at relatively high rates. This genetic recombination is believed to play a role in adaptati...

  11. Early spring mesopelagic carbon remineralization and transfer efficiency in the naturally iron-fertilized Kerguelen area

    NASA Astrophysics Data System (ADS)

    Jacquet, S. H. M.; Dehairs, F.; Lefèvre, D.; Cavagna, A. J.; Planchon, F.; Christaki, U.; Monin, L.; André, L.; Closset, I.; Cardinal, D.

    2015-03-01

    transfer expanding at mesopelagic layers during the cruise. Regarding the deep carbon transfer efficiency, it appeared that above the plateau (A3 site) the mesopelagic remineralization was not a major barrier to the transfer of organic matter to the seafloor (close to 500 m). There, the efficiency of carbon transfer to the bottom waters (>400 m) as assessed by PP, EP and MR fluxes comparisons reached up to 87% of the carbon exported from the upper 150 m. In contrast, at the deeper locations, mesopelagic remineralization clearly limited the transfer of carbon to depths of >400 m. For sites at the margin of the plateau (station E-4W) and the polar front (station F-L), mesopelagic remineralization even exceeded upper 150 m export, resulting in a zero transfer efficiency to depths >800 m. In the polar front meander (time series), the capacity of the meander to transfer carbon to depth >800 m was highly variable (0 to 73%). The highest carbon transfer efficiencies in the meander are furthermore coupled to intense and complete deep (>800 m) remineralization, resulting again in a near-zero, deep (>2000 m) carbon sequestration efficiency there.

  12. Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors.

    PubMed

    Lotery, Andrew J; Derksen, Todd A; Russell, Stephen R; Mullins, Robert F; Sauter, Sybille; Affatigato, Louisa M; Stone, Edwin M; Davidson, Beverly L

    2002-04-10

    We hypothesize that recombinant feline immunodeficiency viral (rFIV) vectors may be useful for gene transfer to the nonhuman primate retina. We performed vitrectomies and subretinal injections in the right eyes of 11 cynomolgus monkeys. Vesicular stomatitis virus glycoprotein-pseudotyped rFIV that expressed the Escherichia coli beta-galactosidase gene was injected into eight eyes. Sham vehicle or lactose buffer injections were also performed in two of these eight study eyes. rFIV pseudotyped with an amphotropic envelope was used in two eyes, and in one animal injections of lactose buffer only were given. After surgery the animals were clinically evaluated by retinal photography and electroretinography. beta-Galactosidase expression was evaluated, at a final end point, in histological sections. We found photoreceptor and Müller cells to have the greatest transgene expression. Focal inflammatory responses localized to the injection site were seen histologically in all eyes. No difference in transduction efficiency was seen between injections near the macula and more peripheral injections. Visual function as assessed by electroretinography was not significantly affected by vector or vehicle injections. We conclude that rFIV vectors administered beneath the retina can transduce a variety of retinal cells in the nonhuman primate retina. rFIV vectors have therapeutic potential and could be exploited to develop gene therapy for the human eye.

  13. Horizontally transferred genes in the genome of Pacific white shrimp, Litopenaeus vannamei

    PubMed Central

    2013-01-01

    Background In recent years, as the development of next-generation sequencing technology, a growing number of genes have been reported as being horizontally transferred from prokaryotes to eukaryotes, most of them involving arthropods. As a member of the phylum Arthropoda, the Pacific white shrimp Litopenaeus vannamei has to adapt to the complex water environments with various symbiotic or parasitic microorganisms, which provide a platform for horizontal gene transfer (HGT). Results In this study, we analyzed the genome-wide HGT events in L. vannamei. Through homology search and phylogenetic analysis, followed by experimental PCR confirmation, 14 genes with HGT event were identified: 12 of them were transferred from bacteria and two from fungi. Structure analysis of these genes showed that the introns of the two fungi-originated genes were substituted by shrimp DNA fragment, two genes transferred from bacteria had shrimp specific introns inserted in them. Furthermore, around other three bacteria-originated genes, there were three large DNA segments inserted into the shrimp genome. One segment was a transposon that fully transferred, and the other two segments contained only coding regions of bacteria. Functional prediction of these 14 genes showed that 6 of them might be related to energy metabolism, and 4 others related to defense of the organism. Conclusions HGT events from bacteria or fungi were happened in the genome of L. vannamei, and these horizontally transferred genes can be transcribed in shrimp. This is the first time to report the existence of horizontally transferred genes in shrimp. Importantly, most of these genes are exposed to a negative selection pressure and appeared to be functional. PMID:23914989

  14. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

    PubMed Central

    Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J.; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

    2009-01-01

    Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton–virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. PMID:19451591

  15. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    PubMed

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

  16. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    NASA Technical Reports Server (NTRS)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  17. A new computational method for the detection of horizontal gene transfer events.

    PubMed

    Tsirigos, Aristotelis; Rigoutsos, Isidore

    2005-01-01

    In recent years, the increase in the amounts of available genomic data has made it easier to appreciate the extent by which organisms increase their genetic diversity through horizontally transferred genetic material. Such transfers have the potential to give rise to extremely dynamic genomes where a significant proportion of their coding DNA has been contributed by external sources. Because of the impact of these horizontal transfers on the ecological and pathogenic character of the recipient organisms, methods are continuously sought that are able to computationally determine which of the genes of a given genome are products of transfer events. In this paper, we introduce and discuss a novel computational method for identifying horizontal transfers that relies on a gene's nucleotide composition and obviates the need for knowledge of codon boundaries. In addition to being applicable to individual genes, the method can be easily extended to the case of clusters of horizontally transferred genes. With the help of an extensive and carefully designed set of experiments on 123 archaeal and bacterial genomes, we demonstrate that the new method exhibits significant improvement in sensitivity when compared to previously published approaches. In fact, it achieves an average relative improvement across genomes of between 11 and 41% compared to the Codon Adaptation Index method in distinguishing native from foreign genes. Our method's horizontal gene transfer predictions for 123 microbial genomes are available online at http://cbcsrv.watson.ibm.com/HGT/.

  18. Boosting the efficiency of quantum dot sensitized solar cells through modulation of interfacial charge transfer.

    PubMed

    Kamat, Prashant V

    2012-11-20

    The demand for clean energy will require the design of nanostructure-based light-harvesting assemblies for the conversion of solar energy into chemical energy (solar fuels) and electrical energy (solar cells). Semiconductor nanocrystals serve as the building blocks for designing next generation solar cells, and metal chalcogenides (e.g., CdS, CdSe, PbS, and PbSe) are particularly useful for harnessing size-dependent optical and electronic properties in these nanostructures. This Account focuses on photoinduced electron transfer processes in quantum dot sensitized solar cells (QDSCs) and discusses strategies to overcome the limitations of various interfacial electron transfer processes. The heterojunction of two semiconductor nanocrystals with matched band energies (e.g., TiO(2) and CdSe) facilitates charge separation. The rate at which these separated charge carriers are driven toward opposing electrodes is a major factor that dictates the overall photocurrent generation efficiency. The hole transfer at the semiconductor remains a major bottleneck in QDSCs. For example, the rate constant for hole transfer is 2-3 orders of magnitude lower than the electron injection from excited CdSe into oxide (e.g., TiO(2)) semiconductor. Disparity between the electron and hole scavenging rate leads to further accumulation of holes within the CdSe QD and increases the rate of electron-hole recombination. To overcome the losses due to charge recombination processes at the interface, researchers need to accelerate electron and hole transport. The power conversion efficiency for liquid junction and solid state quantum dot solar cells, which is in the range of 5-6%, represents a significant advance toward effective utilization of nanomaterials for solar cells. The design of new semiconductor architectures could address many of the issues related to modulation of various charge transfer steps. With the resolution of those problems, the efficiencies of QDSCs could approach those of dye

  19. Thermal metamaterial for convergent transfer of conductive heat with high efficiency

    NASA Astrophysics Data System (ADS)

    Shen, Xiangying; Jiang, Chaoran; Li, Ying; Huang, Jiping

    2016-11-01

    It is crucially important to focus conductive heat in an efficient way, which has received much attention in energy science (say, solar cells), but is still far from being satisfactory due to the diffusive (divergent) nature of the heat. By developing a theory with hybrid transformations (rotation and stretch-compression), here we provide theoretical and experimental evidences for a type of thermal metamaterial called thermal converger. The converger is capable of convergently conducting heat in contrast to the known divergent behavior of heat diffusion, thus yielding a large heating region with high temperatures close to the heat source (high efficiency). The thermal converger further allows us to design a thermal grating—a thermal counterpart of optical grating. This work has relevance to heat focus with high efficiency, and it offers guidance both for efficient heat transfer and for designing thermal-converger-like metamaterials in other fields, such as electrics/magnetics, electromagnetics/optics, acoustics, and particle diffusion.

  20. Effect of Surface Defect States on Valence Band and Charge Separation and Transfer Efficiency

    PubMed Central

    Xu, Juan; Teng, Yiran; Teng, Fei

    2016-01-01

    Both energy band and charge separation and transfer are the crucial affecting factor for a photochemical reaction. Herein, the BiOCl nanosheets without and with surface bismuth vacancy (BOC, V-BOC) are prepared by a simple hydrothermal method. It is found that the new surface defect states caused by bismuth vacancy have greatly up-shifted the valence band and efficiently enhanced the separation and transfer rates of photogenerated electron and hole. It is amazing that the photocatalytic activity of V-BOC is 13.6 times higher than that of BOC for the degradation methyl orange (MO). We can develop an efficient photocatalyst by the introduction of defects. PMID:27586149

  1. Hot-electron-transfer enhancement for the efficient energy conversion of visible light.

    PubMed

    Yu, Sungju; Kim, Yong Hwa; Lee, Su Young; Song, Hyeon Don; Yi, Jongheop

    2014-10-13

    Great strides have been made in enhancing solar energy conversion by utilizing plasmonic nanostructures in semiconductors. However, current generation with plasmonic nanostructures is still somewhat inefficient owing to the ultrafast decay of plasmon-induced hot electrons. It is now shown that the ultrafast decay of hot electrons across Au nanoparticles can be significantly reduced by strong coupling with CdS quantum dots and by a Schottky junction with perovskite SrTiO3 nanoparticles. The designed plasmonic nanostructure with three distinct components enables a hot-electron-assisted energy cascade for electron transfer, CdS→Au→SrTiO3, as demonstrated by steady-state and time-resolved photoluminescence spectroscopy. Consequently, hot-electron transfer enabled the efficient production of H2 from water as well as significant electron harvesting under irradiation with visible light of various wavelengths. These findings provide a new approach for overcoming the low efficiency that is typically associated with plasmonic nanostructures.

