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Sample records for aav2 vector encoding

  1. Diabetes enhances the efficacy of AAV2 vectors in the retina: therapeutic effect of AAV2 encoding vasoinhibin and soluble VEGF receptor 1.

    PubMed

    Díaz-Lezama, Nundehui; Wu, Zhijian; Adán-Castro, Elva; Arnold, Edith; Vázquez-Membrillo, Miguel; Arredondo-Zamarripa, David; Ledesma-Colunga, Maria G; Moreno-Carranza, Bibiana; Martinez de la Escalera, Gonzalo; Colosi, Peter; Clapp, Carmen

    2016-03-01

    Adeno-associated virus (AAV) vector-mediated delivery of inhibitors of blood-retinal barrier breakdown (BRBB) offers promise for the treatment of diabetic macular edema. Here, we demonstrated a reversal of blood-retinal barrier pathology mediated by AAV type 2 (AAV2) vectors encoding vasoinhibin or soluble VEGF receptor 1 (sFlt-1) when administered intravitreally to diabetic rats. Efficacy and safety of the AAV2 vasoinhibin vector were tested by monitoring its effect on diabetes-induced changes in the retinal vascular bed and thickness, and in the electroretinogram (ERG). Also, the transduction of AAV2 vectors and expression of AAV2 receptors and co-receptors were compared between the diabetic and the non-diabetic rat retinas. AAV2 vasoinhibin or AAV2 sFlt-1 vectors were injected intravitreally before or after enhanced BRBB due to diabetes induced by streptozotocin. The BRBB was examined by the Evans blue method, the vascular bed by fluorescein angiography, expression of the AAV2 EGFP reporter vector by confocal microscopy, and the AAV2 genome, expression of transgenes, receptors, and co-receptors by quantitative PCR. AAV2 vasoinhibin and sFlt-1 vectors inhibited the diabetes-mediated increase in BRBB when injected after, but not before, diabetes was induced. The AAV2 vasoinhibin vector decreased retinal microvascular abnormalities and the diabetes-induced reduction of the B-wave of the ERG, but it had no effect in non-diabetic controls. Also, retinal thickness was not altered by diabetes or by the AAV2 vasoinhibin vector. The AAV2 genome, vasoinhibin and sFlt-1 transgenes, and EGFP levels were higher in the retinas from diabetic rats and were associated with an elevated expression of AAV2 receptors (syndecan, glypican, and perlecan) and co-receptors (fibroblast growth factor receptor 1, αvβ5 integrin, and hepatocyte growth factor receptor). We conclude that retinal transduction and efficacy of AAV2 vectors are enhanced in diabetes, possibly due to their elevated

  2. The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

    PubMed Central

    Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

    2004-01-01

    Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

  3. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    SciTech Connect

    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

    2008-11-25

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by {approx} 68% and {approx} 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

  4. Neutralizing antibodies against AAV2, AAV5 and AAV8 in healthy and HIV-1-infected subjects in China: implications for gene therapy using AAV vectors.

    PubMed

    Liu, Q; Huang, W; Zhang, H; Wang, Y; Zhao, J; Song, A; Xie, H; Zhao, C; Gao, D; Wang, Y

    2014-08-01

    Adeno-associated viruses (AAV) have attracted attention as potential vectors for gene therapy and vaccines against several diseases, including HIV-1 infection. However, the presence of neutralizing antibodies (NAbs) after natural AAV infections inhibits their transfection in re-exposed subjects. To identify candidate AAV vectors for therapeutic or prophylactic HIV vaccines, NAbs against AAV2, AAV5 and AAV8 were screened in the sera of healthy individuals in China and 10 developed countries and an HIV-1-infected Chinese population. Seroprevalence was higher for AAV2 (96.6%) and AAV8 (82.0%) than for AAV5 (40.2%) in normal Chinese subjects. Among individuals seropositive for AAV5, >80% had low NAb titers (<1:90). The prevalence and titers of NAbs against the three AAVs were significantly higher in China than in developed countries (P<0.01). The prevalence of NAbs against AAV5 did not differ significantly between healthy and HIV-1-infected Chinese subjects (P=0.39). Co-occurrence of NAbs against AAV2, AAV5, and AAV8 was observed in the healthy population, and 15, 41, and 41% of individuals were AAV2(+), AAV2(+)/AAV8(+), and AAV2(+)/AAV5(+)/AAV8(+), respectively. Therefore, AAV5 exposure is low in healthy and HIV-1-infected populations Chinese individuals, and vectors based on AAV5 may be appropriate for human gene therapy or vaccines. PMID:24849042

  5. Correction of Murine Diabetic Hyperglycaemia With A Single Systemic Administration of An AAV2/8 Vector Containing A Novel Codon Optimized Human Insulin Gene.

    PubMed

    Uin, Gan Shu; Maria, Notaridou; Ying, Fu Zhen; Kok Onn, Lee; Chuan, Sia Kian; Chunilal, Nathwani Amit; Marco, Della Peruta; Yorke, Calne Roy

    2016-01-01

    We report the correction of hyperglycemia of STZ induced diabetic mice using one intravenous systemic administration of a single stranded serotype 8 pseudotyped adeno-associated virus (ssAAV2/8) vector encoding the human proinsulin gene under a constitutive liver specific promoter. In vivo dose titration experiments were carried out and we identified an optimal range that achieved maintenance of euglycaemia or a mild diabetic condition for at least 9 months and ongoing to beyond 1 year for some animals, accompanied by human C-peptide secretion and weight gain. Further DNA codon optimization of the insulin gene construct resulted in approximately 3-10 times more human C-peptide secreted in the blood of codon optimized treated animals thereby reducing the number of vector particles required to achieve the same extent of reduction in blood glucose levels as the non-codon optimized vector. The constitutive secretion of insulin achieved with a single administration of the vector could be of therapeutic value for some diabetic patients. PMID:26795016

  6. Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria

    PubMed Central

    Harding, CO; Gillingham, MB; Hamman, K; Clark, H; Goebel-Daghighi, E; Bird, A; Koeberl, DD

    2009-01-01

    Novel recombinant adeno-associated virus vectors pseudo-typed with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pahenu2 mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pahenu2 mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5±2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism. PMID:16319949

  7. Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria.

    PubMed

    Harding, C O; Gillingham, M B; Hamman, K; Clark, H; Goebel-Daghighi, E; Bird, A; Koeberl, D D

    2006-03-01

    Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pah(enu2) mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pah(enu2) mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5+/-2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism. PMID:16319949

  8. Vitrectomy Before Intravitreal Injection of AAV2/2 Vector Promotes Efficient Transduction of Retinal Ganglion Cells in Dogs and Nonhuman Primates.

    PubMed

    Tshilenge, Kizito-Tshitoko; Ameline, Baptiste; Weber, Michel; Mendes-Madeira, Alexandra; Nedellec, Steven; Biget, Marine; Provost, Nathalie; Libeau, Lyse; Blouin, Véronique; Deschamps, Jack-Yves; Le Meur, Guylène; Colle, Marie-Anne; Moullier, Philippe; Pichard, Virginie; Rolling, Fabienne

    2016-06-01

    Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons. PMID:27229628

  9. AAV2/8 Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Murine Liver, Muscle, and Retina Efficiently

    PubMed Central

    Doria, Monica; Ferrara, Antonella

    2013-01-01

    Abstract During the production of some adeno-associated virus (AAV) serotypes, a large amount of vectors is found in the medium of producing cells. For their purification, previous protocols used tangential flow filtration (TFF) of the medium followed by iodixanol gradient centrifugation. Taking advantage of the higher purity of the medium than the cell-derived material as the source of AAV, we tested a simple method that combines production of large culture medium volumes containing AAV from cell stacks with medium clarification+TFF without further time-consuming and nonscalable centrifugation. To test this, we selected AAV2/8, which is emerging as a favored serotype for transduction of liver, muscle, and retina and abundantly found in the extracellular medium. We show that yields and in vitro infectivity of AAV2/8 vectors produced from the culture medium using this method are higher than those of vectors purified from the same cell lysate using a conventional CsCl2 gradient ultracentrifugation-based method, although purity appears inferior. In addition, we found that the transduction efficiency of AAV2/8 purified from medium was similar to that of AAV2/8 purified from the same cell lysate in the murine liver, muscle, and retina. Considering that the purification protocol from the medium we describe requires 3 hr as opposed to the 63 hr of a conventional two-round CsCl2-gradient ultracentrifugation+desalting, we conclude that TFF of the medium containing AAV2/8 represents a quick and scalable method to purify research-grade vectors for use in animal models. PMID:24116943

  10. Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-CB-hRS1, a Recombinant Adeno-Associated Virus Vector Expressing Retinoschisin

    PubMed Central

    Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Miller, Paul E.; Sharma, Alok K.; Ver Hoeve, James N.; Howard, Kellie; Knop, David R.; Neuringer, Martha; McGill, Trevor; Stoddard, Jonathan; Chulay, Jeffrey D.

    2015-01-01

    Applied Genetic Technologies Corporation is developing rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus (rAAV) vector for treatment of X-linked retinoschisis (XLRS), an inherited retinal disease characterized by splitting (schisis) of retinal layers causing poor vision. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-CB-hRS1 in normal cynomolgus macaques. Three groups of male animals (n = 6 per group) received an intravitreal injection in one eye of either vehicle, or rAAV2tYF-CB-hRS1 at one of two dose levels (4 × 1010 or 4 × 1011 vg/eye). Half the animals were sacrificed after 14 days and the others after 91 or 115 days. The intravitreal injection procedure was well tolerated in all groups. Serial ophthalmic examinations demonstrated a dose-related anterior and posterior segment inflammatory response that improved over time. There were no test article-related effects on intraocular pressure, electroretinography, visual evoked potential, hematology, coagulation, clinical chemistry, or gross necropsy observations. Histopathological examination demonstrated minimal or moderate mononuclear infiltrates in 6 of 12 vector-injected eyes. Immunohistochemical staining showed RS1 labeling of the ganglion cell layer at the foveal slope in vector-injected eyes at both dose levels. Serum anti-AAV antibodies were detected in 4 of 6 vector-injected animals at the day 15 sacrifice and all vector-injected animals at later time points. No animals developed antibodies to RS1. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS. PMID:26390090

  11. Tyrosine triple mutated AAV2-BDNF gene therapy in a rat model of transient IOP elevation

    PubMed Central

    Igarashi, Tsutomu; Kobayashi, Maika; Kameya, Shuhei; Fujimoto, Chiaki; Nakamoto, Kenji; Takahashi, Hisatomo; Igarashi, Toru; Miyake, Noriko; Iijima, Osamu; Hirai, Yukihiko; Shimada, Takashi; Okada, Takashi; Takahashi, Hiroshi

    2016-01-01

    Purpose We examined the neuroprotective effects of exogenous brain-derived neurotrophic factor (BDNF), which provides protection to retinal ganglion cells (RGCs) in rodents, in a model of transient intraocular pressure (IOP) elevation using a mutant (triple Y-F) self-complementary adeno-associated virus type 2 vector encoding BDNF (tm-scAAV2-BDNF). Methods The tm-scAAV2-BDNF or control vector encoding green fluorescent protein (GFP; tm-scAAV2-GFP) was intravitreally administered to rats, which were then divided into four groups: control, ischemia/reperfusion (I/R) injury only, I/R injury with tm-scAAV2-GFP, and tm-scAAV2-BDNF. I/R injury was then induced by transiently increasing IOP, after which the rats were euthanized to measure the inner retinal thickness and cell counts in the RGC layer. Results Intravitreous injection of tm-scAAV2-BDNF resulted in high levels of BDNF expression in the neural retina. Histological analysis showed that the inner retinal thickness and cell numbers in the RGC layer were preserved after transient IOP elevation in eyes treated with tm-scAAV2-BDNF but not in the other I/R groups. Significantly reduced glial fibrillary acidic protein (GFAP) immunostaining after I/R injury in the rats that received tm-scAAV2-BDNF indicated reduced retinal stress, and electroretinogram (ERG) analysis confirmed preservation of retinal function in the tm-scAAV2-BDNF group. Conclusions These results demonstrate the feasibility and effectiveness of neuroprotective gene therapy using tm-scAAV2-BDNF to protect the inner retina from transiently high intraocular pressure. An in vivo gene therapeutic approach to the clinical management of retinal diseases in conditions such as glaucoma, retinal artery occlusion, hypertensive retinopathy, and diabetic retinopathy thus appears feasible. PMID:27440998

  12. High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

    PubMed Central

    Li, Mengxin; Jayandharan, Giridhara R.; Li, Baozheng; Ling, Chen; Ma, Wenqin; Srivastava, Arun

    2010-01-01

    Abstract Adeno-associated virus 2 (AAV2) vectors transduce fibroblasts and mesenchymal stem cells (MSCs) inefficiently, which limits their potential widespread applicability in combinatorial gene and cell therapy. We have reported that AAV2 vectors fail to traffic efficiently to the nucleus in murine fibroblasts. We have also reported that site-directed mutagenesis of surface-exposed tyrosine residues on viral capsids leads to improved intracellular trafficking of the mutant vectors, and the transduction efficiency of the single tyrosine-mutant vectors is ∼10-fold higher in human cells. In the current studies, we evaluated the transduction efficiency of single as well as multiple tyrosine-mutant AAV2 vectors in murine fibroblasts. Our results indicate that the Y444F mutant vectors transduce these cells most efficiently among the seven single-mutant vectors, with >30-fold increase in transgene expression compared with the wild-type vectors. When the Y444F mutation is combined with additional mutations (Y500F and Y730F), the transduction efficiency of the triple-mutant vectors is increased by ∼130-fold and the viral intracellular trafficking is also significant improved. Similarly, the triple-mutant vectors are capable of transducing up to 80–90% of bone marrow-derived primary murine as well as human MSCs. Thus, high-efficiency transduction of fibroblasts with reprogramming genes to generate induced pluripotent stem cells, and the MSCs for delivering therapeutic genes, should now be feasible with the tyrosine-mutant AAV vectors. PMID:20507237

  13. Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans.