  2. Trophic transfer efficiency of mercury to lake whitefish Coregonus clupeaformis from its prey

    USGS Publications Warehouse

    Madenjian, C.P.; O'Connor, D.V.

    2008-01-01

    In the laboratory, net trophic transfer efficiency of mercury to lake whitefish Coregonus clupeaformis from its prey was estimated to be 63.5%. Assuming that gross trophic transfer efficiency of mercury to lake whitefish from its prey was equal to 80%, we estimated that the rate at which lake whitefish eliminated mercury was 0.000730 day-1. Our laboratory estimate of mercury elimination rate was 2.4 times lower than the value predicted by a published regression equation developed from previous studies on mercury elimination rates for fish. Thus, our results suggested that mercury elimination rates for fish have been overestimated in previous studies. ?? 2008 Springer Science+Business Media, LLC.

  3. Tunable and long-range energy transfer efficiency through a graphene nanodisk

    NASA Astrophysics Data System (ADS)

    Karanikolas, Vasilios D.; Marocico, Cristian A.; Bradley, A. Louise

    2016-01-01

    In this paper, we present a theoretical investigation of the energy transfer efficiency between a pair of quantum emitters placed in proximity to a conducting graphene nanodisk. The energy transfer efficiency quantifies the contribution of the energy transfer process to the relaxation of the donor quantum system, as compared to the spontaneous emission rate of the donor in the absence of the acceptor. We use in our calculations the Green's tensor formalism in the electrostatic limit. This approximation works very well for the nanodisks considered here, for which the radius is much smaller than the emission wavelength of the donor. The approximate analytical solutions obtained are used to investigate the decay rate of a single quantum emitter and the energy transfer rate between quantum emitters in the vicinity of the graphene nanodisk. We find that these rates are enhanced several orders of magnitude compared with their free-space values. We determine the resonance frequencies of the spontaneous emission rate of a single quantum emitter to a graphene nanodisk, and the energy transfer rate between a pair of quantum emitters in proximity to a graphene nanodisk. We identify the surface modes which give the largest contributions to the energy transfer function. We connect the resonance frequency values and their surface plasmon wave numbers, which depend on the radius of the graphene nanodisk, with the dispersion relation of an infinite graphene monolayer at the same chemical potential. Analyzing the distance dependence of these rates, we are able to fit the full numerical results with a simple analytical expression which depends only on the geometrical characteristics of the graphene nanodisk, i.e., its radius. We show that the interaction distance depends on the transition dipole moment orientation and the different order resonance frequencies. The interaction distance between a pair of quantum emitters increases from a free-space value of 20 nm to reach values of

  4. Spin-transfer-torque efficiency enhanced by edge-damage of perpendicular magnetic random access memories

    SciTech Connect

    Song, Kyungmi; Lee, Kyung-Jin

    2015-08-07

    We numerically investigate the effect of magnetic and electrical damages at the edge of a perpendicular magnetic random access memory (MRAM) cell on the spin-transfer-torque (STT) efficiency that is defined by the ratio of thermal stability factor to switching current. We find that the switching mode of an edge-damaged cell is different from that of an undamaged cell, which results in a sizable reduction in the switching current. Together with a marginal reduction of the thermal stability factor of an edge-damaged cell, this feature makes the STT efficiency large. Our results suggest that a precise edge control is viable for the optimization of STT-MRAM.

  5. Surface heat transfer coefficient, heat efficiency, and temperature of pulsed solid-state lasers

    SciTech Connect

    Mann, K.; Weber, H.

    1988-08-01

    The temperature of solid-state lasers is a critical parameter. Efficiency and output power are strongly influenced by it. The two parameters which determine the temperature are the heat generation efficiency (HGE) and the surface heat transfer coefficient (SHTC) of the laser rod. These parameters allow the scaling of the rod temperature up to high pumping powers. Moreover, from the temperature inside the rod, the temperature gradients and the mechanical stress can be evaluated. Using transient temperature measurements, the SHTC and the HGE were determined for air- and water-cooled Nd:YAG and alexandrite lasers. The SHTC can be confirmed by theoretical considerations.

  6. Bacteriophage WO Can Mediate Horizontal Gene Transfer in Endosymbiotic Wolbachia Genomes

    PubMed Central

    Wang, Guan H.; Sun, Bao F.; Xiong, Tuan L.; Wang, Yan K.; Murfin, Kristen E.; Xiao, Jin H.; Huang, Da W.

    2016-01-01

    Phage-mediated horizontal gene transfer (HGT) is common in free-living bacteria, and many transferred genes can play a significant role in their new bacterial hosts. However, there are few reports concerning phage-mediated HGT in endosymbionts (obligate intracellular bacteria within animal or plant hosts), such as Wolbachia. The Wolbachia-infecting temperate phage WO can actively shift among Wolbachia genomes and has the potential to mediate HGT between Wolbachia strains. In the present study, we extend previous findings by validating that the phage WO can mediate transfer of non-phage genes. To do so, we utilized bioinformatic, phylogenetic, and molecular analyses based on all sequenced Wolbachia and phage WO genomes. Our results show that the phage WO can mediate HGT between Wolbachia strains, regardless of whether the transferred genes originate from Wolbachia or other unrelated bacteria. PMID:27965627

  7. Genome-wide identification of horizontal gene transfer in Fusarium verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Horizontal gene transfer (HGT), the exchange and stable integration of genetic material between different lineages, breaks species boundaries and generates new biological diversity. In eukaryotes, despite potential barriers, like the nuclear envelope and multicellularity, HGT may be facilitated by t...

  8. An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

    PubMed Central

    Netzker, Tina; Schroeckh, Volker; Gregory, Matthew A.; Flak, Michal; Krespach, Mario K. C.; Leadlay, Peter F.

    2016-01-01

    ABSTRACT Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus. Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca2+ ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate. IMPORTANCE The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species. PMID:27037115

  9. Maximum achievable efficiency in near-field coupled power-transfer systems.

    PubMed

    Zargham, Meysam; Gulak, P Glenn

    2012-06-01

    Wireless power transfer is commonly realized by means of near-field inductive coupling and is critical to many existing and emerging applications in biomedical engineering. This paper presents a closed form analytical solution for the optimum load that achieves the maximum possible power efficiency under arbitrary input impedance conditions based on the general two-port parameters of the network. The two-port approach allows one to predict the power transfer efficiency at any frequency, any type of coil geometry and through any type of media surrounding the coils. Moreover, the results are applicable to any form of passive power transfer such as provided by inductive or capacitive coupling. Our results generalize several well-known special cases. The formulation allows the design of an optimized wireless power transfer link through biological media using readily available EM simulation software. The proposed method effectively decouples the design of the inductive coupling two-port from the problem of loading and power amplifier design. Several case studies are provided for typical applications.

  10. Transfer hydrogenation using recyclable polyurea-encapsulated palladium: efficient and chemoselective reduction of aryl ketones.

    PubMed

    Yu, Jin-Quan; Wu, Hai-Chen; Ramarao, Chandrashekar; Spencer, Jonathan B; Ley, Steven V

    2003-03-21

    A robust and recyclable palladium catalyst [Pd0EnCat] has been prepared by ligand exchange of polyurea-encapsulated palladium(II) acetate with formic acid, resulting in deposition of Pd(0) in the support material; Pd0EnCat is shown to be a highly efficient transfer hydrogenation catalyst for chemoselective reduction of a wide range of aryl ketones to benzyl alcohols.

  11. Lack of glyphosate resistance gene transfer from Roundup Ready soybean to Bradyrhizobium japonicum under field and laboratory conditions.

    PubMed

    Isaza, Laura Arango; Opelt, Katja; Wagner, Tobias; Mattes, Elke; Bieber, Evi; Hatley, Elwood O; Roth, Greg; Sanjuán, Juan; Fischer, Hans-Martin; Sandermann, Heinrich; Hartmann, Anton; Ernst, Dieter

    2011-01-01

    A field study was conducted at the Russell E. Larson Agricultural Research Center to determine the effect of transgenic glyphosate-resistant soybean in combination with herbicide (Roundup) application on its endosymbiont Bradyrhizobium japonicum. DNA of bacteroids from isolated nodules was analysed for the presence of the transgenic 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) DNA sequence using polymerase chain reaction (PCR). To further assess the likelihood that the EPSPS gene may be transferred from the Roundup Ready (RR) soybean to B. japonicum, we have examined the natural transformation efficiency of B. japonicum strain 110spc4. Analyses of nodules showed the presence of the transgenic EPSPS DNA sequence. In bacteroids that were isolated from nodules of transgenic soybean plants and then cultivated in the presence of glyphosate this sequence could not be detected. This indicates that no stable horizontal gene transfer (HGT) of the EPSPS gene had occurred under field conditions. Under laboratory conditions, no natural transformation was detected in B. japonicum strain 110spc4 in the presence of various amounts of recombinant plasmid DNA. Our results indicate that no natural competence state exists in B. japonicum 110spc4. Results from field and laboratory studies indicate the lack of functional transfer of the CP4-EPSPS gene from glyphosate-tolerant soybean treated with glyphosate to root-associated B. japonicum.

  12. Sperm-mediated gene transfer-treated spermatozoa maintain good quality parameters and in vitro fertilization ability in swine.

    PubMed

    Bacci, M L; Zannoni, A; De Cecco, M; Fantinati, P; Bernardini, C; Galeati, G; Spinaci, M; Giovannoni, R; Lavitrano, M; Seren, E; Forni, M

    2009-12-01

    A simple and efficient method for producing multitransgenic animals is required for medical and veterinary applications. Sperm-mediated gene transfer (SMGT) is an effective method for introducing multiple genes into pigs (Sus, Sus scrofa). The major benefits of this technique are the high efficiency, low cost, and ease of use compared with that of other methods: Sperm-mediated gene transfer does not require embryo handling or expensive equipment. The aim of this study was to investigate the influence of SMGT treatment and exogenous DNA uptake on sperm quality. Even after a coincubation with a 20-fold larger amount (100 microg/mL) of DNA than usual (5 microg/mL), sperm quality parameters were not significantly affected, confirming the hypothesis that the SMGT protocol itself or the amount of bound DNA do not compromise the possibility of an extended employment of SMGT. More importantly, we found that semen used for in vitro fertilization 24h after DNA uptake gave good cleavage (60% vs. 58%, treated vs. control) and developmental rates definitely positive (41% vs. 48%, treated vs. control). These good results are connected to a competitive efficiency of transformation (62%) due to the numerous improvements in SMGT technique. We demonstrate that SMGT-treated spermatozoa retain good quality and fertilization potential for at least 24h, expanding the possibility to apply transgenesis in field conditions in swine, where the greatest hurdles are fertilization timing and plain procedure.

  13. Deep ocean nutrients imply large latitudinal variation in particle transfer efficiency.

    PubMed

    Weber, Thomas; Cram, Jacob A; Leung, Shirley W; DeVries, Timothy; Deutsch, Curtis

    2016-08-02

    The "transfer efficiency" of sinking organic particles through the mesopelagic zone and into the deep ocean is a critical determinant of the atmosphere-ocean partition of carbon dioxide (CO2). Our ability to detect large-scale spatial variations in transfer efficiency is limited by the scarcity and uncertainties of particle flux data. Here we reconstruct deep ocean particle fluxes by diagnosing the rate of nutrient accumulation along transport pathways in a data-constrained ocean circulation model. Combined with estimates of organic matter export from the surface, these diagnosed fluxes reveal a global pattern of transfer efficiency to 1,000 m that is high (∼25%) at high latitudes and low (∼5%) in subtropical gyres, with intermediate values in the tropics. This pattern is well correlated with spatial variations in phytoplankton community structure and the export of ballast minerals, which control the size and density of sinking particles. These findings accentuate the importance of high-latitude oceans in sequestering carbon over long timescales, and highlight potential impacts on remineralization depth as phytoplankton communities respond to a warming climate.

  14. Conjugated polymer/porphyrin complexes for efficient energy transfer and improving light-activated antibacterial activity.

    PubMed

    Xing, Chengfen; Xu, Qingling; Tang, Hongwei; Liu, Libing; Wang, Shu

    2009-09-16

    With the increasing antibiotic resistance of microorganisms, there is a growing interest in the design and development of new materials that are effective in killing bacteria to replace conventional antibiotics. Herein, a new anionic water-soluble polythiophene (PTP) and a cationic porphyrin (TPPN) are synthesized and characterized. They can form a complex through electrostatic interactions, and efficient energy transfer from PTP to TPPN occurs upon irradiation under white light (400-800 nm). The energy of TPPN transfers to triplet by intersystem crossing, followed by sensitization of oxygen molecule to enhance the efficiency of singlet oxygen generation related to TPPN itself. The positive charges of PTP/TPPN complex promote its adsorption to the negatively charged bacteria membranes of gram-negative Escherichia coli and gram-positive Bacillus subtilis through electrostatic interactions, and the singlet oxygen effectively kills the bacteria. The photosensitized inactivation of bacteria for the PTP/TPPN complex is efficient, and about 70% reduction of bacterial viability is observed after only 5 min of irradiation with white light at a fluence rate of 90 mW x cm(-2) (27 J x cm(-2)). The technique provides a promising application in photodynamic inactivation of bacteria on the basis of enhanced energy transfer offered by light-harvesting conjugated polymers.

  15. A statistical model for bacterial speciation triggered by lateral gene transfer

    NASA Astrophysics Data System (ADS)

    Sidhu, Sunjeet; Peng, Wequin

    2006-03-01

    The process of bacterial speciation has been a major unresolved issue in the study of bacterial evolution. It has been proposed that lateral gene transfer and homologous recombination play critical and complementary roles in speciation. We introduce a statistical model, of a population, for the evolution under lateral gene transfer and local homologous recombination. We examine the evolutionary dynamics and its dependence on various evolutionary operators. J. G. Lawrence, Theor. Popul. Biol. 61, 449(2002).

  16. Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos

    PubMed Central

    Liu, Tianbin; Dou, Hongwei; Xiang, Xi; Li, Yong; Pang, Xinzhi; Zhang, Yijie; Chen, Yu; Luan, Jing; Xu, Ying; Yang, Zhenzhen; Yang, Wenxian; Liu, Huan; Li, Feida; Wang, Hui; Yang, Huanming; Bolund, Lars; Vajta, Gabor

    2015-01-01

    Abstract Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs. 1.59%, respectively). However, CFFs had an opposite effect on these parameters when compared with CAFs (94.12% vs. 10.87% and 0.31% vs. 1.50%, respectively). Third, effects of genetic modification on the efficiency of SCNT were investigated with transgenic fetal fibroblasts (TFFs) and gene knockout fetal fibroblasts (KOFFs). Genetic modification of FFs increased developmental abnormalities (38.96% and 25.24% vs. 10.87% for KOFFs, TFFs, and FFs, respectively). KOFFs resulted in lower overall efficiency compared to TFFs and FFs (0.68% vs. 1.62% and 1.50%, respectively). In conclusion

  17. Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos.

    PubMed

    Liu, Tianbin; Dou, Hongwei; Xiang, Xi; Li, Lin; Li, Yong; Lin, Lin; Pang, Xinzhi; Zhang, Yijie; Chen, Yu; Luan, Jing; Xu, Ying; Yang, Zhenzhen; Yang, Wenxian; Liu, Huan; Li, Feida; Wang, Hui; Yang, Huanming; Bolund, Lars; Vajta, Gabor; Du, Yutao

    2015-12-01

    Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs. 1.59%, respectively). However, CFFs had an opposite effect on these parameters when compared with CAFs (94.12% vs. 10.87% and 0.31% vs. 1.50%, respectively). Third, effects of genetic modification on the efficiency of SCNT were investigated with transgenic fetal fibroblasts (TFFs) and gene knockout fetal fibroblasts (KOFFs). Genetic modification of FFs increased developmental abnormalities (38.96% and 25.24% vs. 10.87% for KOFFs, TFFs, and FFs, respectively). KOFFs resulted in lower overall efficiency compared to TFFs and FFs (0.68% vs. 1.62% and 1.50%, respectively). In conclusion, this is the

  18. Efficient Approximation of Head-Related Transfer Functions in Subbands for Accurate Sound Localization

    PubMed Central

    Marelli, Damián; Baumgartner, Robert; Majdak, Piotr

    2015-01-01

    Head-related transfer functions (HRTFs) describe the acoustic filtering of incoming sounds by the human morphology and are essential for listeners to localize sound sources in virtual auditory displays. Since rendering complex virtual scenes is computationally demanding, we propose four algorithms for efficiently representing HRTFs in subbands, i.e., as an analysis filterbank (FB) followed by a transfer matrix and a synthesis FB. All four algorithms use sparse approximation procedures to minimize the computational complexity while maintaining perceptually relevant HRTF properties. The first two algorithms separately optimize the complexity of the transfer matrix associated to each HRTF for fixed FBs. The other two algorithms jointly optimize the FBs and transfer matrices for complete HRTF sets by two variants. The first variant aims at minimizing the complexity of the transfer matrices, while the second one does it for the FBs. Numerical experiments investigate the latency-complexity trade-off and show that the proposed methods offer significant computational savings when compared with other available approaches. Psychoacoustic localization experiments were modeled and conducted to find a reasonable approximation tolerance so that no significant localization performance degradation was introduced by the subband representation. PMID:26681930

  19. Efficient preparation of cationized gelatin for gene transduction.

    PubMed

    Fukuyama, Naoto; Onuma, Tsuyoshi; Jujo, Shio; Tamai, Yoshifumi; Suzuki, Takahiro; Myojin, Kazunori; Tabata, Yasuhiko; Ishihara, Yoshimi; Takano, Jiro; Mori, Hidezo

    2006-07-20

    We previously reported gene therapy using cationized gelatin microspheres of φ20-32 μm, prepared from pig skin, as a transducing agent, but although the gelatin offered various advantages, its yield was extremely low (only 0.1%). In this study, we markedly improved the yield of φ20-32 μm cationized gelatin microspheres and prepared a newly less than φ20 μm cationized gelatin. Conventionally, cationized gelatin is prepared by cationization, particulation by agitation, and cross-linking. The yield is determined by the particulation step, for which we had used a three-necked distillation flask of 500 mL and an agitation speed of 420 rpm. The yield was significantly increased from 0.13 ± 0.02% to 8.80 ± 1.90% by using a smaller flask of 300 mL and an agitation speed of 25000 rpm (p < 0.01). We could also prepare cationized gelatin of less than φ20 μm, which had not been possible previously. We confirmed that efficient gene introduction into peritoneal macrophages could be achieved with the new cationized gelatin.

  20. Efficient translation initiation dictates codon usage at gene start

    PubMed Central

    Bentele, Kajetan; Saffert, Paul; Rauscher, Robert; Ignatova, Zoya; Blüthgen, Nils

    2013-01-01

    The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5–10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate. PMID:23774758

  1. A nanoparticle-based epigenetic modulator for efficient gene modulation

    NASA Astrophysics Data System (ADS)

    Pongkulapa, Thanapat

    Modulation of gene expression through chromatin remodeling involves epigenetic mechanisms, such as histone acetylation. Acetylation is tightly regulated by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Molecules that can regulate these enzymes by altering (activating or inhibiting) their functions have become a valuable tool for understanding cell development and diseases. HAT activators, i.e. N-(4-Chloro-(3-trifluoromethyl)phenyl)-2-ethoxybenzamide (CTB), have shown a therapeutic potential for many diseases, including cancer and neurodegeneration. However, these compounds encounter a solubility and a membrane permeability issue, which restricts their full potential for practical usage, especially for in vivo applications. To address this issue, in this work, we developed a nanoparticle-based HAT activator CTB, named Au-CTB, by incorporating a new CTB analogue onto gold nanoparticles (AuNPs) along with a poly(ethylene glycol) moiety and a nuclear localization signal (NLS) peptide to assist with solubility and membrane permeability. We found that our new CTB analogue and Au-CTB could activate HAT activity. Significantly, an increase in potency to activate HAT activity by Au-CTB proved the effectiveness of using the nanoparticle delivery platform. In addition, the versatility of Au-CTB platform permits the attachment of multiple ligands with tunable ratios on the nanoparticle surface via facile surface functionalization of gold nanoparticles. Due to its high delivery efficiency and versatility, Au-CTB can be a powerful platform for applications in epigenetic regulation of gene expression.

  2. Factors that Determine the Efficiency of HIV-1 Strand Transfer Initiated at a Specific Site

    PubMed Central

    Rigby, Sean T.; Van Nostrand, Keith P.; Rose, April E.; Gorelick, Robert J.; Mathews, David H.; Bambara, Robert A.