    PubMed

    Loring, Heather S; ElMallah, Mai K; Flotte, Terence R

    2016-04-01

    The first human gene therapy trials using recombinant adeno-associated virus (rAAV) vectors were performed in cystic fibrosis (CF) patients. Over 100 CF patients were enrolled in 5 separate trials of rAAV2-CFTR administration via nasal, endobronchial, maxillary sinus, and aerosol delivery. Recombinant AAV vectors were designed to deliver the CF transmembrane regulator (CFTR) gene and correct the basic CFTR defect by restoring chloride transport and reverting the upregulation of proinflammatory cytokines. However, vector DNA expression was limited in duration because of the low incidence of integration and natural airway epithelium turnover. In addition, repeated administration of AAV-CFTR vector resulted in a humoral immune response that prevented effective gene transfer from subsequent doses of vector. AAV serotype 2 was used in human trials before the comparison with other serotypes and determination that serotypes 1 and 5 not only possess higher tropism for the airway epithelium, but also are capable of bypassing the binding and trafficking processes-both were important hindrances to the effectiveness of rAAV2. Although rAAV-CFTR gene therapy does not appear likely to supplant newer small-molecule CFTR modulators in the near future, early work with rAAV-CFTR provided an important foundation for later use of rAAV in humans. PMID:26895204

  14. Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy.

    PubMed

    Hareendran, Sangeetha; Ramakrishna, Banumathi; Jayandharan, Giridhara R

    2016-01-01

    Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy. PMID:26443873

  15. rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

    PubMed Central

    2013-01-01

    Background Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. Results We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical

  16. Preclinical Dose-Finding Study With a Liver-Tropic, Recombinant AAV-2/8 Vector in the Mouse Model of Galactosialidosis

    PubMed Central

    Hu, Huimin; Gomero, Elida; Bonten, Erik; Gray, John T; Allay, Jim; Wu, Yanan; Wu, Jianrong; Calabrese, Christopher; Nienhuis, Arthur; d'Azzo, Alessandra

    2012-01-01

    Galactosialidosis (GS) is a lysosomal storage disease linked to deficiency of the protective protein/cathepsin A (PPCA). Similarly to GS patients, Ppca-null mice develop a systemic disease of the reticuloendothelial system, affecting most visceral organs and the nervous system. Symptoms include severe nephropathy, visceromegaly, infertility, progressive ataxia, and shortened life span. Here, we have conducted a preclinical, dose-finding study on a large cohort of GS mice injected intravenously at 1 month of age with increasing doses of a GMP-grade rAAV2/8 vector, expressing PPCA under the control of a liver-specific promoter. Treated mice, monitored for 16 weeks post-treatment, had normal physical appearance and behavior without discernable side effects. Despite the restricted expression of the transgene in the liver, immunohistochemical and biochemical analyses of other systemic organs, serum, and urine showed a dose-dependent, widespread correction of the disease phenotype, suggestive of a protein-mediated mechanism of cross-correction. A notable finding was that rAAV-treated GS mice showed high expression of PPCA in the reproductive organs, which resulted in reversal of their infertility. Together these results support the use of this rAAV-PPCA vector as a viable and safe method of gene delivery for the treatment of systemic disease in non-neuropathic GS patients. PMID:22008912

  17. Preclinical safety evaluation of recombinant adeno-associated virus 2 vector encoding human tumor necrosis factor receptor-immunoglobulin Fc fusion gene.

    PubMed

    Zhou, Xiaobing; Shen, Lianzhong; Liu, Li; Wang, Chao; Qi, Weihong; Zhao, Aizhi; Wu, Xiaobing; Li, Bo

    2016-03-01

    Recombinant adeno-associated virus (rAAV) 2 vector gene therapy offers promise for the healing of Rheumatoid arthritis. To support the clinical development of the candidate gene therapeutic product in China, a comprehensive preclinical safety assessment of rAAV2 encoding human TNF receptor-immunoglobulin Fc fusion gene (rAAV2/human TNFR:Fc), were conducted in 3 species of experimental animals. No abnormal findings were observed in mice following single intravenous administration with test article. Compared with the control group, no differences in mean body weight, food consumption in rats and monkeys following the repeated intraarticular administration with rAAV2/human TNFR:Fc. There were also no significant adverse effects due to treatment noted by clinical chemistry, hematology and pathology assessments. After intraarticular administration with rAAV2/human TNFR:Fc, the vector DNA initially distributed to spleen, lymph nodes, and joint synovium. The vector DNA cleared rapidly as it could be detected mainly at the site of injection by 91 d post-administration (182 d for monkey). Taken together, localized delivery of rAAV2/human TNFR:Fc showed no significant toxicity in mice, rats, and monkeys, which support the planned clinical evaluation of this product. PMID:26837862

  18. Safety and Biodistribution Evaluation in CNGB3-Deficient Mice of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia.

    PubMed

    Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T Michael; Miller, Paul E; McPherson, Leslie; Ver Hoeve, James N; Smith, Leia M; Arndt, Tara; Mandapati, Savitri; Robinson, Paulette M; Calcedo, Roberto; Knop, David R; Hauswirth, William W; Chulay, Jeffrey D

    2016-03-01

    Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice. Three groups of animals (n = 35 males and 35 females per group) received a subretinal injection in one eye of 1 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two dose concentrations (1 × 10(12) or 4.2 × 10(12) vg/ml) and were euthanized 4 or 13 weeks later. There were no test-article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated electroretinography (ERG) responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group and 31% of animals in the lower dose group. Rod-mediated ERG responses were reduced in the treated eye for all groups, with the greatest reduction in males given the higher dose of vector, but returned to normal by the end of the study. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in nonocular tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations. PMID:27003752

  19. Vector Encoding in Biochemical Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    Encoding of environmental cues via biochemical signaling pathways is of vital importance in the transmission of information for cells in a network. The current literature assumes a single cell state is used to encode information, however, recent research suggests the optimal strategy utilizes a vector of cell states sampled at various time points. To elucidate the optimal sampling strategy for vector encoding, we take an information theoretic approach and determine the mutual information of the calcium signaling dynamics obtained from fibroblast cells perturbed with different concentrations of ATP. Specifically, we analyze the sampling strategies under the cases of fixed and non-fixed vector dimension as well as the efficiency of these strategies. Our results show that sampling with greater frequency is optimal in the case of non-fixed vector dimension but that, in general, a lower sampling frequency is best from both a fixed vector dimension and efficiency standpoint. Further, we find the use of a simple modified Ornstein-Uhlenbeck process as a model qualitatively captures many of our experimental results suggesting that sampling in biochemical networks is based on a few basic components.

  20. The impact of minimally oversized adeno-associated viral vectors encoding human factor VIII on vector potency in vivo

    PubMed Central

    Kyostio-Moore, Sirkka; Berthelette, Patricia; Piraino, Susan; Sookdeo, Cathleen; Nambiar, Bindu; Jackson, Robert; Burnham, Brenda; O’Riordan, Catherine R; Cheng, Seng H; Armentano, Donna

    2016-01-01

    Recombinant adeno-associated viral (rAAV) vectors containing oversized genomes provide transgene expression despite low efficiency packaging of complete genomes. Here, we characterized the properties of oversized rAAV2/8 vectors (up to 5.4 kb) encoding human factor VIII (FVIII) under the transcriptional control of three liver promoters. All vectors provided sustained production of active FVIII in mice for 7 months and contained comparable levels of vector genomes and complete expression cassettes in liver. Therefore, for the 5.4 kb genome size range, a strong expression cassette was more important for FVIII production than the vector genome size. To evaluate the potency of slightly oversized vectors, a 5.1 kb AAVrh8R/FVIII vector was compared to a 4.6 kb (wild-type size) vector with an identical expression cassette (but containing a smaller C1-domain deleted FVIII) for 3 months in mice. The 5.1 kb vector had twofold to threefold lower levels of plasma FVIII protein and liver vector genomes than that obtained with the 4.6 kb vector. Vector genomes for both vectors persisted equally and existed primarily as high molecular weight concatemeric circular forms in liver. Taken together, these results indicate that the slightly oversized vectors containing heterogeneously packaged vector genomes generated a functional transgene product but exhibited a twofold to threefold lower in vivo potency. PMID:26958574

  1. Intravitreal Injection of AAV2 Transduces Macaque Inner Retina

    PubMed Central

    Yin, Lu; Greenberg, Kenneth; Hunter, Jennifer J.; Dalkara, Deniz; Kolstad, Kathleen D.; Masella, Benjamin D.; Wolfe, Robert; Visel, Meike; Stone, Daniel; Libby, Richard T.; DiLoreto, David; Schaffer, David; Flannery, John; Williams, David R.

    2011-01-01

    Purpose. Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans. Methods. In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters. Results. AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-β-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters—human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)—AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis. Conclusions. The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea. PMID:21310920

  2. Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II.

    PubMed

    Sun, Baodong; Zhang, Haoyue; Franco, Luis M; Young, Sarah P; Schneider, Ayn; Bird, Andrew; Amalfitano, Andrea; Chen, Y-T; Koeberl, Dwight D

    2005-01-01

    Glycogen storage disease type II (GSD-II; Pompe disease) causes death in infancy from cardiorespiratory failure. The underlying deficiency of acid alpha-glucosidase (GAA; acid maltase) can be corrected by liver-targeted gene therapy in GSD-II, if secretion of GAA is accompanied by receptor-mediated uptake in cardiac and skeletal muscle. An adeno-associated virus (AAV) vector encoding human (h) GAA was pseudotyped as AAV8 (AAV2/8) and injected intravenously into immunodeficient GSD-II mice. High levels of hGAA were maintained in plasma for 24 weeks following AAV2/8 vector administration. A marked increase in vector copy number in the liver was demonstrated for the AAV2/8 vector compared to the analogous AAV2/2 vector. GAA deficiency in the heart and skeletal muscle was corrected with the AAV2/8 vector in male GSD-II mice, consistent with receptor-mediated uptake of hGAA. Male GSD-II mice demonstrated complete correction of glycogen storage in heart and diaphragm with the AAV2/8 vector, while female GSD-II mice had correction only in the heart. A biomarker for GSD-II was reduced in both sexes following AAV2/8 vector administration. Therefore, GAA production with an AAV2/8 vector in a depot organ, the liver, generated evidence for efficacious gene therapy in a mouse model for GSD-II. PMID:15585406

  3. Enhanced efficacy of an AAV vector encoding chimeric, highly secreted acid alpha-glucosidase in glycogen storage disease type II.

    PubMed

    Sun, Baodong; Zhang, Haoyue; Benjamin, Daniel K; Brown, Talmage; Bird, Andrew; Young, Sarah P; McVie-Wylie, Alison; Chen, Y-T; Koeberl, Dwight D

    2006-12-01

    Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is an inherited muscular dystrophy caused by deficiency in the activity of the lysosomal enzyme acid alpha-glucosidase (GAA). We hypothesized that chimeric GAA containing an alternative signal peptide could increase the secretion of GAA from transduced cells and enhance the receptor-mediated uptake of GAA in striated muscle. The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold. Receptor-mediated uptake of secreted, chimeric GAA corrected cultured GSD-II patient cells. High-level hGAA was sustained in the plasma of GSD-II mice for 24 weeks following administration of an AAV2/8 vector encoding chimeric GAA; furthermore, GAA activity was increased and glycogen content was significantly reduced in striated muscle and in the brain. Administration of only 1 x 10(10) vector particles increased GAA activity in the heart and diaphragm for >18 weeks, whereas 3 x 10(10) vector particles increased GAA activity and reduced glycogen content in the heart, diaphragm, and quadriceps. Furthermore, an AAV2/2 vector encoding chimeric GAA produced secreted hGAA for >12 weeks in the majority of treated GSD-II mice. Thus, chimeric, highly secreted GAA enhanced the efficacy of AAV vector-mediated gene therapy in GSD-II mice. PMID:16987711

  4. Local and systemic responses following intravitreous injection of AAV2-encoded modified Volvox channelrhodopsin-1 in a genetically blind rat model.

    PubMed

    Sugano, E; Tabata, K; Takahashi, M; Nishiyama, F; Shimizu, H; Sato, M; Tamai, M; Tomita, H

    2016-02-01

    We previously designed a modified channelrhodopsin-1 (mVChR1) protein chimera with a broader action than that of Chlamydomonas channelrhodopsin-2 and reported that its transduction into retinal ganglion cells can restore visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats, with photostimuli ranging from 486 to 640 nm. In the current study, we sought to investigate the safety and influence of mVChR1 transgene expression. Adeno-associated virus type 2 encoding mVChR1 was administered by intravitreous injection into dystrophic RCS rats. Reverse-transcription PCR was used to monitor virus and transgene dissemination and the results demonstrated that their expression was restricted specifically within the eye tissues, and not in non-target organs. Moreover, examination of the blood, plasma and serum revealed that no excess immunoreactivity was present, as determined using standard clinical hematological parameters. Serum antibodies targeting the recombinant adeno-associated virus (rAAV) capsid increased after the injection; however, no increase in mVChR1 antibody was detected during the observation period. In addition, retinal histological examination showed no signs of inflammation in rAAV-injected rats. In conclusion, our results demonstrate that mVChR1 can be exogenously expressed without harmful immunological reactions in vivo. These findings will aid in studies of AAV gene transfer to restore vision in late-stage retinitis pigmentosa. PMID:26440056

  5. Hepatorenal correction in murine glycogen storage disease type I with a double-stranded adeno-associated virus vector.

    PubMed

    Luo, Xiaoyan; Hall, Gentzon; Li, Songtao; Bird, Andrew; Lavin, Peter J; Winn, Michelle P; Kemper, Alex R; Brown, Talmage T; Koeberl, Dwight D

    2011-11-01

    Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (-/-) mice. Hypoglycemia during fasting (plasma glucose <100 mg/dl) was prevented for >6 months by the dsAAV2/7, dsAAV2/8, and dsAAV2/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2/7 and dsAAV2/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (-/-) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2/9 vector. Hepatorenal correction in G6pase (-/-) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism. PMID:21730973

  6. Hepatorenal Correction in Murine Glycogen Storage Disease Type I With a Double-stranded Adeno-associated Virus Vector

    PubMed Central

    Luo, Xiaoyan; Hall, Gentzon; Li, Songtao; Bird, Andrew; Lavin, Peter J; Winn, Michelle P; Kemper, Alex R; Brown, Talmage T; Koeberl, Dwight D

    2011-01-01

    Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (−/−) mice. Hypoglycemia during fasting (plasma glucose <100 mg/dl) was prevented for >6 months by the dsAAV2/7, dsAAV2/8, and dsAAV2/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2/7 and dsAAV2/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (−/−) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2/9 vector. Hepatorenal correction in G6pase (−/−) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism. PMID:21730973

  7. Pulse Vector-Excitation Speech Encoder

    NASA Technical Reports Server (NTRS)

    Davidson, Grant; Gersho, Allen

    1989-01-01

    Proposed pulse vector-excitation speech encoder (PVXC) encodes analog speech signals into digital representation for transmission or storage at rates below 5 kilobits per second. Produces high quality of reconstructed speech, but with less computation than required by comparable speech-encoding systems. Has some characteristics of multipulse linear predictive coding (MPLPC) and of code-excited linear prediction (CELP). System uses mathematical model of vocal tract in conjunction with set of excitation vectors and perceptually-based error criterion to synthesize natural-sounding speech.

  8. Vector Adaptive/Predictive Encoding Of Speech

    NASA Technical Reports Server (NTRS)

    Chen, Juin-Hwey; Gersho, Allen

    1989-01-01

    Vector adaptive/predictive technique for digital encoding of speech signals yields decoded speech of very good quality after transmission at coding rate of 9.6 kb/s and of reasonably good quality at 4.8 kb/s. Requires 3 to 4 million multiplications and additions per second. Combines advantages of adaptive/predictive coding, and code-excited linear prediction, yielding speech of high quality but requires 600 million multiplications and additions per second at encoding rate of 4.8 kb/s. Vector adaptive/predictive coding technique bridges gaps in performance and complexity between adaptive/predictive coding and code-excited linear prediction.

  9. Enhanced Efficacy of an AAV Vector Encoding Chimeric, Highly-Secreted Acid α-glucosidase in Glycogen Storage Disease Type II

    PubMed Central

    Sun, Baodong; Zhang, Haoyue; Benjamin, Daniel K.; Brown, Talmage; Bird, Andrew; Young, Sarah P.; McVie-Wylie, Alison; Chen, Y-T; Koeberl, Dwight D.

    2009-01-01

    Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is an inherited muscular dystrophy caused by deficiency in the activity of the lysosomal enzyme acid α-glucosidase (GAA). We hypothesized that chimeric GAA containing an alternative signal peptide could increase the secretion of GAA from transduced cells and enhance the receptor-mediated uptake of GAA in striated muscle. The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold. Receptor-mediated uptake of secreted, chimeric GAA corrected cultured GSD-II patient cells. High-level hGAA was sustained in the plasma of GSD-II mice for 24 weeks following administration of an AAV2/8 vector encoding chimeric GAA; furthermore, GAA activity was increased and glycogen content was significantly reduced in striated muscle and in the brain. Administration of only 1×1010 vector particles increased GAA activity in the heart and diaphragm for >18 weeks, whereas 3×1010 vector particles increased GAA activity and reduced glycogen content in the heart, diaphragm, and quadriceps. Furthermore, an AAV2/2 vector encoding chimeric GAA produced secreted hGAA for >12 weeks in the majority of treated GSD-II mice. Thus, chimeric, highly secreted GAA enhanced the efficacy of AAV vector-mediated gene therapy in GSD-II mice. PMID:16987711

  10. Enhancement of flap survival and changes in angiogenic gene expression after AAV2-mediated VEGF gene transfer to rat ischemic flaps.

    PubMed

    Wang, Xiao Tian; Avanessian, Bella; Ma, Qiangzhong; Durfee, Heather; Tang, Yu Qing; Liu, Paul Y

    2011-01-01

    Necrosis of surgically transferred flaps due to ischemia is a serious wound problem. We evaluated the improvement of flap survival and changes in angiogenic gene expression profiles after transfer of the VEGF gene by means of adeno-associated virus type 2 (AAV2) vector to rat ischemic flaps. Thirty rats were divided into one experimental group, one AAV2-GFP group, and one saline group. AAV2-VEGF or AAV2-GFP were injected intradermally into the rat dorsum in the AAV2-VEGF or AAV2-GFP group. The saline group received saline injection. A 3 × 10 cm flap was raised in each rat two weeks post-injection. One week after surgery, flap viability was evaluated. Angiogenesis real-time PCR array was performed to analyze the expression of angiogenesis-associated genes. The AAV2-VEGF treatment significantly improved flap survival (p<0.05). Immunohistochemical staining showed increased VEGF expression in AAV2-VEGF treated flaps. The PCR array identified remarkable changes in 6 out of the 84 angiogenesis-associated genes in AAV2-VEGF treated flaps. Particularly, EGF, PDGF-A and VEGF-B genes were up-regulated in these flaps. In contrast, FGF2 gene expression was down-regulated. In conclusion, AAV2-VEGF improves flap survival and affects the expression of a series of endogenous growth factor genes, which likely play critical roles in the enhancement of ischemic flap survival. PMID:21649787

  11. Slow AAV2 clearance from the brain of nonhuman primates and anti-capsid immune response.

    PubMed

    Samaranch, L; Hadaczek, P; Kells, A P; Bringas, J R; Stockinger, D; San Sebastian, W; Macayan, M; Samineni, S; Pivirotto, P; Forsayeth, J; Bankiewicz, K S

    2016-04-01

    Adeno-associated virus serotype 2 (AAV2) has previously been reported to be a slowly uncoating virus in peripheral tissues, but persistence of intact vector in primate brain has not been explored. Because some neurological gene therapies may require re-administration of the same vector to patients, it seems important to understand the optimal timeframe in which to consider such repeat intervention. Surprisingly, convection-enhanced delivery of AAV2 into the thalamus of nonhuman primates (NHPs) resulted in robust staining of neurons with A20 antibody that detected intact AAV2 particles at ∼1.5 months after infusion. However, by 2.5 months, no A20 staining was visible. These data confirmed earlier findings of persistence of intact AAV2 particles in ocular and hepatic tissues. In order to probe the potential consequences of this persistence, we infused AAV2-human aromatic L-amino acid decarboxylase into left and right thalamus of three NHPs, with a 3-month delay between infusions. During that interval, we immunized each animal subcutaneously with AAV2 virus-like particles (empty vector) in order to induce strong anti-capsid humoral immunity. Various high neutralizing antibody titers were achieved. The lowest titer animal showed infiltration of B lymphocytes and CD8(+) T cells into both the secondary and primary infusion sites. In the other two animals, extremely high titers resulted in no transduction of the second site and, therefore, no lymphocytic infiltration. However, such infiltration was prominent at the primary infusion site in each animal and was associated with overt neuronal loss and inflammation. PMID:26510688

  12. Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes.

    PubMed

    Xu, R; Janson, C G; Mastakov, M; Lawlor, P; Young, D; Mouravlev, A; Fitzsimons, H; Choi, K L; Ma, H; Dragunow, M; Leone, P; Chen, Q; Dicker, B; During, M J

    2001-09-01

    This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette. PMID:11571569

  13. Impact of Age at Administration, Lysosomal Storage, and Transgene Regulatory Elements on AAV2/8-Mediated Rat Liver Transduction

    PubMed Central

    Cotugno, Gabriella; Annunziata, Patrizia; Barone, Maria Vittoria; Karali, Marianthi; Banfi, Sandro; Auricchio, Alberto

    2012-01-01

    Liver-directed gene transfer is being investigated for the treatment of systemic or liver-specific diseases. Recombinant vectors based on adeno-associated virus serotype 8 (AAV2/8) efficiently transduce liver cells allowing long term transgene expression after a single administration in animal models and in patients. We evaluated the impact on AAV2/8-mediated rat liver transduction of the following variables: i) age at vector administration, ii) presence of lysosomal storage in liver cells, and iii) regulatory elements included in the transgene expression cassette. We found that systemic administration of AAV2/8 to newborn rats results in vector genome dilution and reduced transduction efficacy when compared to adult injected animals, presumably due to hepatocyte proliferation. Accumulation of glycosaminoglycans in lysosomes does not impact on levels and distribution of AAV2/8-mediated liver transduction. Transgene expression occurs in hepatocytes but not in Kupffer or liver endothelial cells when the liver-specific thyroxine-binding-globulin promoter is used. However, extra-hepatic transduction is observed in the spleen and kidney of animals injected at birth. The use of target sequences for the hematopoietic-specific microRNA miR142-3p does not improve liver transduction efficacy neither reduce immune responses to the lysosomal enzyme arylsulfatase B. The inclusion of a variant of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE-m) decreases AAV2/8-mediated liver transduction levels. As AAV2/8-mediated liver gene transfer is entering in the clinical arena, these data will provide relevant information to the design of efficient AAV2/8-based therapeutic strategies. PMID:22428010

  14. Viral and Cellular Components of AAV2 Replication Compartments.

    PubMed

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  15. Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient

    PubMed Central

    Cereso, Nicolas; Pequignot, Marie O; Robert, Lorenne; Becker, Fabienne; De Luca, Valerie; Nabholz, Nicolas; Rigau, Valerie; De Vos, John; Hamel, Christian P; Kalatzis, Vasiliki

    2014-01-01

    Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM-/y patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC–derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model. PMID:26015956

  16. Method and system for efficiently searching an encoded vector index

    DOEpatents

    Bui, Thuan Quang; Egan, Randy Lynn; Kathmann, Kevin James

    2001-09-04

    Method and system aspects for efficiently searching an encoded vector index are provided. The aspects include the translation of a search query into a candidate bitmap, and the mapping of data from the candidate bitmap into a search result bitmap according to entry values in the encoded vector index. Further, the translation includes the setting of a bit in the candidate bitmap for each entry in a symbol table that corresponds to candidate of the search query. Also included in the mapping is the identification of a bit value in the candidate bitmap pointed to by an entry in an encoded vector.

  17. Efficacy and Safety of rAAV2-ND4 Treatment for Leber's Hereditary Optic Neuropathy.

    PubMed

    Wan, Xing; Pei, Han; Zhao, Min-Jian; Yang, Shuo; Hu, Wei-Kun; He, Heng; Ma, Si-Qi; Zhang, Ge; Dong, Xiao-Yan; Chen, Chen; Wang, Dao-Wen; Li, Bin

    2016-01-01

    Leber's hereditary optic neuropathy (LHON) is a mitochondrially inherited disease leading to blindness. A mitochondrial DNA point mutation at the 11778 nucleotide site of the NADH dehydrogenase subunit 4 (ND4) gene is the most common cause. The aim of this study was to evaluate the efficacy and safety of a recombinant adeno-associated virus 2 (AAV2) carrying ND4 (rAAV2-ND4) in LHON patients carrying the G11778A mutation. Nine patients were administered rAAV2-ND4 by intravitreal injection to one eye and then followed for 9 months. Ophthalmologic examinations of visual acuity, visual field, and optical coherence tomography were performed. Physical examinations included routine blood and urine. The visual acuity of the injected eyes of six patients improved by at least 0.3 log MAR after 9 months of follow-up. In these six patients, the visual field was enlarged but the retinal nerve fibre layer remained relatively stable. No other outcome measure was significantly changed. None of the nine patients had local or systemic adverse events related to the vector during the 9-month follow-up period. These findings support the feasible use of gene therapy for LHON. PMID:26892229

  18. Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization

    PubMed Central

    Wang, Haibo; Han, Xiaokun; Bretz, Colin A; Becker, Silke; Gambhir, Deeksha; Smith, George W; Samulski, R Jude; Wittchen, Erika S; Quilliam, Lawrence A; Chrzanowska-Wodnicka, Magdalena; Hartnett, M Elizabeth

    2016-01-01

    To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 108 viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes. PMID:27606349

  19. Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization.

    PubMed

    Wang, Haibo; Han, Xiaokun; Bretz, Colin A; Becker, Silke; Gambhir, Deeksha; Smith, George W; Samulski, R Jude; Wittchen, Erika S; Quilliam, Lawrence A; Chrzanowska-Wodnicka, Magdalena; Hartnett, M Elizabeth

    2016-01-01

    To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 10(8) viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes. PMID:27606349

  20. Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

    PubMed Central

    Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K; Jou, William; Pack, Stephanie; Liu, Eric; Pechhold, Klaus; Schmidt, Michael; McAlister, Victor J; Chiorini, John A; Blanchette-Mackie, E Joan; Harlan, David M; Owens, Roland A

    2009-01-01

    Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation. PMID:19450275

  1. Intravitreal AAV2.COMP-Ang1 Prevents Neurovascular Degeneration in a Murine Model of Diabetic Retinopathy.

    PubMed

    Cahoon, Judd M; Rai, Ruju R; Carroll, Lara S; Uehara, Hironori; Zhang, Xiaohui; O'Neil, Christina L; Medina, Reinhold J; Das, Subtrata K; Muddana, Santosh K; Olson, Paul R; Nielson, Spencer; Walker, Kortnie; Flood, Maggie M; Messenger, Wyatt B; Archer, Bonnie J; Barabas, Peter; Krizaj, David; Gibson, Christopher C; Li, Dean Y; Koh, Gou Y; Gao, Guangping; Stitt, Alan W; Ambati, Balamurali K

    2015-12-01

    Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR. PMID:26340930

  2. Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.