    2010-01-01

    Summary HIV-1 employs strand transfer for recombination between the two viral genomes. We previously provided evidence that strand transfer proceeds by an invasion-mediated mechanism, in which a DNA segment on the original RNA template is invaded by a second RNA template at a gap site. The initial RNA-DNA hybrid then expands until the DNA is fully transferred. Ribonuclease H (RNase H) cleavages and nucleocapsid protein (NC) were required for long distance propagation of the hybrid. The evaluation was performed on a unique substrate with a short gap serving as a pre-created invasion site (PCIS). In our current work, this substrate provided the opportunity to test what factors influence a specific invasion site to support transfer, and distinguish factors that influence invasion site creation from those that impact later steps. RNase H can act in a polymerization-dependent or -independent mode. Polymerization-dependent and -independent RNase H were found to be important to create efficiently-used invasion sites in the primer-donor complex, with or without NC. Propagation and terminus transfer steps, emanating from a PCIS in the presence of NC, were stimulated by polymerization-dependent but not -independent RNase H. RNase H can carry out primary and secondary cleavages during synthesis. While both modes of cleavage promoted invasion, only primary cleavage promoted propagation in the presence of NC in our system. These observations suggest that once invasion is initiated at a short gap, it can propagate through an adjacent region interrupted only by nicks, with help by NC. We considered the possibility that propagation solely by strand exchange was a significant contributor to transfers. However, it did not promote transfer, even if synthetic progress of the RT was intentionally slowed, which is consistent with strand exchange by random walk in which rate declines precipitously with distance. PMID:19853618

  3. Immune deficiency enhances expression of recombinant human antibody in mice after nonviral in vivo gene transfer.

    PubMed

    Kitaguchi, Kohji; Toda, Mikako; Takekoshi, Masataka; Maeda, Fumiko; Muramatsu, Tatsuo; Murai, Atsushi

    2005-10-01

    A cDNA encoding human antibody against hepatitis B virus was expressed in normal and severe combined immune deficiency (SCID) mice to clarify whether or not host immune status affects circulating levels of the recombinant human antibody (RhAb) after nonviral in vivo gene transfer. For transferring genes, either electroporation (EP) or hydrodynamics-based transfection (HD) was employed. The former was applied to the leg muscle to express the gene, while the latter primarily targeted foreign gene expression in the liver. The expressed RhAb was secreted into the blood circulation, and its existence was assayed by ELISA. Prior to the investigation of host immune status, suitable forms of plasmid expression vectors and types of electrodes were determined in normal mice. Results showed that the vector encoding both the light and heavy chains driven by the CMV promoter had the highest plasma RhAb concentrations, and a pair of pincette-type electrodes conferred the best performance. In both EP and HD, the SCID state showed an increased and prolonged RhAb production in the blood circulation due probably to suppressed recognition of RhAb as a foreign protein to the host animal. The difference in gene transfer methods demonstrated a characteristic pattern: an early and sharp rise followed by a relatively rapid decrease in HD, in contrast to a gradual rise followed by a plateau level maintained in EP. As a result, with the same amount of gene transferred, the plasma RhAb concentrations for the first 7 or 8 weeks were higher in HD than EP, while the reverse was true for the latter period. Multiple gene transfer contributed to maintaining and prolonging high RhAb concentrations in plasma by both methods with similar characteristic patterns accompanying the respective gene transfer method. These results suggest the importance of host immunological potency for maintaining plasma RhAb concentrations if these gene transfer technologies are used for clinical and therapeutic purposes.

  4. Plasmid-mediated transfer of the bla(NDM-1) gene in Gram-negative rods.

    PubMed

    Potron, Anaïs; Poirel, Laurent; Nordmann, Patrice

    2011-11-01

    The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase NDM-1 (New Delhi metallo-β-lactamase) producers, mostly in Enterobacteriacae. Five bla(NDM) (-1) -positive plasmids of different incompatibility groups (IncL/M, FII, A/C and two untypeable plasmids) from clinical Enterobacteriaceae were evaluated for conjugation properties and host specificity. Successful conjugative transfers were obtained using all tested enterobacterial species as recipients (Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Proteus mirabilis) and all plasmid types. Conjugation frequencies varied from 1 × 10(-4) to 6 × 10(-8) transconjugants per donor. Higher conjugation rates were obtained for two plasmids at 30 °C compared with that observed at 25 and 37 °C. Carbapenems used as selector did not lead to higher conjugation frequencies. None of the five plasmids was transferable to Acinetobacter baumannii or Pseudomonas aeruginosa by conjugation. This work underlines how efficient the spread of the carbapenemase bla(NDM) (-1) gene could be among Enterobacteriaceae.

  5. CREDVW-Linked Polymeric Micelles As a Targeting Gene Transfer Vector for Selective Transfection and Proliferation of Endothelial Cells.

    PubMed

    Hao, Xuefang; Li, Qian; Lv, Juan; Yu, Li; Ren, Xiangkui; Zhang, Li; Feng, Yakai; Zhang, Wencheng

    2015-06-10

    Nowadays, gene transfer technology has been widely used to promote endothelialization of artificial vascular grafts. However, the lack of gene vectors with low cytotoxicity and targeting function still remains a pressing challenge. Herein, polyethylenimine (PEI, 1.8 kDa or 10 kDa) was conjugated to an amphiphilic and biodegradable diblock copolymer poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-b-PLGA) to prepare mPEG-b-PLGA-g-PEI copolymers with the aim to develop gene vectors with low cytotoxicity while high transfection efficiency. The micelles were prepared from mPEG-b-PLGA-g-PEI copolymers by self-assembly method. Furthermore, Cys-Arg-Glu-Asp-Val-Trp (CREDVW) peptide was linked to micelle surface to enable the micelles with special recognition for endothelial cells (ECs). In addition, pEGFP-ZNF580 plasmids were condensed into these CREDVW-linked micelles to enhance the proliferation of ECs. These CREDVW-linked micelle/pEGFP-ZNF580 complexes exhibited low cytotoxicity by MTT assay. The cell transfection results demonstrated that pEGFP-ZNF580 could be transferred into ECs efficiently by these micelles. The results of Western blot analysis showed that the relative ZNF580 protein level in transfected ECs increased to 76.9%. The rapid migration of transfected ECs can be verified by wound healing assay. These results indicated that CREDVW-linked micelles could be a suitable gene transfer vector with low cytotoxicity and high transfection efficiency, which has great potential for rapid endothelialization of artificial blood vessels.

  6. An XMRV Derived Retroviral Vector as a Tool for Gene Transfer

    PubMed Central

    2011-01-01

    Background Retroviral vectors are widely used tools for gene delivery and gene therapy. They are useful for gene expression studies and genetic manipulation in vitro and in vivo. Many retroviral vectors are derived from the mouse gammaretrovirus, murine leukemia virus (MLV). These vectors have been widely used in gene therapy clinical trials. XMRV, initially found in prostate cancer tissue, was the first human gammaretrovirus described. Findings We developed a new retroviral vector based on XMRV called pXC. It was developed for gene transfer to human cells and is produced by transient cotransfection of LNCaP cells with pXC and XMRV-packaging plasmids. Conclusions We demonstrated that pXC mediates expression of inserted transgenes in cell lines. This new vector will be a useful tool for gene transfer in human and non-human cell lines, including gene therapy studies. PMID:21651801

  7. Horizontal gene transfer from diverse bacteria to an insect genome enables a tripartite nested mealybug symbiosis.

    PubMed

    Husnik, Filip; Nikoh, Naruo; Koga, Ryuichi; Ross, Laura; Duncan, Rebecca P; Fujie, Manabu; Tanaka, Makiko; Satoh, Nori; Bachtrog, Doris; Wilson, Alex C C; von Dohlen, Carol D; Fukatsu, Takema; McCutcheon, John P

    2013-06-20

    The smallest reported bacterial genome belongs to Tremblaya princeps, a symbiont of Planococcus citri mealybugs (PCIT). Tremblaya PCIT not only has a 139 kb genome, but possesses its own bacterial endosymbiont, Moranella endobia. Genome and transcriptome sequencing, including genome sequencing from a Tremblaya lineage lacking intracellular bacteria, reveals that the extreme genomic degeneracy of Tremblaya PCIT likely resulted from acquiring Moranella as an endosymbiont. In addition, at least 22 expressed horizontally transferred genes from multiple diverse bacteria to the mealybug genome likely complement missing symbiont genes. However, none of these horizontally transferred genes are from Tremblaya, showing that genome reduction in this symbiont has not been enabled by gene transfer to the host nucleus. Our results thus indicate that the functioning of this three-way symbiosis is dependent on genes from at least six lineages of organisms and reveal a path to intimate endosymbiosis distinct from that followed by organelles.

  8. Herpes simplex virus type 1 (HSV-1)-derived recombinant vectors for gene transfer and gene therapy.

    PubMed

    Marconi, Peggy; Fraefel, Cornel; Epstein, Alberto L

    2015-01-01

    Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination, either in eukaryotic cells or in bacteria.

  9. EV Charging Through Wireless Power Transfer: Analysis of Efficiency Optimization and Technology Trends

    SciTech Connect

    Miller, John M; Rakouth, Heri; Suh, In-Soo

    2012-01-01

    This paper is aimed at reviewing the technology trends for wireless power transfer (WPT) for electric vehicles (EV). It also analyzes the factors affecting its efficiency and describes the techniques currently used for its optimization. The review of the technology trends encompasses both stationary and moving vehicle charging systems. The study of the stationary vehicle charging technology is based on current implementations and on-going developments at WiTricity and Oak Ridge National Lab (ORNL). The moving vehicle charging technology is primarily described through the results achieved by the Korean Advanced Institute of Technology (KAIST) along with on-going efforts at Stanford University. The factors affecting the efficiency are determined through the analysis of the equivalent circuit of magnetic resonant coupling. The air gap between both transmitting and receiving coils along with the magnetic field distribution and the relative impedance mismatch between the related circuits are the primary factors affecting the WPT efficiency. Currently the industry is looking at an air gap of 25 cm or below. To control the magnetic field distribution, Kaist has recently developed the Shaped Magnetic Field In Resonance (SMFIR) technology that uses conveniently shaped ferrite material to provide low reluctance path. The efficiency can be further increased by means of impedance matching. As a result, Delphi's implementation of the WiTricity's technology exhibits a WPT efficiency above 90% for stationary charging while KAIST has demonstrated a maximum efficiency of 83% for moving vehicle with its On Line Vehicle (OLEV) project. This study is restricted to near-field applications (short and mid-range) and does not address long-range technology such as microwave power transfer that has low efficiency as it is based on radiating electromagnetic waves. This paper exemplifies Delphi's work in powertrain electrification as part of its innovation for the real world program geared

  10. Light, nutrients, and food-chain length constrain planktonic energy transfer efficiency across multiple trophic levels.