    PubMed

    Nault, Jean-Charles; Datta, Shalini; Imbeaud, Sandrine; Franconi, Andrea; Mallet, Maxime; Couchy, Gabrielle; Letouzé, Eric; Pilati, Camilla; Verret, Benjamin; Blanc, Jean-Frédéric; Balabaud, Charles; Calderaro, Julien; Laurent, Alexis; Letexier, Mélanie; Bioulac-Sage, Paulette; Calvo, Fabien; Zucman-Rossi, Jessica

    2015-10-01

    Hepatocellular carcinomas (HCCs) are liver tumors related to various etiologies, including alcohol intake and infection with hepatitis B (HBV) or C (HCV) virus. Additional risk factors remain to be identified, particularly in patients who develop HCC without cirrhosis. We found clonal integration of adeno-associated virus type 2 (AAV2) in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2 (cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes. Tumors with viral integration mainly developed in non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11 cases), suggesting a pathogenic role for AAV2 in these patients. In conclusion, AAV2 is a DNA virus associated with oncogenic insertional mutagenesis in human HCC. PMID:26301494

  3. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington's disease.

    PubMed

    Hadaczek, Piotr; Stanek, Lisa; Ciesielska, Agnieszka; Sudhakar, Vivek; Samaranch, Lluis; Pivirotto, Philip; Bringas, John; O'Riordan, Catherine; Mastis, Bryan; San Sebastian, Waldy; Forsayeth, John; Cheng, Seng H; Bankiewicz, Krystof S; Shihabuddin, Lamya S

    2016-01-01

    Huntington's disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP), with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease. PMID:27408903

  4. Carbidopa-Based Modulation of the Functional Effect of the AAV2-hAADC Gene Therapy in 6-OHDA Lesioned Rats

    PubMed Central

    Forsayeth, John; Bankiewicz, Krystof

    2015-01-01

    Progressively blunted response to L-DOPA in Parkinson’s disease (PD) is a critical factor that complicates long-term pharmacotherapy in view of the central importance of this drug in management of the PD-related motor disturbance. This phenomenon is likely due to progressive loss of one of the key enzymes involved in the biosynthetic pathway for dopamine in the basal ganglia: aromatic L-amino acid decarboxylase (AADC). We have developed a gene therapy based on an adeno-associated virus encoding human AADC (AAV2-hAADC) infused into the Parkinsonian striatum. Although no adverse clinical effects of the AAV2-hAADC gene therapy have been observed so far, the ability to more precisely regulate transgene expression or transgene product activity could be an important long-term safety feature. The present study was designed to define pharmacological regulation of the functional activity of AAV2-hAADC transgene product by manipulating L-DOPA and carbidopa (AADC inhibitor) administration in hemi-parkinsonian rats. Thirty days after unilateral striatal infusion of AAV2-hAADC, animals displayed circling behavior and acceleration of dopamine metabolism in the lesioned striatum after administration of a low dose of L-DOPA (5 mg/kg) co-administered with 1.25 mg/kg of carbidopa. This phenomenon was not observed in control AAV2-GFP-treated rats. Withdrawal of carbidopa from a daily L-DOPA regimen decreased the peripheral L-DOPA pool, resulting in almost total loss of L-DOPA-induced behavioral response in AAV2-hAADC rats and a significant decline in striatal dopamine turnover. The serum L-DOPA level correlated with the magnitude of circling behavior in AAV2-hAADC rats. Additionally, AADC activity in homogenates of lesioned striata transduced by AAV2-AADC was 10-fold higher when compared with AAV2-GFP-treated control striata, confirming functional transduction. Our data suggests that the pharmacological regulation of circulating L-DOPA might be effective in the controlling of

  5. Carbidopa-based modulation of the functional effect of the AAV2-hAADC gene therapy in 6-OHDA lesioned rats.

    PubMed

    Ciesielska, Agnieszka; Sharma, Nitasha; Beyer, Janine; Forsayeth, John; Bankiewicz, Krystof

    2015-01-01

    Progressively blunted response to L-DOPA in Parkinson's disease (PD) is a critical factor that complicates long-term pharmacotherapy in view of the central importance of this drug in management of the PD-related motor disturbance. This phenomenon is likely due to progressive loss of one of the key enzymes involved in the biosynthetic pathway for dopamine in the basal ganglia: aromatic L-amino acid decarboxylase (AADC). We have developed a gene therapy based on an adeno-associated virus encoding human AADC (AAV2-hAADC) infused into the Parkinsonian striatum. Although no adverse clinical effects of the AAV2-hAADC gene therapy have been observed so far, the ability to more precisely regulate transgene expression or transgene product activity could be an important long-term safety feature. The present study was designed to define pharmacological regulation of the functional activity of AAV2-hAADC transgene product by manipulating L-DOPA and carbidopa (AADC inhibitor) administration in hemi-parkinsonian rats. Thirty days after unilateral striatal infusion of AAV2-hAADC, animals displayed circling behavior and acceleration of dopamine metabolism in the lesioned striatum after administration of a low dose of L-DOPA (5 mg/kg) co-administered with 1.25 mg/kg of carbidopa. This phenomenon was not observed in control AAV2-GFP-treated rats. Withdrawal of carbidopa from a daily L-DOPA regimen decreased the peripheral L-DOPA pool, resulting in almost total loss of L-DOPA-induced behavioral response in AAV2-hAADC rats and a significant decline in striatal dopamine turnover. The serum L-DOPA level correlated with the magnitude of circling behavior in AAV2-hAADC rats. Additionally, AADC activity in homogenates of lesioned striata transduced by AAV2-AADC was 10-fold higher when compared with AAV2-GFP-treated control striata, confirming functional transduction. Our data suggests that the pharmacological regulation of circulating L-DOPA might be effective in the controlling of

  6. Injection of AAV2-BMP2 and AAV2-TIMP1 into the nucleus pulposus slows the course of intervertebral disc degeneration in an in vivo rabbit model

    PubMed Central

    Leckie, Steven K.; Bechara, Bernard P.; Hartman, Robert A.; Sowa, Gwendolyn A.; Woods, Barrett I.; Coelho, Joao P.; Witt, William T.; Dong, Qing D.; Bowman, Brent W.; Bell, Kevin M.; Vo, Nam V.; Wang, Bing; Kang, James D.

    2016-01-01

    BACKGROUND CONTEXT Intervertebral disc degeneration (IDD) is a common cause of back pain. Patients who fail conservative management may face the morbidity of surgery. Alternative treatment modalities could have a significant impact on disease progression and patients’ quality of life. PURPOSE To determine if the injection of a virus vector carrying a therapeutic gene directly into the nucleus pulposus improves the course of IDD. STUDY DESIGN Prospective randomized controlled animal study. METHODS Thirty-four skeletally mature New Zealand white rabbits were used. In the treatment group, L2–L3, L3–L4, and L4–L5 discs were punctured in accordance with a previously validated rabbit annulotomy model for IDD and then subsequently treated with adeno-associated virus serotype 2 (AAV2) vector carrying genes for either bone morphogenetic protein 2 (BMP2) or tissue inhibitor of metalloproteinase 1 (TIMP1). A nonoperative control group, nonpunctured sham surgical group, and punctured control group were also evaluated. Serial magnetic resonance imaging (MRI) studies at 0, 6, and 12 weeks were obtained, and a validated MRI analysis program was used to quantify degeneration. The rabbits were sacrificed at 12 weeks, and L4–L5 discs were analyzed histologically. Viscoelastic properties of the L3–L4 discs were analyzed using uniaxial load normalized displacement testing. Creep curves were mathematically modeled according to a previously validated two-phase exponential model. Serum samples obtained at 0, 6, and 12 weeks were assayed for biochemical evidence of degeneration. RESULTS The punctured group demonstrated MRI and histologic evidence of degeneration as expected. The treatment groups demonstrated less MRI and histologic evidence of degeneration than the punctured group. The serum biochemical marker C-telopeptide of collagen type II increased rapidly in the punctured group, but the treated groups returned to control values by 12 weeks. The treatment groups

  7. Multilineage transduction of resident lung cells in vivo by AAV2/8 for α1-antitrypsin gene therapy

    PubMed Central

    Payne, Julia G; Takahashi, Ayuko; Higgins, Michelle I; Porter, Emily L; Suki, Bela; Balazs, Alejandro; Wilson, Andrew A

    2016-01-01

    In vivo gene delivery has long represented an appealing potential treatment approach for monogenic diseases such as α1-antitrypsin deficiency (AATD) but has proven challenging to achieve in practice. Alternate pseudotyping of recombinant adeno-associated virus (AAV) vectors is producing vectors with increasingly heterogeneous tropic specificity, giving researchers the ability to target numerous end-organs affected by disease. Herein, we describe sustained pulmonary transgene expression for at least 52 weeks after a single intratracheal instillation of AAV2/8 and characterize the multiple cell types transduced within the lung utilizing this approach. We demonstrate that lung-directed AAV2/8 is able to achieve therapeutic α-1 antitrypsin (AAT) protein levels within the lung epithelial lining fluid and that AAT gene delivery ameliorates the severity of experimental emphysema in mice. We find that AAV2/8 efficiently transduces hepatocytes in vivo after intratracheal administration, a finding that may have significance for AAV-based human gene therapy studies. These results support direct transgene delivery to the lung as a potential alternative approach to achieve the goal of developing a gene therapy for AATD. PMID:27408904

  8. CNS-restricted Transduction and CRISPR/Cas9-mediated Gene Deletion with an Engineered AAV Vector.

    PubMed

    Murlidharan, Giridhar; Sakamoto, Kensuke; Rao, Lavanya; Corriher, Travis; Wang, Dan; Gao, Guangping; Sullivan, Patrick; Asokan, Aravind

    2016-01-01

    Gene therapy using recombinant adeno-associated viral (AAV) vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9), which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137) in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities. PMID:27434683

  9. Loss-insensitive vector encoding with two-uniform frames

    NASA Astrophysics Data System (ADS)

    Bodmann, Bernhard G.; Paulsen, Vern I.

    2005-08-01

    The central topic of this paper is the linear, redundant encoding of vectors using frames for the purpose of loss-insensitive data transmission. Our goal is to minimize the reconstruction error when frame coefficients are accidentally erased. Two-uniform frames are known to be optimal for handling up to two erasures, in the sense that they minimize the largest Euclidean error norm when up to two frame coefficients are set to zero. Here, we consider the case when an arbitrary number of the frame coefficients of a vector is lost. We derive general error bounds and apply these to concrete examples. We show that among the 227 known equivalence classes of two-uniform (36,15)-frames arising from Hadamard matrices, there are 5 that give smallest error bounds for up to 8 erasures.

  10. Safety in nonhuman primates of ocular AAV2-RPE65, a candidate treatment for blindness in Leber congenital amaurosis.

    PubMed

    Jacobson, Samuel G; Boye, Sanford L; Aleman, Tomas S; Conlon, Thomas J; Zeiss, Caroline J; Roman, Alejandro J; Cideciyan, Artur V; Schwartz, Sharon B; Komaromy, Andras M; Doobrajh, Michelle; Cheung, Andy Y; Sumaroka, Alexander; Pearce-Kelling, Susan E; Aguirre, Gustavo D; Kaushal, Shalesh; Maguire, Albert M; Flotte, Terence R; Hauswirth, William W

    2006-08-01

    Leber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by subretinal delivery of AAV-RPE65 vector. Human ocular gene transfer trials are being considered. Our safety studies of subretinal AAV-2/2.RPE65 in RPE65-mutant dogs showed evidence of modest photoreceptor loss in the injection region in some animals at higher vector doses. We now test the hypothesis that there can be vectorrelated toxicity to the normal monkey, with its human-like retina. Good Laboratory Practice safety studies following single intraocular injections of AAV-2/2.RPE65 in normal cynomolgus monkeys were performed for 1-week and 3-month durations. Systemic toxicity was not identified. Ocular-specific studies included clinical examinations, electroretinography, and retinal histopathology. Signs of ocular inflammation postinjection had almost disappeared by 1 week. At 3 months, electroretinography in vector-injected eyes was no different than in vehicle-injected control eyes or compared with presurgical recordings. Healed sites of retinal perforation from subretinal injections were noted clinically and by histopathology. Foveal architecture in subretinally injected eyes, vector or vehicle, could be abnormal. Morphometry of central retina showed no photoreceptor layer thickness abnormalities occurring in a dose-dependent manner. Vector sequences were present in the injected retina, vitreous, and optic nerve at 1 week but not consistently in the brain. At 3 months, there were no vector sequences in optic nerve and brain. The results allow for consideration of an upper range for no observed adverse effect level in future human trials of subretinal AAV-2/2.RPE65. The potential value of foveal treatment for LCA and other retinal degenerations warrants further research into how to achieve gene transfer without retinal injury from surgical detachment of the retina

  11. Efficacy and Safety of rAAV2-ND4 Treatment for Leber’s Hereditary Optic Neuropathy

    PubMed Central

    Wan, Xing; Pei, Han; Zhao, Min-jian; Yang, Shuo; Hu, Wei-kun; He, Heng; Ma, Si-qi; Zhang, Ge; Dong, Xiao-yan; Chen, Chen; Wang, Dao-wen; Li, Bin

    2016-01-01

    Leber’s hereditary optic neuropathy (LHON) is a mitochondrially inherited disease leading to blindness. A mitochondrial DNA point mutation at the 11778 nucleotide site of the NADH dehydrogenase subunit 4 (ND4) gene is the most common cause. The aim of this study was to evaluate the efficacy and safety of a recombinant adeno-associated virus 2 (AAV2) carrying ND4 (rAAV2-ND4) in LHON patients carrying the G11778A mutation. Nine patients were administered rAAV2-ND4 by intravitreal injection to one eye and then followed for 9 months. Ophthalmologic examinations of visual acuity, visual field, and optical coherence tomography were performed. Physical examinations included routine blood and urine. The visual acuity of the injected eyes of six patients improved by at least 0.3 log MAR after 9 months of follow-up. In these six patients, the visual field was enlarged but the retinal nerve fibre layer remained relatively stable. No other outcome measure was significantly changed. None of the nine patients had local or systemic adverse events related to the vector during the 9-month follow-up period. These findings support the feasible use of gene therapy for LHON. PMID:26892229

  12. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington’s disease

    PubMed Central

    Hadaczek, Piotr; Stanek, Lisa; Ciesielska, Agnieszka; Sudhakar, Vivek; Samaranch, Lluis; Pivirotto, Philip; Bringas, John; O’Riordan, Catherine; Mastis, Bryan; San Sebastian, Waldy; Forsayeth, John; Cheng, Seng H; Bankiewicz, Krystof S; Shihabuddin, Lamya S

    2016-01-01

    Huntington’s disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP), with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease. PMID:27408903

  13. Intracisternal delivery of NFκB-inducible scAAV2/9 reveals locoregional neuroinflammation induced by systemic kainic acid treatment.

    PubMed

    Bockstael, Olivier; Tenenbaum, Liliane; Dalkara, Deniz; Melas, Catherine; De Witte, Olivier; Levivier, Marc; Chtarto, Abdelwahed

    2014-01-01

    We have previously demonstrated disease-dependent gene delivery in the brain using an AAV vector responding to NFκB activation as a probe for inflammatory responses. This vector, injected focally in the parenchyma prior to a systemic kainic acid (KA) injection mediated inducible transgene expression in the hippocampus but not in the cerebellum, regions, respectively, known to be affected or not by the pathology. However, such a focal approach relies on previous knowledge of the model parameters and does not allow to predict the whole brain response to the disease. Global brain gene delivery would allow to predict the regional distribution of the pathology as well as to deliver therapeutic factors in all affected brain regions. We show that self-complementary AAV2/9 (scAAV2/9) delivery in the adult rat cisterna magna allows a widespread but not homogenous transduction of the brain. Indeed, superficial regions, i.e., cortex, hippocampus, and cerebellum were more efficiently transduced than deeper regions, such as striatum, and substantia nigra. These data suggest that viral particles penetration from the cerebrospinal fluid (CSF) into the brain is a limiting factor. Interestingly, AAV2/9-2YF a rationally designed capsid mutant (affecting surface tyrosines) increased gene transfer efficiency approximately fivefold. Neurons, astrocytes, and oligodendrocytes, but not microglia, were transduced in varying proportions depending on the brain region and the type of capsid. Finally, after a single intracisternal injection of scAAV2/9-2YF using the NFκB-inducible promoter, KA treatment induced transgene expression in the hippocampus and cortex but not in the cerebellum, corresponding to the expression of the CD11b marker of microglial activation. These data support the use of disease-inducible vectors administered in the cisterna magna as a tool to characterize the brain pathology in systemic drug-induced or transgenic disease models. However, further improvements are

  14. Humoral and cell-mediated immune response, and growth factor synthesis after direct intraarticular injection of rAAV2-IGF-I and rAAV5-IGF-I in the equine middle carpal joint.

    PubMed

    Ortved, Kyla; Wagner, Bettina; Calcedo, Roberto; Wilson, James; Schaefer, Deanna; Nixon, Alan

    2015-03-01

    Intraarticular (IA) administration of viral vectors expressing a therapeutic transgene is an attractive treatment modality for osteoarthritis (OA) as the joint can be treated as a contained unit. Humoral and cell-mediated immune responses in vivo can limit vector effectiveness. Transduction of articular tissues has been investigated; however, the immune response to IA vectors remains largely unknown. We hypothesized that IA rAAV2 and rAAV5 overexpressing insulin-like growth factor-I (IGF-I) would result in long-term IGF-I formation but would also induce neutralizing antibodies (NAb) and anti-capsid effector T cells. Twelve healthy horses were assigned to treatment (rAAV2 or rAAV5) or control (saline) groups. Middle carpal joints were injected with 5×10(11) vector genomes/joint. Synovial fluid was analyzed for changes in composition, NAb titers, immunoglobulin isotypes, proinflammatory cytokines, and IGF-I. Serum was analyzed for antibody titers and cytokines. A T cell restimulation assay was used to assess T cell responses. Injection of rAAV2- or rAAV5-IGF-I did not induce greater inflammation compared with saline. Synovial fluid IGF-I was significantly increased in both rAAV2- and rAAV5-IGF-I joints by day 14 and remained elevated until day 56; however, rAAV5 achieved the highest concentrations. A capsid-specific T cell response was not noted although all virus-treated horses had increased NAbs in serum and synovial fluid after treatment. Taken together, our data show that IA injection of rAAV2- or rAAV5-IGF-I does not incite a clinically detectable inflammatory or cell-mediated immune response and that IA gene therapy using minimally immunogenic vectors represents a clinically relevant tool for treating articular disorders including OA. PMID:25705927

  15. Humoral and Cell-Mediated Immune Response, and Growth Factor Synthesis After Direct Intraarticular Injection of rAAV2-IGF-I and rAAV5-IGF-I in the Equine Middle Carpal Joint

    PubMed Central

    Wagner, Bettina; Calcedo, Roberto; Wilson, James; Schaefer, Deanna; Nixon, Alan

    2015-01-01

    Abstract Intraarticular (IA) administration of viral vectors expressing a therapeutic transgene is an attractive treatment modality for osteoarthritis (OA) as the joint can be treated as a contained unit. Humoral and cell-mediated immune responses in vivo can limit vector effectiveness. Transduction of articular tissues has been investigated; however, the immune response to IA vectors remains largely unknown. We hypothesized that IA rAAV2 and rAAV5 overexpressing insulin-like growth factor-I (IGF-I) would result in long-term IGF-I formation but would also induce neutralizing antibodies (NAb) and anti-capsid effector T cells. Twelve healthy horses were assigned to treatment (rAAV2 or rAAV5) or control (saline) groups. Middle carpal joints were injected with 5×1011 vector genomes/joint. Synovial fluid was analyzed for changes in composition, NAb titers, immunoglobulin isotypes, proinflammatory cytokines, and IGF-I. Serum was analyzed for antibody titers and cytokines. A T cell restimulation assay was used to assess T cell responses. Injection of rAAV2- or rAAV5-IGF-I did not induce greater inflammation compared with saline. Synovial fluid IGF-I was significantly increased in both rAAV2- and rAAV5-IGF-I joints by day 14 and remained elevated until day 56; however, rAAV5 achieved the highest concentrations. A capsid-specific T cell response was not noted although all virus-treated horses had increased NAbs in serum and synovial fluid after treatment. Taken together, our data show that IA injection of rAAV2- or rAAV5-IGF-I does not incite a clinically detectable inflammatory or cell-mediated immune response and that IA gene therapy using minimally immunogenic vectors represents a clinically relevant tool for treating articular disorders including OA. PMID:25705927

  16. Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

    PubMed

    Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

    2012-03-01

    Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration. PMID:21695398

  17. Human melanopsin-AAV2/8 transfection to retina transiently restores visual function in rd1 mice

    PubMed Central

    Liu, Ming-Ming; Dai, Jia-Man; Liu, Wen-Yi; Zhao, Cong-Jian; Lin, Bin; Yin, Zheng-Qin

    2016-01-01

    AIM To explore whether ectopic expression of human melanopsin can effectively and safely restore visual function in rd1 mice. METHODS Hematoxylin-eosin staining of retinal sections from rd1 mice was used to detect the thickness of the outer nuclear layer to determine the timing of surgery. We constructed a human melanopsin-AAV2/8 viral vector and injected it into the subretinal space of rd1 mice. The Phoenix Micron IV system was used to exclude the aborted injections, and immunohistochemistry was used to validate the ectopic expression of human melanopsin. Furthermore, visual electrophysiology and behavioral tests were used to detect visual function 30 and 45d after the injection. The structure of the retina was compared between the human melanopsin-injected group and phosphate buffer saline (PBS)-injected group. RESULTS Retinas of rd1 mice lost almost all of their photoreceptors on postnatal day 28 (P28). We therefore injected the human melanopsin-adeno-associated virus (AAV) 2/8 viral vector into P30 rd1 mice. After excluding aborted injections, we used immunohistochemistry of the whole mount retina to confirm the ectopic expression of human melanopsin by co-expression of human melanopsin and YFP that was carried by a viral vector. At 30d post-injection, visual electrophysiology and the behavioral test significantly improved. However, restoration of vision disappeared 45d after human melanopsin injection. Notably, human melanopsin-injected mice did not show any structural differences in their retinas compared with PBS-injected mice. CONCLUSION Ectopic expression of human melanopsin effectively and safely restores visual function in rd1 mice. PMID:27275417

  18. Inducible scAAV2.GRE.MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy.

    PubMed

    Borrás, T; Buie, L K; Spiga, M G

    2016-05-01

    Current treatment of glaucoma relies on administration of daily drops or eye surgery. A gene therapy approach to treat steroid-induced glaucoma would bring a resolution to millions of people worldwide who depend on glucocorticoid therapy for a myriad of inflammatory disorders. Previously, we had characterized a short-term Adh.GRE.MMP1 gene vector for the production of steroid-induced MMP1 in the trabecular meshwork and tested reduction of elevated intraocular pressure (IOP) in a sheep model. Here we conducted a trial transferring the same transgene cassette to a clinically safe vector (scAAV2), and extended the therapeutic outcome to longer periods of times. No evidence of ocular and/or systemic toxicity was observed. Viral genome distributions showed potential reinducible vector DNAs in the trabecular meshwork (0.4 v.g. per cell) and negligible copies in six major internal organs (0.00002-0.005 v.g. per cell). Histological sections confirmed successful transduction of scAAV2.GFP to the trabecular meshwork. Optimization of the sheep steroid-induced hypertensive model revealed that topical ophthalmic drug difluprednate 0.05% (durezol) induced the highest IOP elevation in the shortest time. This is the first efficacy/toxicity study of a feasible gene therapy treatment of steroid-induced hypertension using clinically accepted self-complementary adeno-associated vectors (scAAV) vectors in a large animal model. PMID:26855269

  19. Hydrostatic Isolated Limb Perfusion with Adeno-associated Virus Vectors Enhances Correction of Skeletal Muscle in Pompe Disease

    PubMed Central

    Sun, Baodong; Li, Songtao; Bird, Andrew; Koeberl, Dwight D.

    2010-01-01

    Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) stems from the inherited deficiency of acid-α-glucosidase (GAA; acid maltase; EC 3.2.1.20), which primarily involves cardiac and skeletal muscles. We hypothesized that hydrostatic isolated limb perfusion (ILP) administration of an adeno-associated virus (AAV) vector containing a muscle specific promoter could achieve relatively higher transgene expression in the hindlimb muscles of GAA-knockout (GAA-KO) mice, in comparison with intravenous (IV) administration. ILP adminstration of AAV2/8 vectors encoding alkaline phosphatase or human GAA transduced skeletal muscles of the hindlimb widely, despite the relatively low number of vector particles administered (1×1011), and IV administration of an equivalent vector dose failed to transduce skeletal muscle detectably. Similarly, ILP administration of fewer vector particles of the AAV2/9 vector encoding human GAA (3×1010) transduced skeletal muscles of the hindlimb widely and significantly reduced glycogen content to, in comparison with IV administration. The only advantage for IV administration was moderately high level transduction of cardiac muscle, which demonstrated compellingly that ILP administration sequestered vector particles within the perfused limb. Reduction of glycogen storage in the extensor digitorum longus demonstrated the potential advantage of ILP-mediated delivery of AAV vectors in Pompe disease, because type II myofibers are resistant to enzyme replacement therapy. Thus, ILP will enhance AAV transduction of multiple skeletal muscles while reducing the required dosages in terms of vector particle numbers. PMID:20686508

  20. Inducible scAAV2.GRE.MMP1 lowers IOP long-term in a large animal model for steroid-induced glaucoma gene therapy

    PubMed Central

    Borrás, Teresa; Buie, LaKisha K.; Spiga, Maria Grazia

    2016-01-01

    Current treatment of glaucoma relies on administration of daily drops or eye surgery. A gene therapy approach to treat steroid-induced glaucoma would bring a resolution to millions of people worldwide that depend on glucocorticoid therapy for a myriad of inflammatory disorders. Previously, we had characterized a short-term Adh.GRE.MMP1 gene vector for the production of steroid-induced MMP1 in the trabecular meshwork and tested reduction of elevated intraocular pressure (IOP) in a sheep model. Here we conducted a trial transferring the same transgene cassette to a clinically safe vector (scAAV2), and extended the therapeutic outcome to longer periods of times. No evidence of ocular and/or systemic toxicity was observed. Viral genome distributions showed potential re-inducible vector DNAs in the trabecular meshwork (0.4 vg/cell) and negligible copies in six major internal organs (0.00002-0.005 vg/cell). Histological sections confirmed successful transduction of scAAV2.GFP to the trabecular meshwork. Optimization of the sheep steroid–induced hypertensive model revealed that topical ophthalmic drug difluprednate 0.05% (durezol) induced the highest IOP elevation in the shortest time. This is the first efficacy/toxicity study of a feasible gene therapy treatment of steroid-induced hypertension using clinically accepted scAAV vectors in a large animal model. PMID:26855269

  1. Safety and Tolerability of Magnetic Resonance Imaging-Guided Convection-Enhanced Delivery of AAV2-hAADC with a Novel Delivery Platform in Nonhuman Primate Striatum

    PubMed Central

    San Sebastian, Waldy; Richardson, R. Mark; Kells, Adrian P.; Lamarre, Clementine; Bringas, John; Pivirotto, Philip; Salegio, Ernesto A.; DeArmond, Stephen J.; Forsayeth, John