    PubMed

    Dickman, Elizabeth M; Newell, Jennifer M; González, María J; Vanni, Michael J

    2008-11-25

    The efficiency of energy transfer through food chains [food chain efficiency (FCE)] is an important ecosystem function. It has been hypothesized that FCE across multiple trophic levels is constrained by the efficiency at which herbivores use plant energy, which depends on plant nutritional quality. Furthermore, the number of trophic levels may also constrain FCE, because herbivores are less efficient in using plant production when they are constrained by carnivores. These hypotheses have not been tested experimentally in food chains with 3 or more trophic levels. In a field experiment manipulating light, nutrients, and food-chain length, we show that FCE is constrained by algal food quality and food-chain length. FCE across 3 trophic levels (phytoplankton to carnivorous fish) was highest under low light and high nutrients, where algal quality was best as indicated by taxonomic composition and nutrient stoichiometry. In 3-level systems, FCE was constrained by the efficiency at which both herbivores and carnivores converted food into production; a strong nutrient effect on carnivore efficiency suggests a carryover effect of algal quality across 3 trophic levels. Energy transfer efficiency from algae to herbivores was also higher in 2-level systems (without carnivores) than in 3-level systems. Our results support the hypothesis that FCE is strongly constrained by light, nutrients, and food-chain length and suggest that carryover effects across multiple trophic levels are important. Because many environmental perturbations affect light, nutrients, and food-chain length, and many ecological services are mediated by FCE, it will be important to apply these findings to various ecosystem types.

  11. Interleukin-6 gene transfer reverses body weight gain and fatty liver in obese mice.

    PubMed

    Ma, Yongjie; Gao, Mingming; Sun, Hao; Liu, Dexi

    2015-05-01

    Interleukin-6 (IL-6) is a multifunctional protein and has a major influence on energy metabolism. The current study was designed to assess the therapeutic effect of overexpression of Il-6 gene through gene transfer on high fat diet-induced obese mice. Hydrodynamic delivery of 1 μg pLIVE-IL6 plasmid per mouse into C57BL/6 obese mice resulted in peak level at 10 ng/ml of circulating IL-6 1 day after gene transfer and above 1n g/ml thereafter for a period of 6 weeks. Persistent Il-6 gene expression did not affect food intake but induced a significant reduction in body weight and improved obesity-associated hepatic steatosis. Il-6 gene delivery enhanced thermogenic gene expression and elevated protein levels of phosphorylated STAT3, PGC1α and UCP1 in brown adipose tissue. Il-6 overexpression elevated mRNA levels of lipolysis genes, triggered phosphorylation of STAT3, AMPK, and ACC, and increased expression of genes involved in fatty acid oxidation in skeletal muscle. IL-6 did not affect macrophage infiltration but maintained the M2 macrophage population in adipose tissue. Collectively, these results suggest that overexpression of the Il-6 gene by hydrodynamic gene delivery induces weight loss and alleviates obesity-induced fatty liver and insulin resistance, supporting the notion that gene transfer is a valid approach in managing obesity epidemics.

  12. Evolutionary transfers of mitochondrial genes to the nucleus in the Populus lineage and coexpression of nuclear and mitochondrial Sdh4 genes.

    PubMed

    Choi, Catherine; Liu, Zhenlan; Adams, Keith L

    2006-01-01

    The transfer of mitochondrial genes to the nucleus is an ongoing evolutionary process in flowering plants. Evolutionarily recent gene transfers provide insights into the evolutionary dynamics of the process and the way in which transferred genes become functional in the nucleus. Genes that are present in the mitochondrion of some angiosperms but have been transferred to the nucleus in the Populus lineage were identified by searches of Populus sequence databases. Sequence analyses and expression experiments were used to characterize the transferred genes. Two succinate dehydrogenase genes and six mitochondrial ribosomal protein genes have been transferred to the nucleus in the Populus lineage and have become expressed. Three transferred genes have gained an N-terminal mitochondrial targeting presequence from other pre-existing genes and two of the transferred genes do not contain an N-terminal targeting presequence. Intact copies of the succinate dehydrogenase gene Sdh4 are present in both the mitochondrion and the nucleus. Both copies of Sdh4 are expressed in multiple organs of two Populus species and RNA editing occurs in the mitochondrial copy. These results provide a genome-wide perspective on mitochondrial genes that were transferred to the nucleus and became expressed, functional genes during the evolutionary history of Populus.

  13. Chemoreceptor Gene Loss and Acquisition via Horizontal Gene Transfer in Escherichia coli

    PubMed Central

    Borziak, Kirill; Fleetwood, Aaron D.

    2013-01-01

    Chemotaxis allows bacteria to more efficiently colonize optimal microhabitats within their larger environment. Chemotaxis in Escherichia coli is the best-studied model system, and a large number of E. coli strains have been sequenced. The Escherichia/Shigella genus encompasses a great variety of commensal and pathogenic strains, but the role of chemotaxis in their association with the host remains poorly understood. Here we show that the core chemotaxis genes are lost in many, but not all, nonmotile strains but are well preserved in all motile strains. The genes encoding the Tar, Tsr, and Aer chemoreceptors, which mediate chemotaxis to a broad spectrum of chemical and physical cues, are also nearly uniformly conserved in motile strains. In contrast, the clade of extraintestinal pathogenic E. coli strains apparently underwent an ancestral loss of Trg and Tap chemoreceptors, which sense sugars, dipeptides, and pyrimidines. The broad range of time estimated for the loss of these genes (1 to 3 million years ago) corresponds to the appearance of the genus Homo. PMID:23749975

  14. Efficient and persistent expression of beta-glucuronidase gene in CD34+ cells from human umbilical cord blood by retroviral vector.

    PubMed

    Ohashi, T; Iizuka, S; Sly, W S; Machiki, K; Eto, Y

    1998-10-01

    We succeeded in efficiently transferring the beta-glucuronidase gene in a retroviral vector to human hematopoietic progenitor cells using a centrifugation enhancement protocol. The transduction efficiency in CFU-GM was highly variable (23-100%) with an average of 66.8%. In the case of BFU-E, efficiency was 83% and 76% in 2 separate experiments. In LTCIC (long-term culture-initiating cell), transduction efficiency were 20% and 50% in 2 experiments. The enzymatic activity of beta-glucuronidase in transduced cells were increased above the control level up to 5 wk. Considering that correction of the enzyme deficiency in a small number of hematopoietic cells can be therapeutic for the Sly disease mouse, our data provide encouragement that human trials of gene therapy based on transferring beta-glucuronidase gene to hematopoietic cells may be efficacious.

  15. Exploration of horizontal gene transfer between transplastomic tobacco and plant-associated bacteria.

    PubMed

    Demanèche, Sandrine; Monier, Jean-Michel; Dugat-Bony, Eric; Simonet, Pascal

    2011-10-01

    The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD-aadA-rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes.

  16. Estimate of net trophic transfer efficiency of PCBs to Lake Michigan lake trout from their prey

    USGS Publications Warehouse

    Madenjian, Charles P.; Hesselberg, Robert J.; DeSorcie, Timothy J.; Schmidt, Larry J.; Stedman, Ralph M.; Quintal, Richard T.; Begnoche, Linda J.; Passino-Reader, Dora R.

    1998-01-01

    Most of the polychlorinated biphenyl (PCB) body burden accumulated by lake trout (Salvelinus namaycush) from the Laurentian Great Lakes is from their food. We used diet information, PCB determinations in both lake trout and their prey, and bioenergetics modeling to estimate the efficiency with which Lake Michigan lake trout retain PCBs from their food. Our estimates were the most reliable estimates to date because (a) the lake trout and prey fish sampled during our study were all from the same vicinity of the lake, (b) detailed measurements were made on the PCB concentrations of both lake trout and prey fish over wide ranges in fish size, and (c) lake trout diet was analyzed in detail over a wide range of lake trout size. Our estimates of net trophic transfer efficiency of PCBs to lake trout from their prey averaged from 0.73 to 0.89 for lake trout between the ages of 5 and 10 years old. There was no evidence of an upward or downward trend in our estimates of net trophic transfer efficiency for lake trout between the ages of 5 and 10 years old, and therefore this efficiency appeared to be constant over the duration of the lake trout's adult life in the lake. On the basis of our estimtes, lake trout retained 80% of the PCBs that are contained within their food.

  17. Modulating the electronic structure of chromophores by chemical substituents for efficient energy transfer: application to fluorone.

    PubMed

    Sand, Andrew M; Liu, Claire; Valentine, Andrew J S; Mazziotti, David A

    2014-08-07

    Strong electron correlation within a quasi-spin model of chromophores was recently shown to enhance exciton energy transfer significantly. Here we investigate how the modulation of the electronic structure of the chromophores by chemical substitution can enhance energy-transfer efficiency. Unlike previous work that does not consider the direct effect of the electronic structure on exciton dynamics, we add chemical substituents to the fluorone dimer to study the effect of electron-donating and electron-withdrawing substituents on exciton energy transfer. The exciton dynamics are studied from the solution of a quantum Liouville equation for an open system whose model Hamiltonian is derived from excited-state electronic structure calculations. Both van der Waals energies and coupling energies, arising from the Hellmann-Feynman force generated upon transferring the dimers from infinity to a finite separation, are built into the model Hamiltonian. Though these two effects are implicitly treated in dipole-based models, their explicit and separate treatment as discussed here is critical to forging the correct connection with the electronic structure calculations. We find that the addition of electron-donating substituents to the fluorone system results in an increase in exciton-transfer rates by factors ranging from 1.3-1.9. The computed oscillator strength is consistent with the recent experimental results on a larger heterodimer system containing fluorone. The oscillator strength increases with the addition of electron-donating substituents. Our results indicate that the study of chromophore networks via electronic structure will help in the future design of efficient synthetic light-harvesting systems.