    2012-01-01

    Abstract Degeneration of nigrostriatal neurons in Parkinson's disease (PD) causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa (l-DOPA) into dopamine in the striatum. Because loss of this enzyme appears to be a major driver of progressive impairment of response to the mainstay drug, l-DOPA, one promising approach has been to use gene therapy to restore AADC activity in the human putamen and thereby restore normal l-DOPA response in patients with PD. An open-label phase I clinical trial of this approach in patients with PD provided encouraging signs of improvement in Unified Parkinson's Disease Rating Scale scores and reductions in antiparkinsonian medications. However, such improvement was modest compared with the results previously reported in parkinsonian rhesus macaques. The reason for this discrepancy may have been that the relatively small volume of vector infused in the clinical study restricted the distribution of AADC expression, such that only about 20% of the postcommissural putamen was covered, as revealed by l-[3-18F]-α-methyltyrosine-positron emission tomography. To achieve more quantitative distribution of vector, we have developed a visual guidance system for parenchymal infusion of AAV2. The purpose of the present study was to evaluate the combined magnetic resonance imaging-guided delivery system with AAV2-hAADC under conditions that approximate the intended clinical protocol. Our data indicate that this approach directed accurate cannula placement and effective vector distribution without inducing any untoward effects in nonhuman primates infused with a high dose of AAV2-hAADC. PMID:22017504

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

    PubMed Central

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

    2013-01-01

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

  3. Integrin alphaVbeta5 is not involved in adeno-associated virus type 2 (AAV2) infection.

    PubMed

    Qiu, J; Brown, K E

    1999-11-25

    alphaVbeta5 integrin was recently proposed as a coreceptor for adeno-associated virus type 2 (AAV2) infection (Summerford et al., 1999, Nat. Med. 5, 78-82), based mainly on the direct binding of AAV2 to denatured beta5 by virus overlay assay. In studies using purified natural or recombinant human integrin alphaVbeta5 we were unable to demonstrate AAV2 binding, either by virus overlay or by liquid binding assay. Furthermore, neither purified integrin alphaVbeta5, nor RGD peptides, nor functional blocking monoclonal antibody blocked rAAV2 transduction. These data strongly suggest that integrin alphaVbeta5 is not involved in AAV2 infection. PMID:10562505

  4. AAV2 Delivery of Flt23k Intraceptors Inhibits Murine Choroidal Neovascularization

    PubMed Central

    Zhang, Xiaohui; Das, Subrata K; Passi, Samuel F; Uehara, Hironori; Bohner, Austin; Chen, Marcus; Tiem, Michelle; Archer, Bonnie; Ambati, Balamurali K

    2015-01-01

    Long-term inhibition of extracellular vascular endothelial growth factor (VEGF) in the treatment of age-related macular degeneration (AMD) may induce retinal neuronal toxicity and risk other side effects. We developed a novel strategy which inhibits retinal pigment epithelium (RPE)-derived VEGF, sparing other highly sensitive retinal tissues. Flt23k, an intraceptor inhibitor of VEGF, was able to inhibit VEGF in vitro. Adeno-associated virus type 2 (AAV2)–mediated expression of Flt23k was maintained for up to 6 months postsubretinal injection in mice. Flt23k was able to effectively inhibit laser-induced murine choroidal neovascularization (CNV). VEGF levels in the RPE/choroid complex decreased significantly in AAV2.Flt23k treated eyes. Neither retinal structure detected by Heidelberg Spectralis nor function measured by electroretinography (ERG) was adversely affected by treatment with AAV2.Flt23k. Hence AAV2.Flt23k can effectively maintain long-term expression and inhibit laser-induced CNV in mice through downregulation of VEGF while maintaining a sound retinal safety profile. These findings suggest a promising novel approach for the treatment of CNV. PMID:25306972

  5. Noninvasive Imaging Reveals Stable Transgene Expression in Mouse Airways After Delivery of a Nonintegrating Recombinant Adeno-Associated Viral Vector.

    PubMed

    Vidović, Dragana; Gijsbers, Rik; Jimenez, Ana Quiles; Dooley, James; Van den Haute, Chris; Van der Perren, Anke; Liston, Adrian; Baekelandt, Veerle; Debyser, Zeger; Carlon, Marianne Sylvia

    2016-01-01

    Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV approach for pulmonary gene transfer. Only limited clinical successes have been achieved for airway gene transfer so far, underscoring the need for further preclinical development of rAAV-based gene therapy for pulmonary disorders. We sought to determine the preclinical potential of an airway-tropic serotype, rAAV2/5, encoding reporter genes when delivered to mouse airways. Although several groups have assessed the stability of gene transfer using a nonintegrating rAAV in mouse airways, long-term stability for more than a year has not been reported. Additionally, an extensive quantitative analysis of the specific cell types targeted by rAAV2/5 using cell-specific markers is lacking. We obtained sustained gene expression in upper and lower airways up to 15 months after vector administration, a substantial proportion of the lifespan of a laboratory mouse. In addition, we demonstrated that readministration of rAAV2/5 to the airways is feasible and increases gene expression 14 months after primary vector administration, despite the presence of circulating neutralizing antibodies. Finally, identification of transduced cell types revealed different subpopulations being targeted by rAAV2/5, with 64% of β-galactosidase-positive cells being ciliated cells, 34% club cells in the conducting airways, and 75% alveolar type II cells in the alveoli at 1 month postinjection. This underscores the therapeutic potential of a nonintegrating rAAV vector to develop a gene therapeutic drug for a variety of pulmonary disorders, such as cystic fibrosis, primary ciliary dyskinesia, and surfactant deficiencies. PMID:26567984

  6. Fast Encoding Method for Image Vector Quantization Based on Multiple Appropriate Features to Estimate Euclidean Distance

    NASA Astrophysics Data System (ADS)

    Pan, Zhibin; Kotani, Koji; Ohmi, Tadahiro

    The encoding process of finding the best-matched codeword (winner) for a certain input vector in image vector quantization (VQ) is computationally very expensive due to a lot of k-dimensional Euclidean distance computations. In order to speed up the VQ encoding process, it is beneficial to firstly estimate how large the Euclidean distance is between the input vector and a candidate codeword by using appropriate low dimensional features of a vector instead of an immediate Euclidean distance computation. If the estimated Euclidean distance is large enough, it implies that the current candidate codeword could not be a winner so that it can be rejected safely and thus avoid actual Euclidean distance computation. Sum (1-D), L2 norm (1-D) and partial sums (2-D) of a vector are used together as the appropriate features in this paper because they are the first three simplest features. Then, four estimations of Euclidean distance between the input vector and a codeword are connected to each other by the Cauchy-Schwarz inequality to realize codeword rejection. For typical standard images with very different details (Lena, F-16, Pepper and Baboon), the final remaining must-do actual Euclidean distance computations can be eliminated obviously and the total computational cost including all overhead can also be reduced obviously compared to the state-of-the-art EEENNS method meanwhile keeping a full search (FS) equivalent PSNR.

  7. Pulse Code Modulation (PCM) encoder handbook for Aydin Vector MMP-900 series system

    NASA Technical Reports Server (NTRS)

    Raphael, David

    1995-01-01

    This handbook explicates the hardware and software properties of a time division multiplex system. This system is used to sample analog and digital data. The data is then merged with frame synchronization information to produce a serial pulse coded modulation (PCM) bit stream. Information in this handbook is required by users to design congruous interface and attest effective utilization of this encoder system. Aydin Vector provides all of the components for these systems to Goddard Space Flight Center/Wallops Flight Facility.

  8. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

    PubMed Central

    2009-01-01

    Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T

  9. AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons

    PubMed Central

    Elsworth, JD; Redmond, DE; Leranth, C; Bjugstad, KB; Sladek, JR; Collier, TJ; Foti, SB; Samulski, RJ; Vives, KP; Roth, RH

    2009-01-01

    Neural transplantation offers the potential of treating Parkinson’s disease by grafting fetal dopamine neurons to depleted regions of the brain. However, clinical studies of neural grafting in Parkinson’s disease have produced only modest improvements. One of the main reasons for this is the low survival rate of transplanted neurons. The inadequate supply of critical neurotrophic factors in the adult brain is likely to be a major cause of early cell death and restricted outgrowth of fetal grafts placed into the mature striatum. Glial derived neurotrophic factor (GDNF) is a potent neurotrophic factor that is crucial to the survival, outgrowth and maintenance of dopamine neurons, and so is a candidate for protecting grafted fetal dopamine neurons in the adult brain. We found that implantation of adeno-associated virus type 2 encoding GDNF (AAV2-GDNF) in the normal monkey caudate nucleus induced over-expression of GDNF that persisted for at least 6 months after injection. In a 6-month within-animal controlled study, AAV2-GDNF enhanced the survival of fetal dopamine neurons by 4-fold, and increased the outgrowth of grafted fetal dopamine neurons by almost 3-fold in the caudate nucleus of MPTP-treated monkeys, compared with control grafts in the other caudate nucleus. Thus, the addition of GDNF gene therapy to neural transplantation may be a useful strategy to improve treatment for Parkinson’s disease. PMID:18346734

  10. Efficient Gene Transfer Into the Mouse Lung by Fetal Intratracheal Injection of rAAV2/6.2

    PubMed Central

    Carlon, Marianne; Toelen, Jaan; Van der Perren, Anke; Vandenberghe, Luk H; Reumers, Veerle; Sbragia, Lourenço; Gijsbers, Rik; Baekelandt, Veerle; Himmelreich, Uwe; Wilson, James M; Deprest, Jan; Debyser, Zeger

    2010-01-01

    Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for β-galactosidase (β-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect. PMID:20664525

  11. PiggyBac transposon vectors: the tools of the human gene encoding

    PubMed Central

    Zhao, Shuang; Jiang, Enze; Chen, Shuangshuang; Gu, Yuan; Shangguan, Anna Junjie; Lv, Tangfeng

    2016-01-01

    A transposon is a DNA segment, which is able to change its relative position within the entire genome of a cell. The piggyBac (PB) transposon is a movable genetic element that efficiently transposes between vectors and chromosomes through a “cut-and-paste” mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeats (ITRs) sequences located on both ends of the transposon vector and eight efficiently moves the contents from its original positions and efficiently integrates them into TTAA chromosomal sites. PB has drawn much attention because of its transposition efficiency, safety and stability. Due to its priorities, PB can be used as a new genetic vehicle, a new tool for oncogene screening and a new method for gene therapy. PB has created a new outlook for human gene encoding. PMID:26958506

  12. Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells

    PubMed Central

    Gruenert, Anja K.; Czugala, Marta; Mueller, Chris; Schmeer, Marco; Schleef, Martin; Kruse, Friedrich E.; Fuchsluger, Thomas A.

    2016-01-01

    Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic. PMID:27023329

  13. Pulse Code Modulation (PCM) encoder handbook for Aydin Vector MMP-600 series system

    NASA Technical Reports Server (NTRS)

    Currier, S. F.; Powell, W. R.

    1986-01-01

    The hardware and software characteristics of a time division multiplex system are described. The system is used to sample analog and digital data. The data is merged with synchronization information to produce a serial pulse coded modulation (PCM) bit stream. Information presented herein is required by users to design compatible interfaces and assure effective utilization of this encoder system. GSFC/Wallops Flight Facility has flown approximately 50 of these systems through 1984 on sounding rockets with no inflight failures. Aydin Vector manufactures all of the components for these systems.

  14. Flagellin Encoded in Gene-Based Vector Vaccines Is a Route-Dependent Immune Adjuvant.

    PubMed

    Rady, Hamada F; Dai, Guixiang; Huang, Weitao; Shellito, Judd E; Ramsay, Alistair J

    2016-01-01

    Flagellin has been tested as a protein-based vaccine adjuvant, with the majority of studies focused on antibody responses. Here, we evaluated the adjuvant activity of flagellin for both cellular and humoral immune responses in BALB/c mice in the setting of gene-based immunization, and have made several novel observations. DNA vaccines and adenovirus (Ad) vectors were engineered to encode mycobacterial protein Ag85B, with or without flagellin of Salmonella typhimurium (FliC). DNA-encoded flagellin given IM enhanced splenic CD4+ and CD8+ T cell responses to co-expressed vaccine antigen, including memory responses. Boosting either IM or intranasally with Ad vectors expressing Ag85B without flagellin led to durable enhancement of Ag85B-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with significantly improved protection against challenge with pathogenic aerosolized M. tuberculosis. However, inclusion of flagellin in both DNA prime and Ad booster vaccines induced localized pulmonary inflammation and transient weight loss, with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in levels of mucosal CD4+ and CD8+ T cell responses following IM DNA/IN Ad mucosal prime-boosting, although antibody responses were not diminished. These findings indicate that flagellin has differential and route-dependent adjuvant activity when included as a component of systemic or mucosally-delivered gene-based prime-boost immunization. Clear adjuvant activity for both T and B cell responses was observed when flagellin was included in the DNA priming vaccine, but side effects occurred when given in an Ad boosting vector, particularly via the pulmonary route. PMID:26844553

  15. Flagellin Encoded in Gene-Based Vector Vaccines Is a Route-Dependent Immune Adjuvant

    PubMed Central

    Rady, Hamada F.; Dai, Guixiang; Huang, Weitao; Shellito, Judd E.; Ramsay, Alistair J.