  18. Nuclear transfer from sexed parent embryos in cattle: efficiency and birth of offspring.

    PubMed

    Le Bourhis, D; Chesne, P; Nibart, M; Marchal, J; Humblot, P; Renard, J P; Heyman, Y

    1998-07-01

    The objectives of this study were to evaluate the efficiency and reliability of embryo sexing from isolated single blastomeres, and after nuclear transfer to examine the influence of the sex of donor embryos on development in vitro and in vivo up to calving. The sex of the donor embryo was determined by revealing a specific Y DNA sequence by PCR and electrophoresis after isolation of one, two, three, or more than five cells. The efficiency of sex determination was over 90% and reliability was 100% independent of the number of blastomeres used. In a second experiment, sex was determined from a single cell and the other cells were used for nuclear transfer. The effect of sex on in vitro development was studied in 386 male and 314 female reconstructed embryos derived from 19 male and 14 female parent embryos, respectively. Developmental competence in vitro of male and female constructs over 7 days was not statistically different (25.2 and 23.1% blastocysts on day 7, respectively; P > 0.05). After the transfer of predetermined male (n = 30) and female (n = 27) cloned embryos into recipient heifers, no effect of sex was observed on pregnancy rates at day 21, 35 and 90, or on calving rates (P > 0.05). These rates did not differ between single and twin transfer (P > 0.05). The sex of the calves born always corresponded to that determined from a single blastomere. These results show that sex can be determined accurately when using a single blastomere before nuclear transfer and that the sex of the parent embryo does not affect in vitro development or in vivo survival rates of cloned embryos.

  19. Horizontal transfers of transposable elements in eukaryotes: The flying genes.

    PubMed

    Panaud, Olivier

    2016-01-01

    Transposable elements (TEs) are the major components of eukaryotic genomes. Their propensity to densely populate and in some cases invade the genomes of plants and animals is in contradiction with the fact that transposition is strictly controlled by several molecular pathways acting at either transcriptional or post-transcriptional levels. Horizontal transfers, defined as the transmission of genetic material between sexually isolated species, have long been considered as rare phenomena. Here, we show that the horizontal transfers of transposable elements (HTTs) are very frequent in ecosystems. The exact mechanisms of such transfers are not well understood, but species involved in close biotic interactions, like parasitism, show a propensity to exchange genetic material horizontally. We propose that HTTs allow TEs to escape the silencing machinery of their host genome and may therefore be an important mechanism for their survival and their dissemination in eukaryotes.

  20. Effects of Tip Clearance and Casing Recess on Heat Transfer and Stage Efficiency in Axial Turbines

    NASA Technical Reports Server (NTRS)

    Ameri, A. A.; Steinthorsson, E.; Rigby, David L.

    1998-01-01

    Calculations were performed to assess the effect of the tip leakage flow on the rate of heat transfer to blade, blade tip and casing. The effect on exit angle and efficiency was also examined. Passage geometries with and without casing recess were considered. The geometry and the flow conditions of the GE-E 3 first stage turbine, which represents a modem gas turbine blade were used for the analysis. Clearance heights of 0%, 1%, 1.5% and 3% of the passage height were considered. For the two largest clearance heights considered, different recess depths were studied. There was an increase in the thermal load on all the heat transfer surfaces considered due to enlargement of the clearance gap. Introduction of recessed casing resulted in a drop in the rate of heat transfer on the pressure side but the picture on the suction side was found to be more complex for the smaller tip clearance height considered. For the larger tip clearance height the effect of casing recess was an orderly reduction in the suction side heat transfer as the casing recess height was increased. There was a marked reduction of heat load and peak values on the blade tip upon introduction of casing recess, however only a small reduction was observed on the casing itself. It was reconfirmed that there is a linear relationship between the efficiency and the tip gap height. It was also observed that the recess casing has a small effect on the efficiency but can have a moderating effect on the flow underturning at smaller tip clearances.

  1. Resurrection of an ancestral gene: functional and evolutionary analyses of the Ngrol genes transferred from Agrobacterium to Nicotiana.

    PubMed

    Aoki, Seishiro

    2004-08-01

    The Ng rol genes, which have high similarity in sequence to the rol genes of Agrobacterium rhizogenes, are present in the genome of untransformed plants of Nicotiana glauca. It is thought that bacterial infection resulted in the transfer of the Ng rol genes to plants early in the evolution of the genus Nicotiana, since several species in this genus contain rol-like sequences but others do not. Plants transformed with the bacterial rol genes exhibit various developmental and morphological changes. The presence of rol-like sequences in plant genomes is therefore thought to have contributed to the evolution of Nicotiana species. This paper focuses on studies of the Ng rol genes in present-day plants and during the evolution of the genus Nicotiana. The functional sequences of several Ng rol genes may have been conserved after their ancient introduction from a bacterium to the plant. Resurrection of an ancestral function of one of the Ng rol genes, as examined by physiological and evolutionary analyses, is also described. The origin of the Ng rol genes is then considered, based on results of molecular phylogenetic analyses. The effects of the horizontal transfer of the Ng rol genes and mutations in the genes are discussed on the plants of the genus Nicotiana during evolution.

  2. Modeling the efficiency of Förster resonant energy transfer from energy relay dyes in dye-sensitized solar cells.

    PubMed

    Hoke, Eric T; Hardin, Brian E; McGehee, Michael D

    2010-02-15

    Förster resonant energy transfer can improve the spectral breadth, absorption and energy conversion efficiency of dye sensitized solar cells. In this design, unattached relay dyes absorb the high energy photons and transfer the excitation to sensitizing dye molecules by Förster resonant energy transfer. We use an analytic theory to calculate the excitation transfer efficiency from the relay dye to the sensitizing dye accounting for dynamic quenching and relay dye diffusion. We present calculations for pores of cylindrical and spherical geometry and examine the effects of the Förster radius, the pore size, sensitizing dye surface concentration, collisional quenching rate, and relay dye lifetime. We find that the excitation transfer efficiency can easily exceed 90% for appropriately chosen dyes and propose two different strategies for selecting dyes to achieve record power conversion efficiencies.

  3. Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae.

    PubMed

    Urbanczyk, Henryk; Ast, Jennifer C; Kaeding, Allison J; Oliver, James D; Dunlap, Paul V

    2008-05-01

    Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib(2) operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib(2

  4. Phylogenetic Analysis of the Incidence of lux Gene Horizontal Transfer in Vibrionaceae▿ †

    PubMed Central

    Urbanczyk, Henryk; Ast, Jennifer C.; Kaeding, Allison J.; Oliver, James D.; Dunlap, Paul V.

    2008-01-01

    Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib2 operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib2

  5. Lightning-triggered electroporation and electrofusion as possible contributors to natural horizontal gene transfer.

    PubMed

    Kotnik, Tadej

    2013-09-01

    Phylogenetic studies show that horizontal gene transfer (HGT) is a significant contributor to genetic variability of prokaryotes, and was perhaps even more abundant during the early evolution. Hitherto, research of natural HGT has mainly focused on three mechanisms of DNA transfer: conjugation, natural competence, and viral transduction. This paper discusses the feasibility of a fourth such mechanism--cell electroporation and/or electrofusion triggered by atmospheric electrostatic discharges (lightnings). A description of electroporation as a phenomenon is followed by a review of experimental evidence that electroporation of prokaryotes in aqueous environments can result in release of non-denatured DNA, as well as uptake of DNA from the surroundings and transformation. Similarly, a description of electrofusion is followed by a review of experiments showing that prokaryotes devoid of cell wall can electrofuse into hybrids expressing the genes of their both precursors. Under sufficiently fine-tuned conditions, electroporation and electrofusion are efficient tools for artificial transformation and hybridization, respectively, but the quantitative analysis developed here shows that conditions for electroporation-based DNA release, DNA uptake and transformation, as well as for electrofusion are also present in many natural aqueous environments exposed to lightnings. Electroporation is thus a plausible contributor to natural HGT among prokaryotes, and could have been particularly important during the early evolution, when the other mechanisms might have been scarcer or nonexistent. In modern prokaryotes, natural absence of the cell wall is rare, but it is reasonable to assume that the wall has formed during a certain stage of evolution, and at least prior to this, electrofusion could also have contributed to natural HGT. The concluding section outlines several guidelines for assessment of the feasibility of lightning-triggered HGT.

  6. High excitation transfer efficiency from energy relay dyes in dye-sensitized solar cells.

    PubMed

    Hardin, Brian E; Yum, Jun-Ho; Hoke, Eric T; Jun, Young Chul; Péchy, Peter; Torres, Tomás; Brongersma, Mark L; Nazeeruddin, Md Khaja; Grätzel, Michael; McGehee, Michael D

    2010-08-11

    The energy relay dye, 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM), was used with a near-infrared sensitizing dye, TT1, to increase the overall power conversion efficiency of a dye-sensitized solar cell (DSC) from 3.5% to 4.5%. The unattached DCM dyes exhibit an average excitation transfer efficiency (ETE) of 96% inside TT1-covered, mesostructured TiO(2) films. Further performance increases were limited by the solubility of DCM in an acetonitrile based electrolyte. This demonstration shows that energy relay dyes can be efficiently implemented in optimized dye-sensitized solar cells, but also highlights the need to design highly soluble energy relay dyes with high molar extinction coefficients.

  7. Construction of a Helicobacter pylori-Escherichia coli shuttle vector for gene transfer in Helicobacter pylori.

    PubMed Central

    Lee, W K; An, Y S; Kim, K H; Kim, S H; Song, J Y; Ryu, B D; Choi, Y J; Yoon, Y H; Baik, S C; Rhee, K H; Cho, M J

    1997-01-01

    In this study, a Helicobacter pylori-Escherichia coli shuttle vector was constructed for transferring DNA into H. pylori. The smallest cryptic plasmid (1.2 kb), pHP489, among those harbored by 77 H. pylori isolates was selected as a base replicon for constructing vectors. HindIII-digested pHP489 was ligated with a kanamycin resistance gene [aph(3')-III], which originated from Campylobacter jejuni, to produce the recombinant plasmid pHP489K. pHP489K was efficiently transformed into and stably maintained in H. pylori strains. The shuttle vector pBHP489K (3.6 kb) was constructed by the recombination of pHP489, ColE1, and aph(3')-III sequences. pBHP489K was reciprocally transformed into and maintained in both H. pylori and E. coli. Introduction of the shuttle vector clone DNA (pBHP489K/AB; 6.7 kb), containing the ureA and ureB genes of H. pylori, into urease-negative mutants of H. pylori led to the restoration of their urease activity. The transformants were confirmed to contain the incoming plasmid DNA. pBHP489K satisfied the requirements for an H. pylori-E. coli shuttle vector, implying that it might be a useful vector for investigating pathogenicity and restriction-modification systems of H. pylori. PMID:9406406

  8. Hepatic gene transfer in neonatal mice by adeno-associated virus serotype 8 vector.

    PubMed

    Wang, Lili; Wang, Huan; Bell, Peter; McMenamin, Deirdre; Wilson, James M

    2012-05-01

    For genetic diseases that manifest at a young age with irreversible consequences, early treatment is critical and essential. Neonatal gene therapy has the advantages of achieving therapeutic effects before disease manifestation, a low vector requirement and high vector-to-cell ratio, and a relatively immature immune system. Therapeutic effects or long-term rescue of neonatal lethality have been demonstrated in several animal models. However, vigorous cell proliferation in the newborn stage is a significant challenge for nonintegrating vectors, such as adeno-associated viral (AAV) vector. Slightly delaying the injection age, and readministration at a later time, are two of the alternative strategies to solve this problem. In this study, we demonstrated robust and efficient hepatic gene transfer by self-complementary AAV8 vector in neonatal mice. However, transduction quickly decreased over a few weeks because of vector dilution caused by fast proliferation. Delaying the injection age improved sustained expression, although it also increased neutralizing antibody (NAb) responses to AAV capsid. This approach can be used to treat genetic diseases with slow progression. For genetic diseases with early onset and severe consequences, early treatment is essential. A second injection of vector of a different serotype at a later time may overcome preexisting NAb and achieve sustained therapeutic effects.

  9. Human β-NGF gene transferred to cat corneal endothelial cells

    PubMed Central

    Luo, Wen-Juan; Liu, Min; Zhao, Gui-Qiu; Wang, Chuan-Fu; Hu, Li-Ting; Liu, Xiang-Ping

    2016-01-01

    AIM To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. METHODS The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco's modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. RESULTS The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result

  10. Lentivirus-mediated gene transfer of uroporphyrinogen III synthase fully corrects the porphyric phenotype in human cells.

    PubMed

    Géronimi, F; Richard, E; Lamrissi-Garcia, I; Lalanne, M; Ged, C; Redonnet-Vernhet, I; Moreau-Gaudry, F; de Verneuil, H

    2003-05-01

    Congenital erythropoietic porphyria (CEP) is an inherited disease due to a deficiency in the uroporphyrinogen III synthase, the fourth enzyme of the heme biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor, with death often occurring early in adult life. For severe transfusion-dependent cases, when allogeneic cell transplantation cannot be performed, the autografting of genetically modified primitive/stem cells may be the only alternative. In vitro gene transfer experiments have documented the feasibility of gene therapy via hematopoietic cells to treat this disease. In the present study lentiviral transduction of porphyric cell lines and primary CD34(+) cells with the therapeutic human uroporphyrinogen III synthase (UROS) cDNA resulted in both enzymatic and metabolic correction, as demonstrated by the increase in UROS activity and the suppression of porphyrin accumulation in transduced cells. Very high gene transfer efficiency (up to 90%) was achieved in both cell lines and CD34(+) cells without any selection. Expression of the transgene remained stable over long-term liquid culture. Furthermore, gene expression was maintained during in vitro erythroid differentiation of CD34(+) cells. Therefore the use of lentiviral vectors is promising for the future treatment of CEP patients by gene therapy.

  11. Impact of deletion of envelope-related genes of recombinant Sendai viruses on immune responses following pulmonary gene transfer of neonatal mice.

    PubMed

    Tanaka, S; Yonemitsu, Y; Yoshida, K; Okano, S; Kondo, H; Inoue, M; Hasegawa, M; Masumoto, K; Suita, S; Taguchi, T; Sueishi, K

    2007-07-01

    We demonstrated previously that the additive-type recombinant Sendai virus (rSeV) is highly efficient for use in pulmonary gene transfer; however, rSeV exhibits inflammatory responses. To overcome this problem, we tested newly developed non-transmissible constructs, namely, temperature-sensitive F-deleted vector, rSeV/dF (ts-rSeV/dF) and a rSeV with all the envelope-related genes deleted (rSeV/dFdMdHN), for pulmonary gene transfer in neonatal mice, by assessing their toxicity and immune responses. The gene expression in the lungs of neonatal ICR mice peaked on day 2, then gradually decreased until almost disappearing at 14 days after infection in all constructs. Loss of body weight and mortality rate, however, were dramatically improved in mice treated with SeV/dFdMdHN (mortality=0%, n=41) and ts-rSeV/dF (24.2%, n=33) compared with additive rSeV (70.7%, n=58). Although the deletion of envelope-related genes of SeV had a small impact on the production of antibody and cytotoxic T-lymphocyte activity in both adults and neonates, a dramatic reduction was found in the events related to innate responses, including the production of proinflammatory cytokines, particularly in the case of neonates. These results indicate that pulmonary gene transfer using SeV/dFdMdHN warrants further investigation for its possible use in developing safer therapeutics for neonatal lung diseases, including cystic fibrosis.

  12. The effect of RAFT-derived cationic block copolymer structure on gene silencing efficiency.

    PubMed

    Hinton, Tracey M; Guerrero-Sanchez, Carlos; Graham, Janease E; Le, Tam; Muir, Benjamin W; Shi, Shuning; Tizard, Mark L V; Gunatillake, Pathiraja A; McLean, Keith M; Thang, San H

    2012-10-01

    In this work a series of ABA tri-block copolymers was prepared from oligo(ethylene glycol) methyl ether methacrylate (OEGMA(475)) and N,N-dimethylaminoethyl methacrylate (DMAEMA) to investigate the effect of polymer composition on cell viability, siRNA uptake, serum stability and gene silencing. Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization was used as the method of polymer synthesis as this technique allows the preparation of well-defined block copolymers with low polydispersity. Eight block copolymers were prepared by systematically varying the central cationic block (DMAEMA) length from 38 to 192 monomer units and the outer hydrophilic block (OEGMA(475)) from 7 to 69 units. The polymers were characterized using size exclusion chromatography and (1)H NMR. Chinese Hamster Ovary-GFP and Human Embryonic Kidney 293 cells were used to assay cell viability while the efficiency of block copolymers to complex with siRNA was evaluated by agarose gel electrophoresis. The ability of the polymer-siRNA complexes to enter into cells and to silence the targeted reporter gene enhanced green fluorescent protein (EGFP) was measured by using a CHO-GFP silencing assay. The length of the central cationic block appears to be the key structural parameter that has a significant effect on cell viability and gene silencing efficiency with block lengths of 110-120 monomer units being the optimum. The ABA block copolymer architecture is also critical with the outer hydrophilic blocks contributing to serum stability and overall efficiency of the polymer as a delivery system.

  13. Interleukin 10 gene transfer prevents experimental colitis in rats

    PubMed Central

    Barbara, G; Xing, Z; Hogaboam, C; Gauldie, J; Collins, S

    2000-01-01

    BACKGROUND—The development of colitis in interleukin 10 (IL-10) deficient mice, together with the known anti-inflammatory and immunomodulatory properties of this cytokine have prompted consideration of IL-10 as a treatment for inflammatory bowel disease (IBD). However, studies using hrIL-10 in IBD models have yielded inconsistent results.
AIMS—To examine the therapeutic potential of overexpressing the IL-10 gene before and after the induction of experimental colitis in rats.
METHODS—Gene transfer was achieved by intraperitoneal injection of non-replicating human type 5 adenovirus bearing the IL-10 gene, either 24 hours before or one hour after intrarectal administration of dinitrobenzene sulphonic acid in rats. Colonic damage and inflammation was assessed macroscopically and by measuring myeloperoxidase activity and leukotriene B4 concentrations.
RESULTS—Gene transfer increased IL-10 protein in serum for up to six days. IL-10 gene transfer prior to colitis improved colitis macroscopically and histologically, and significantly reduced colonic myeloperoxidase activity and leukotriene B4 concentrations. In contrast, IL-10 gene transfer after the onset of colitis had no beneficial effect.
CONCLUSIONS—Gene therapy using an adenovirus-IL-10 construct was successful in preventing but not in reversing experimental colitis in the rat.


Keywords: gene therapy; colitis; interleukin 10; adenovirus vector; leukotrienes; inflammatory bowel disease; maintenance therapy PMID:10673295

  14. Multilevel populations and the evolution of antibiotic resistance through horizontal gene transfer.

    PubMed

    Andam, Cheryl P; Fournier, Gregory P; Gogarten, Johann Peter

    2011-09-01

    Horizontal gene transfer (HGT) can create diversity in the genetic repertoire of a lineage. Successful gene transfer likely occurs more frequently between more closely related organisms, leading to the formation of higher-level exchange groups that in some respects are comparable to single-species populations. Genes that appear fixed in a single species can be replaced through distant homologs or iso-functional analogs acquired through HGT. These genes may originate from other species or they may be acquired by an individual strain from the species pan-genome. Because of their similarity to alleles in a population, we label these gene variants that are exchanged between related species as homeoalleles. In a case study, we show that biased gene transfer plays an important role in the evolution of aminoacyl-tRNA synthetases (aaRS). Many microorganisms make use of these genes against naturally occurring antibiotics. We suggest that the resistance against naturally occurring antibiotics is the likely driving force behind the frequent switching between divergent aaRS types and the reason for the maintenance of these homeoalleles in higher-level exchange groups. Resistance to naturally occurring antibiotics may lead to the maintenance of different types of aminoacyl-tRNA synthetases in Bacteria through gene transfer.

  15. Studies on the transfer techniques of three maize genes.

    PubMed

    Wang, G; Zhang, H; Ding, Q; Xie, Y; Dai, J

    1996-01-01

    Maize transformation has been carried out through microprojectile bombardment, ultrasonication in a DNA buffer, and ovary-injection with a self-made microinjector. The plasmid pB48.415, which carries a 3'-truncated Bt-toxin protein gene and a hygromycin phosphotransferase (hpt) gene, was used in the transformation. Transgenic maize plants were obtained from immature embryos and embryogenic calli bombarded with a particle gun, embryogenic calli ultrasonicated under different conditions of ovaries injected 10-20 hours after pollination. The results of Dot blotting and Southern blotting analyses proved the integration of the Bt gene into maize genome.