    2016-01-01

    Flagellin has been tested as a protein-based vaccine adjuvant, with the majority of studies focused on antibody responses. Here, we evaluated the adjuvant activity of flagellin for both cellular and humoral immune responses in BALB/c mice in the setting of gene-based immunization, and have made several novel observations. DNA vaccines and adenovirus (Ad) vectors were engineered to encode mycobacterial protein Ag85B, with or without flagellin of Salmonella typhimurium (FliC). DNA-encoded flagellin given IM enhanced splenic CD4+ and CD8+ T cell responses to co-expressed vaccine antigen, including memory responses. Boosting either IM or intranasally with Ad vectors expressing Ag85B without flagellin led to durable enhancement of Ag85B-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with significantly improved protection against challenge with pathogenic aerosolized M. tuberculosis. However, inclusion of flagellin in both DNA prime and Ad booster vaccines induced localized pulmonary inflammation and transient weight loss, with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in levels of mucosal CD4+ and CD8+ T cell responses following IM DNA/IN Ad mucosal prime-boosting, although antibody responses were not diminished. These findings indicate that flagellin has differential and route-dependent adjuvant activity when included as a component of systemic or mucosally-delivered gene-based prime-boost immunization. Clear adjuvant activity for both T and B cell responses was observed when flagellin was included in the DNA priming vaccine, but side effects occurred when given in an Ad boosting vector, particularly via the pulmonary route. PMID:26844553

  16. Successful Interference with Cellular Immune Responses to Immunogenic Proteins Encoded by Recombinant Viral Vectors

    PubMed Central

    Sarukhan, Adelaida; Camugli, Sabine; Gjata, Bernard; von Boehmer, Harald; Danos, Olivier; Jooss, Karin

    2001-01-01

    Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4+ T-cell activation were markedly delayed when AAV vectors were used. Upon recombinant Ad (rAd) gene transfer, T cells were activated both by direct transduction of dendritic cells and by cross-presentation of the transgene product, while upon rAAV gene transfer T cells were only activated by the latter mechanism. These results suggested that activation of the immune system by the transgene product following rAAV-mediated gene transfer might be easier to control than that following rAd-mediated gene transfer. Therefore, we tested protocols aimed at interfering with either antigen presentation by blocking the CD40/CD40L pathway or with the T-cell response by inducing transgene-specific tolerance. Long-term expression of the AAV-HA was achieved in both cases, whereas immune responses against Ad-HA could not be prevented. These data clearly underline the importance of understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to specifically interfere with them and

  17. Using support vector machine and dynamic parameter encoding to enhance global optimization

    NASA Astrophysics Data System (ADS)

    Zheng, Z.; Chen, X.; Liu, C.; Huang, K.

    2016-05-01

    This study presents an approach which combines support vector machine (SVM) and dynamic parameter encoding (DPE) to enhance the run-time performance of global optimization with time-consuming fitness function evaluations. SVMs are used as surrogate models to partly substitute for fitness evaluations. To reduce the computation time and guarantee correct convergence, this work proposes a novel strategy to adaptively adjust the number of fitness evaluations needed according to the approximate error of the surrogate model. Meanwhile, DPE is employed to compress the solution space, so that it not only accelerates the convergence but also decreases the approximate error. Numerical results of optimizing a few benchmark functions and an antenna in a practical application are presented, which verify the feasibility, efficiency and robustness of the proposed approach.

  18. Comparative assessment of vaccine vectors encoding ten malaria antigens identifies two protective liver-stage candidates

    PubMed Central

    Longley, Rhea J.; Salman, Ahmed M.; Cottingham, Matthew G.; Ewer, Katie; Janse, Chris J.; Khan, Shahid M.; Spencer, Alexandra J.; Hill, Adrian V. S.

    2015-01-01

    The development of an efficacious Plasmodium falciparum malaria vaccine remains a top priority for global health. Vaccination with irradiated sporozoites is able to provide complete sterile protection through the action of CD8+ T cells at the liver-stage of infection. However, this method is currently unsuitable for large-scale deployment and focus has instead turned to the development of sub-unit vaccines. Sub-unit vaccine efforts have traditionally focused on two well-known pre-erythrocytic antigens, CSP and TRAP, yet thousands of genes are expressed in the liver-stage. We sought to assess the ability of eight alternative P. falciparum pre-erythrocytic antigens to induce a high proportion of CD8+ T cells. We show that all antigens, when expressed individually in the non-replicating viral vectors ChAd63 and MVA, are capable of inducing an immune response in mice. Furthermore, we also developed chimeric P. berghei parasites expressing the cognate P. falciparum antigen to enable assessment of efficacy in mice. Our preliminary results indicate that vectors encoding either PfLSA1 or PfLSAP2 are capable of inducing sterile protection dependent on the presence of CD8+ T cells. This work has identified two promising P. falciparum liver-stage candidate antigens that will now undergo further testing in humans. PMID:26139288

  19. Hydrostatic isolated limb perfusion with adeno-associated virus vectors enhances correction of skeletal muscle in Pompe disease.

    PubMed

    Sun, B; Li, S; Bird, A; Koeberl, D D

    2010-12-01

    Glycogen storage disease type II (Pompe disease; MIM 232300) stems from the inherited deficiency of acid-α-glucosidase (GAA; acid maltase; EC 3.2.1.20), which primarily involves cardiac and skeletal muscles. We hypothesized that hydrostatic isolated limb perfusion (ILP) administration of an adeno-associated virus (AAV) vector containing a muscle-specific promoter could achieve relatively higher transgene expression in the hindlimb muscles of GAA-knockout (GAA-KO) mice, in comparison with intravenous (IV) administration. ILP administration of AAV2/8 vectors encoding alkaline phosphatase or human GAA-transduced skeletal muscles of the hindlimb widely, despite the relatively low number of vector particles administered (1 × 10¹¹), and IV administration of an equivalent vector dose failed to transduce skeletal muscle detectably. Similarly, ILP administration of fewer vector particles of the AAV2/9 vector encoding human GAA (3 × 10¹⁰) transduced skeletal muscles of the hindlimb widely and significantly reduced glycogen content to, in comparison with IV administration. The only advantage for IV administration was moderately high-level transduction of cardiac muscle, which demonstrated compellingly that ILP administration sequestered vector particles within the perfused limb. Reduction of glycogen storage in the extensor digitorum longus demonstrated the potential advantage of ILP-mediated delivery of AAV vectors in Pompe disease, because type II myofibers are resistant to enzyme replacement therapy. Thus, ILP will enhance AAV transduction of multiple skeletal muscles while reducing the required dosages in terms of vector particle numbers. PMID:20686508

  20. Developing Adenoviral Vectors Encoding Therapeutic Genes Toxic to Host Cells: Comparing Binary and Single Inducible Vectors Expressing Truncated E2F-1

    PubMed Central

    Gomez-Gutierrez, Jorge G.; Rao, Xiao-Mei; Garcia-Garcia, Aracely; Hao, Hongying; McMasters, Kelly M.; Zhou, H. Sam

    2010-01-01

    Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation. PMID:20003994

  1. Safety and biodistribution assessment of sc-rAAV2.5IL-1Ra administered via intra-articular injection in a mono-iodoacetate-induced osteoarthritis rat model

    PubMed Central

    Wang, Gensheng; Evans, Christopher H; Benson, Janet M; Hutt, Julie A; Seagrave, JeanClare; Wilder, Julie A; Grieger, Joshua C; Samulski, R Jude; Terse, Pramod S

    2016-01-01

    Interleukin-1 (IL-1) plays an important role in the pathophysiology of osteoarthritis (OA), and gene transfer of IL-1 receptor antagonist (IL-1Ra) holds promise for OA treatment. A preclinical safety and biodistribution study evaluated a self-complementary adeno-associated viral vector carrying rat IL-1Ra transgene (sc-rAAV2.5rIL-1Ra) at 5 × 108, 5 × 109, or 5 × 1010 vg/knee, or human IL-1Ra transgene (sc-rAAV2.5hIL-1Ra) at 5 × 1010 vg/knee, in Wistar rats with mono-iodoacetate (MIA)–induced OA at days 7, 26, 91, 180, and 364 following intra-articular injection. The MIA-induced OA lesions were consistent with the published data on this model. The vector genomes persisted in the injected knees for up to a year with only limited vector leakage to systemic circulation and uptake in tissues outside the knee. Low levels of IL-1Ra expression and mitigation of OA lesions were observed in the vector-injected knees, albeit inconsistently. Neutralizing antibodies against the vector capsid developed in a dose-dependent manner, but only the human vector induced a small splenic T-cell immune response to the vector capsid. No local or systemic toxicity attributable to vector administration was identified in the rats as indicated by clinical signs, body weight, feed consumption, clinical pathology, and gross and microscopic pathology through day 364. Taken together, the gene therapy vector demonstrated a favorable safety profile. PMID:26817025

  2. Development of Novel Magnetic Encoder and Vector Control Method for Servo Control of High-Speed Motor

    NASA Astrophysics Data System (ADS)

    Hao, Shuanghui; Liu, Jie; Mizugaki, Yoshio

    This paper presents a novel unipolar magnetic encoder and a new Vector control method of AC servo which can enable the feedback of the rotor position with high accuracy in high speed occasion. The signal generator of encoder consists of three pairs of semiconductor hall elements and a rotor of alnico magnet with one pair of poles. This encoder is featured with the operation module and the communication module to transmit an angular data in the form of asynchronous serial transfer protocol. Besides, a delay compensation algorithm is proposed to ensure the accurate estimation of instant angular position and the estimation error is also analyzed. At last, an experimental apparatus was built for a 600W, 4 pairs of pole, at rated speed 20,000min-1, permanent-magnet synchronous motor drive and the proposed vector control scheme has been implemented. The experiment results supported the validity of the proposed magnetic encoder and vector control method for high-speed AC servo control within 0.05 % error of revolving speed.

  3. Adeno-associated virus 2-mediated antiangiogenic cancer gene therapy: long-term efficacy of a vector encoding angiostatin and endostatin over vectors encoding a single factor.

    PubMed

    Ponnazhagan, Selvarangan; Mahendra, Gandham; Kumar, Sanjay; Shaw, Denise R; Stockard, Cecil R; Grizzle, William E; Meleth, Sreelatha

    2004-03-01

    Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)-mediated gene transfer. Both vectors provided significant protective efficacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiostatin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy. PMID:14996740

  4. Correction of glycogen storage disease type II by an adeno-associated virus vector containing a muscle-specific promoter.

    PubMed

    Sun, Baodong; Zhang, Haoyue; Franco, Luis M; Brown, Talmage; Bird, Andrew; Schneider, Ayn; Koeberl, Dwight D

    2005-06-01

    Glycogen storage disease type II (Pompe disease) causes death in infancy from cardiorespiratory failure due to acid alpha-glucosidase (GAA; acid maltase) deficiency. An AAV2 vector pseudotyped as AAV6 (AAV2/6 vector) transiently expressed high-level human GAA in GAA-knockout (GAA-KO) mice without reducing glycogen storage; however, in immunodeficient GAA-KO/SCID mice the AAV2/6 vector expressed high-level GAA and reduced the glycogen content of the injected muscle for 24 weeks. A CD4+/CD8+ lymphocytic infiltrate was observed in response to the AAV2/6 vector in immunocompetent GAA-KO mice. When a muscle-specific creatine kinase promoter was substituted for the CB promoter (AAV-MCKhGAApA), that AAV2/6 vector expressed high-level GAA and reduced glycogen content in immunocompetent GAA-KO mice. Muscle-restricted expression of hGAA provoked only a humoral (not cellular) immune response. Intravenous administration of a high number of particles of AAV-MCKhGAApA as AAV2/7 reduced the glycogen content of the heart and skeletal muscle and corrected individual myofibers in immunocompetent GAA-KO mice 24 weeks postinjection. In summary, persistent correction of muscle glycogen content was achieved with an AAV vector containing a muscle-specific promoter in GAA-KO mice, and this approach should be considered for muscle-targeted gene therapy in Pompe disease. PMID:15922959

  5. Comparative insecticidal properties of two nucleopolyhedrovirus vectors encoding a similar toxin gene chimer.

    PubMed

    Treacy, M F; Rensner, P E; All, J N

    2000-08-01

    Laboratory, greenhouse and field studies were conducted to characterize the insecticidal properties of genetically altered forms of Autographa californica (Speyer) nucleopolyhedrovirus (AcNPV) and Helicoverpa zea (Boddie) NPV (HzNPV) against selected heliothine species. The altered viruses each contained a chimeric 0.8-kb fragment encoding the insect-specific, sodium channel neurotoxin from the Algerian scorpion Androctonus australis Hector (AaIT, hence recombinant viruses designated Ac-AaIT and Hz-AaIT). Based on LD50 values, results from diet-overlay bioassays showed Ac-AaIT and Hz-AaIT to be equally virulent against larval tobacco budworm, Heliothis virescens (F.), but Hz-AaIT averaged 1,335-fold greater bioactivity than Ac-AaIT against larval cotton bollworm, Helicoverpa zea (Boddie). Hz-AaIT killed larvae of both heliothine species at rates significantly faster than those imparted by HzNPV (viral LT50 values averaged 2.5 and 5.6 d, respectively). In greenhouse studies, foliar sprays of Ac-AaIT and Hz-AaIT were equally effective in controlling H. virescens on cotton; however, Hz-AaIT provided control of H. zea on cotton at a level superior to that of Ac-AaIT. For example, after three weekly sessions of foliar application and H. zea artificial infestation, cotton treated with Ac-AaIT or Hz-AaIT at 10 x 10(11) occulsion bodies (OB)/ha averaged 2.5 and 16.2 nondamaged flower buds per plant, respectively. Another greenhouse study conducted against heliothine species on cotton showed that the quicker killing speed exhibited by Hz-AaIT led to improved plant protection versus HzNPV. Finally, results from three field trials demonstrated that Hz-AaIT at 5-12 x 10(11) OB/ha provided control of the heliothine complex in cotton at levels slightly better than Bacillus thuringiensis, equal to the macrolide, spinosad, and only slightly less than that of selected pyrethroid and carbamate insecticides. Overall, results from these studies indicate that, because of host range

  6. Analysis of T cell responses to chimpanzee adenovirus vectors encoding HIV gag–pol–nef antigen

    PubMed Central

    Herath, S.; Le Heron, A.; Colloca, S.; Bergin, P.; Patterson, S.; Weber, J.; Tatoud, R.; Dickson, G.

    2015-01-01

    Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene. PMID:26546736

  7. Analysis of T cell responses to chimpanzee adenovirus vectors encoding HIV gag-pol-nef antigen.

    PubMed

    Herath, S; Le Heron, A; Colloca, S; Bergin, P; Patterson, S; Weber, J; Tatoud, R; Dickson, G

    2015-12-16

    Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene. PMID:26546736

  8. Long-term efficacy following readministration of an adeno-associated virus vector in dogs with glycogen storage disease type Ia.