  16. Horizontal Gene Transfers from Bacteria to Entamoeba Complex: A Strategy for Dating Events along Species Divergence

    PubMed Central

    Romero, Miguel; Ximenez, Cecilia

    2016-01-01

    Horizontal gene transfer has proved to be relevant in eukaryotic evolution, as it has been found more often than expected and related to adaptation to certain niches. A relatively large list of laterally transferred genes has been proposed and evaluated for the parasite Entamoeba histolytica. The goals of this work were to elucidate the importance of lateral gene transfer along the evolutionary history of some members of the genus Entamoeba, through identifying donor groups and estimating the divergence time of some of these events. In order to estimate the divergence time of some of the horizontal gene transfer events, the dating of some Entamoeba species was necessary, following an indirect dating strategy based on the fossil record of plausible hosts. The divergence between E. histolytica and E. nuttallii probably occurred 5.93 million years ago (Mya); this lineage diverged from E. dispar 9.97 Mya, while the ancestor of the latter separated from E. invadens 68.18 Mya. We estimated times for 22 transferences; the most recent occurred 31.45 Mya and the oldest 253.59 Mya. Indeed, the acquisition of genes through lateral transfer may have triggered a period of adaptive radiation, thus playing a major role in the evolution of the Entamoeba genus. PMID:27239333

  17. Cre Recombinase Gene Transfer In Vitro and Detection of loxP-Dependent Recombination.

    PubMed

    Ohtsubo, Kazuaki; Marth, Jamey D

    2007-07-01

    INTRODUCTIONAltering the genome of intact cells and organisms by site-specific DNA recombination has become an important gene-transfer methodology. The inclusion of exogenous recombinase target sequences within transferred DNA segments allows subsequent modifications to previously altered genomic structure that increase the utility of gene transfer and enhance experimental design. In this protocol, correctly targeted mouse embryonic stem (ES) cell clones bearing the F[tkneo] allele (containing several loxP sites) are subjected to in vitro Cre gene transfer to generate ES cell subclones bearing either Type 1 (Δ) or Type 2 (F) alleles. Type 2 ES cells are used to generate chimeric mice that are then crossed to germ-line Cre-expressing mice, such as ZP3-Cre transgenic mates. The additional time needed to breed the mice (~2-3 mo) is typically less troublesome than the cost and effort of maintaining multiple clone-derived lines of mice.

  18. Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene therapy of hemophilia B

    SciTech Connect

    Thompson, A.R. Puget Sound Blood Center, Seattle, WA ); Darlington, G. ); Armentano, D.; Woo, S.L.C.

    1990-08-01

    Hemophilia B (Christmas disease) is a chromosome X-linked blood clotting disorder which results when factor IX is deficient or functionally defective. The enzyme is synthesized in the liver, and the existence of animal models for this genetic disease will permit the development of somatic gene therapy protocols aimed at transfer of the functional gene into the liver. The authors report the construction of an N2-based recombinant retroviral vector, NCMVFIX, for efficient transfer and expression of human factor IX cDNA in primary rabbit hepatocytes. In this construct the human cytomegalovirus immediate early promoter directs the expression of factor IX. Hepatocytes were isolated from 3-week-old New Zealand White rabbits, infected with the recombinant virus, and analyzed for secretion of active factor IX. The infected rabbit hepatocytes produced human factor IX that is indistinguishable from enzyme derived from normal human plasma. The recombinant protein is sufficiently {gamma}-carboxylated and is functionally active in clotting assays. These results establish the feasibility of using infected hepatocytes for the expression of this protein and are a step toward the goal of correcting hemophilia B by hepatic gene transfer.

  19. Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

    PubMed Central

    Midoux, P; Mendes, C; Legrand, A; Raimond, J; Mayer, R; Monsigny, M; Roche, A C

    1993-01-01

    Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine. Images PMID:8383843

  20. Deep ocean nutrients imply large latitudinal variation in particle transfer efficiency

    NASA Astrophysics Data System (ADS)

    Weber, Thomas; Cram, Jacob A.; Leung, Shirley W.; DeVries, Timothy; Deutsch, Curtis

    2016-08-01

    The “transfer efficiency” of sinking organic particles through the mesopelagic zone and into the deep ocean is a critical determinant of the atmosphere-ocean partition of carbon dioxide (CO2). Our ability to detect large-scale spatial variations in transfer efficiency is limited by the scarcity and uncertainties of particle flux data. Here we reconstruct deep ocean particle fluxes by diagnosing the rate of nutrient accumulation along transport pathways in a data-constrained ocean circulation model. Combined with estimates of organic matter export from the surface, these diagnosed fluxes reveal a global pattern of transfer efficiency to 1,000 m that is high (˜25%) at high latitudes and low (˜5%) in subtropical gyres, with intermediate values in the tropics. This pattern is well correlated with spatial variations in phytoplankton community structure and the export of ballast minerals, which control the size and density of sinking particles. These findings accentuate the importance of high-latitude oceans in sequestering carbon over long timescales, and highlight potential impacts on remineralization depth as phytoplankton communities respond to a warming climate.

  1. Improving the heat transfer efficiency of synthetic oil with silica nanoparticles.

    PubMed

    Timofeeva, Elena V; Moravek, Michael R; Singh, Dileep

    2011-12-01

    The heat transfer properties of synthetic oil (Therminol 66) used for high temperature applications was improved by introducing 15 nm silicon dioxide nanoparticles. Stable suspensions of inorganic nanoparticles in the non-polar fluid were prepared using a cationic surfactant (benzalkonium chloride). The effects of nanoparticle and surfactant concentrations on thermo-physical properties (viscosity, thermal conductivity and total heat absorption) of these nanofluids were investigated in a wide temperature range. The surfactant-to-nanoparticle (SN) ratio was optimized for higher thermal conductivity and lower viscosity, which are both critical for the efficiency of heat transfer. The rheological behavior of SiO(2)/TH66 nanofluids was correlated to average agglomerate sizes, which were shown to vary with SN ratio and temperature. The conditions of ultrasonic treatment were studied and the temporary decrease of agglomerate size from an equilibrium size (characteristic to SN ratio) was demonstrated. The heat transfer efficiencies were estimated for the formulated nanofluids for both turbulent and laminar flow regimes and were compared to the performance of the base fluid.

  2. DNA Base Pair Resolution Measurements Using Resonance Energy Transfer Efficiency in Lanthanide Doped Nanoparticles

    PubMed Central

    Delplanque, Aleksandra; Wawrzynczyk, Dominika; Jaworski, Pawel; Matczyszyn, Katarzyna; Pawlik, Krzysztof; Buckle, Malcolm; Nyk, Marcin; Nogues, Claude; Samoc, Marek

    2015-01-01

    Lanthanide-doped nanoparticles are of considerable interest for biodetection and bioimaging techniques thanks to their unique chemical and optical properties. As a sensitive luminescence material, they can be used as (bio) probes in Förster Resonance Energy Transfer (FRET) where trivalent lanthanide ions (La3+) act as energy donors. In this paper we present an efficient method to transfer ultrasmall (ca. 8 nm) NaYF4 nanoparticles dispersed in organic solvent to an aqueous solution via oxidation of the oleic acid ligand. Nanoparticles were then functionalized with single strand DNA oligomers (ssDNA) by inducing covalent bonds between surface carboxylic groups and a 5’ amine modified-ssDNA. Hybridization with the 5’ fluorophore (Cy5) modified complementary ssDNA strand demonstrated the specificity of binding and allowed the fine control over the distance between Eu3+ ions doped nanoparticle and the fluorophore by varying the number of the dsDNA base pairs. First, our results confirmed nonradiative resonance energy transfer and demonstrate the dependence of its efficiency on the distance between the donor (Eu3+) and the acceptor (Cy5) with sensitivity at a nanometre scale. PMID:25748446

  3. Deep ocean nutrients imply large latitudinal variation in particle transfer efficiency

    PubMed Central

    Weber, Thomas; Cram, Jacob A.; Leung, Shirley W.; DeVries, Timothy; Deutsch, Curtis

    2016-01-01

    The “transfer efficiency” of sinking organic particles through the mesopelagic zone and into the deep ocean is a critical determinant of the atmosphere−ocean partition of carbon dioxide (CO2). Our ability to detect large-scale spatial variations in transfer efficiency is limited by the scarcity and uncertainties of particle flux data. Here we reconstruct deep ocean particle fluxes by diagnosing the rate of nutrient accumulation along transport pathways in a data-constrained ocean circulation model. Combined with estimates of organic matter export from the surface, these diagnosed fluxes reveal a global pattern of transfer efficiency to 1,000 m that is high (∼25%) at high latitudes and low (∼5%) in subtropical gyres, with intermediate values in the tropics. This pattern is well correlated with spatial variations in phytoplankton community structure and the export of ballast minerals, which control the size and density of sinking particles. These findings accentuate the importance of high-latitude oceans in sequestering carbon over long timescales, and highlight potential impacts on remineralization depth as phytoplankton communities respond to a warming climate. PMID:27457946

  4. Development of an Autologous Macrophage-based Adoptive Gene Transfer Strategy to Treat Posttraumatic Osteoarthritis (PTOA) and Osteoarithritis (OA)

    DTIC Science & Technology

    2016-05-01

    AWARD NUMBER: W81XWH-13-1-0228 TITLE: Development of an Autologous Macrophage-based Adoptive Gene Transfer Strategy to Treat Posttraumatic...Final 3. DATES COVERED 1 Sep 2013 - 28 Feb 2016 4. TITLE AND SUBTITLE Development of an Autologous Macrophage-based Adoptive Gene Transfer Strategy to...autologous macrophage-based adoptive gene transfer strategy can effectively deliver and confine expression of an anti-catabolic gene (IL-1ra or IL-1β

  5. Horizontal Gene Transfer and the Evolution of Bacterial and Archaeal Population Structure

    PubMed Central

    Alm, Eric J.; Hanage, William P.

    2013-01-01

    Many bacterial and archaeal lineages have a history of extensive and ongoing horizontal gene transfer and loss, as evidenced by the large differences in genome content even among otherwise closely related isolates. How ecologically cohesive populations might evolve and be maintained under such conditions of rapid gene turnover has remained controversial. Here we synthesize recent literature demonstrating the importance of habitat and niche in structuring horizontal gene transfer. This leads to a model of ecological speciation via gradual genetic isolation triggered by differential habitat association of nascent populations. Further, we hypothesize that subpopulations can evolve through local gene exchange networks by tapping into a gene pool that is adaptive towards local, continuously changing organismic interactions and is, to a large degree, responsible for the observed rapid gene turnover. Overall, these insights help explain how bacteria and archaea form populations that display both ecological cohesion and high genomic diversity. PMID:23332119

  6. Synergistic "ping-pong" energy transfer for efficient light activation in a c