    PubMed

    Demaster, Amanda; Luo, Xiaoyan; Curtis, Sarah; Williams, Kyha D; Landau, Dustin J; Drake, Elizabeth J; Kozink, Daniel M; Bird, Andrew; Crane, Bayley; Sun, Francis; Pinto, Carlos R; Brown, Talmage T; Kemper, Alex R; Koeberl, Dwight D

    2012-04-01

    Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV vector

  9. Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus.

    PubMed

    Romanutti, Carina; D'Antuono, Alejandra; Palacios, Carlos; Quattrocchi, Valeria; Zamorano, Patricia; La Torre, Jose; Mattion, Nora

    2013-08-30

    The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV. PMID:23683999

  10. Construction and application of the vectors to identify genes encoding exported proteins of Escherichia coli.

    PubMed

    Niu, Dong; Shen, Qinfang; Zhu, Junli; Liu, Jiangmei; Yuan, Jiajie; Tan, Shuang; Yu, Xuping

    2013-10-01

    In order to clone genes having signal sequences of Escherichia coli, four vectors with or without Lac or Ara promoter were constructed using a leaderless β-lactamase as reporter. Fragments of tetracycline resistance gene (Tet) with or without promoter were used to confirm the vectors' ability to clone and report signal sequences. The minimum inhibitory concentration of ampicillin of the transformants was measured to detect the expression and secretion efficiency of the vectors. The results showed that the β-lactamase could be co-expressed and secreted with Tet protein. The Lac or Ara promoter in the vectors could be regulated by different inducers, and the Ara promoter showed higher regulative efficiency than the Lac. The best induction dose of L-arabinose for the Ara promoter is 1.25 %. All the four vectors were stably maintained in host after being inoculated for 20 passages in antibiotics-free media. Genomic library of an avian pathogenic strain, E. coli O2, was constructed using the pMB-Ara-T vector we developed. 318 clones were obtained from the genomic library of E. coli strain O2, and the inserts in these clones represented 276 genes based on sequence analysis. Among the 276 cloned fragments, only 128 had complete promoter sequence. For the 128 fragments with promoter, only 27 could be expressed under LB culture condition without inducer, the other 101 were only expressed under induction. The results showed our constructed vectors could efficiently capture all kinds of exported protein genes in vitro, including the ones without promoter or with inactive promoter. PMID:24052231

  11. Long-Term Efficacy Following Readministration of an Adeno-Associated Virus Vector in Dogs with Glycogen Storage Disease Type Ia

    PubMed Central

    Demaster, Amanda; Luo, Xiaoyan; Curtis, Sarah; Williams, Kyha D.; Landau, Dustin J.; Drake, Elizabeth J.; Kozink, Daniel M.; Bird, Andrew; Crane, Bayley; Sun, Francis; Pinto, Carlos R.; Brown, Talmage T.; Kemper, Alex R.

    2012-01-01

    Abstract Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV

  12. Novel Recombinant Hepatitis B Virus Vectors Efficiently Deliver Protein and RNA Encoding Genes into Primary Hepatocytes

    PubMed Central

    Hong, Ran; Bai, Weiya; Zhai, Jianwei; Liu, Wei; Li, Xinyan; Zhang, Jiming; Cui, Xiaoxian; Zhao, Xue; Ye, Xiaoli; Deng, Qiang; Tiollais, Pierre; Wen, Yumei

    2013-01-01

    Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection. PMID:23552416

  13. Recombinant baculoviruses as vectors for identifying proteins encoded by intron-containing members of complex multigene families.

    PubMed Central

    Iatrou, K; Meidinger, R G; Goldsmith, M R

    1989-01-01

    Using a transfer vector derived from Bombyx mori nuclear polyhedrosis virus (BmNPV), we have constructed recombinant baculoviruses that contain complete silk moth chorion chromosomal genes encoding high-cysteine proteins under the control of the polyhedrin promoter. Silk moth tissue culture cells infected with these recombinant viruses were found to contain abundant RNA sequences of sizes similar to those of the authentic chorion mRNAs. Chorion transcripts present in infected cells were initiated almost exclusively at the cap site of the polyhedrin start site. Primer extension and RNase protection experiments revealed that a considerable proportion of the resultant transcripts were spliced at the same sites as those utilized in follicular cells for the production of functional chorion mRNA. Electrophoretic analysis and immunoprecipitation of the proteins of host cells infected with the recombinant viruses revealed the presence of the corresponding chorion proteins. We conclude that baculovirus vectors can be used for expressing efficiently not only cDNAs or simple genes devoid of intervening sequences but also intron-containing chromosomal genes. Thus, recombinant baculoviruses offer a powerful alternative to hybrid-selected translation, particularly when the identification of proteins encoded by members of complex multigene families is required. Images PMID:2556701

  14. A 110-kDa nuclear shuttle protein, nucleolin, specifically binds to adeno-associated virus type 2 (AAV-2) capsid.

    PubMed

    Qiu, J; Brown, K E

    1999-05-10

    A 110-kDa protein was copurified with adeno-associated virus type 2 (AAV-2) virions after CsCl density gradient isopycnic centrifugation. Amino acid sequence of peptides derived from this protein after tryptic digestion, monoclonal antibody production, and Western blot analysis showed that the copurified protein was the major nucleolar phosphoprotein, human nucleolin. Virus overlay assays demonstrated that AAV-2 capsid specifically bound to the human nucleolin, and immunoprecipitation studies confirmed the in vitro binding of nucleolin and intact AAV-2 capsids but not denatured viral proteins. Double-immunofluorescence staining of infected cells showed that AAV capsid and nucleolin were colocalized in both cytoplasm and nucleus. In addition, when cytoplasmic and nuclear fractions were extracted from AAV-infected KB cells at different time points postinfection, immunoprecipitation data and Western blotting showed that AAV capsid formation and nucleolin interact specifically and share their subcellular localization in infected cells. With the known functions of nucleolin in the synthesis of rRNA and ribosome assembly, binding to single-stranded DNA, and acting as a shuttle between cytoplasm and nucleolus, our data showing that AAV-2 capsid binds specifically to nucleolin both in vitro and in vivo suggest a key role of nucleolin in AAV-2 replication, particularly in capsid assembly. PMID:10329548

  15. Reversion of the biochemical defects in murine embryonic Sandhoff neurons using a bicistronic lentiviral vector encoding hexosaminidase alpha and beta.

    PubMed

    Arfi, Audrey; Zisling, Rivka; Richard, Emmanuel; Batista, Lionel; Poenaru, Livia; Futerman, Anthony H; Caillaud, Catherine

    2006-03-01

    Sandhoff disease, a neurodegenerative disorder characterized by the intracellular accumulation of GM2 ganglioside, is caused by mutations in the hexosaminidase beta-chain gene resulting in a hexosaminidase A (alphabeta) and B (betabeta) deficiency. A bicistronic lentiviral vector encoding both the hexosaminidase alpha and beta chains (SIV.ASB) has previously been shown to correct the beta-hexosaminidase deficiency and to reduce GM2 levels both in transduced and cross-corrected human Sandhoff fibroblasts. Recent advances in determining the neuropathophysiological mechanisms in Sandhoff disease have shown a mechanistic link between GM2 accumulation, neuronal cell death, reduction of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activity, and axonal outgrowth. To examine the ability of the SIV.ASB vector to reverse these pathophysiological events, hippocampal neurons from embryonic Sandhoff mice were transduced with the lentivector. Normal axonal growth rates were restored, as was the rate of Ca(2+) uptake via the SERCA and the sensitivity of the neurons to thapsigargin-induced cell death, concomitant with a decrease in GM2 and GA2 levels. Thus, we have demonstrated that the bicistronic vector can reverse the biochemical defects and down-stream consequences in Sandhoff neurons, reinforcing its potential for Sandhoff disease in vivo gene therapy. PMID:16441513

  16. Biodistribution of rAAV vectors following intraocular administration: evidence for the presence and persistence of vector DNA in the optic nerve and in the brain.

    PubMed

    Provost, Nathalie; Le Meur, Guylène; Weber, Michel; Mendes-Madeira, Alexandra; Podevin, Guillaume; Cherel, Yan; Colle, Marie-Anne; Deschamps, Jack-Yves; Moullier, Philippe; Rolling, Fabienne

    2005-02-01

    The purpose of our study was to evaluate the biodistribution of rAAV vectors following subretinal or intravitreal injection. In rats, we performed subretinal or intravitreal injections of rAAV-2/2.CMV.gfp. In large animals, rAAV-2/4.CMV.gfp or rAAV-2/5.CMV.gfp was delivered into the subretinal space while rAAV-2/2.CMV.gfp was delivered either to the subretinal space or to the vitreous. In euthanized animals, we undertook a complete necropsy. In animals maintained alive, we collected blood and tissue samples from the submandibular lymph node, liver, and gonads. We analyzed total DNA, extracted from various tissue samples and peripheral blood mononuclear cells (PBMC), by PCR. Following subretinal or intravitreal injections in rats and in large animals, vector sequences were not detected in the liver or in the gonads but were occasionally found in PBMC. An unexpected result was the detection of rAAV sequences in the optic nerve following subretinal injection. The most striking finding was the detection of vector sequences in the brain, along the visual pathway, in rAAV-2/2 intravitreally injected dogs. These findings raise safety concerns regarding intraocular administration of rAAV vectors and will have an impact on the development of future gene therapy trials for retinal diseases. PMID:15668139

  17. The effect of recombinant adeno-associated virus-adiponectin (rAAV2/1-Acrp30) on glycolipid dysmetabolism and liver morphology in diabetic rats.

    PubMed

    Long, Wen; Hui Ju, Zhong; Fan, Zhang; Jing, Wang; Qiong, Li

    2014-09-15

    Adiponectin is an adipocytokine derived from adipocytes with insulin resistance-improving and anti-inflammatory activities. The level of Adiponectin is decreased in obesity, insulin resistance and Type 2 diabetes mellitus. The administration of recombinant adiponectin has been shown to improve hyperglycemia and insulin resistance in diabetic mice. Therefore, we investigated the effects of recombinant adeno-associated virus-adiponectin (rAAV2/1-Acrp30) on the glycolipid profile and liver morphology in streptozotocin-induced diabetic rats. Animals were fed a high-fat/high-glucose diet for 4weeks and diabetes induced by intraperitoneal administration of streptozotocin. The animals were divided randomly into four groups: diabetes control group, rAAV2/1-Acrp30 treatment group, vacuity virus group, and normal control group. Compared with diabetic rats and those in the vacuity virus group, animals treated with rAAV2/1-Acrp30 exhibited significantly lower values for glycaemic and lipidic profiles, and significantly higher levels of HDL. Although APN expression increased in the liver tissue, serum levels were not significantly increased. However, the rAAV2/1-Acrp30 treated animals showed amelioration of hepatic disease, accompanied by marked reduction in the expression of NF-κBp65 and IκBα. The results suggest that rAAV2/1-Acrp30 ameliorates glycolipid dysmetabolism and hepatic disease in streptozotocin-induced diabetic rats. These observations indicate that the function of rAAV2/1-Acrp30 is mediated by downregulated expression of NF-κBp65 and IκBα. PMID:25019654

  18. Selectable retrovirus vectors encoding Friend virus gp55 or erythropoietin induce polycythemia with different phenotypic expression and disease progression.

    PubMed Central

    Ahlers, N; Hunt, N; Just, U; Laker, C; Ostertag, W; Nowock, J

    1994-01-01

    The Friend spleen focus-forming virus induces a massive expansion of erythroid progenitor cells resulting in polycythemia and splenomegaly. The pathogenic agent is the membrane glycoprotein gp55, encoded by the env gene. Recent evidence indicates that gp55 binds to and activates the erythropoietin (Epo) receptor. It is not clear, however, whether gp55 completely mimics the natural receptor ligand (Epo). To directly compare both effectors, we constructed selectable retroviral vectors which carry either the env or the Epo gene. The selection marker allowed for clonal analysis of infected cells. After infection of DBA/2J mice, the spleen weight, hematological indices, and Epo titer of peripheral blood were monitored. Although both viruses induced an acute erythrocytosis, there were significant differences in disease phenotype and progression. The Epo virus caused an enhanced increase of hematocrit and erythrocytes, whereas with the env virus the pool of late progenitors (CFU-erythroid) was dramatically expanded, resulting in a more severe splenomegaly. The distribution of cytologically recognizable erythroid precursors was shifted towards immature cell types by the env vector compared with Epo. These data suggest that Epo and gp55 differentially affect proliferation and differentiation. Gp55 appears to promote proliferation over differentiation, whereas Epo preferentially drives differentiation. Images PMID:7933106

  19. Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys

    PubMed Central

    Mockenhaupt, Stefan; Grosse, Stefanie; Rupp, Daniel; Bartenschlager, Ralf; Grimm, Dirk

    2015-01-01

    Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antisense strand that binds and silences designated target mRNAs. However, in principle, the sense strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces sense strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA sense strands in human cells selectively without affecting desired antisense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA sense strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA sense strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans. PMID:26170322

  20. Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys.

    PubMed

    Mockenhaupt, Stefan; Grosse, Stefanie; Rupp, Daniel; Bartenschlager, Ralf; Grimm, Dirk

    2015-07-28

    Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antisense strand that binds and silences designated target mRNAs. However, in principle, the sense strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces sense strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA sense strands in human cells selectively without affecting desired antisense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA sense strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA sense strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans. PMID:26170322

  1. Systemic Administration of a Recombinant AAV1 Vector Encoding IGF-1 Improves Disease Manifestations in SMA Mice

    PubMed Central

    Tsai, Li-Kai; Chen, Chien-Lin; Ting, Chen-Hung; Lin-Chao, Sue; Hwu, Wuh-Liang; Dodge, James C; Passini, Marco A; Cheng, Seng H

    2014-01-01

    Spinal muscular atrophy is a progressive motor neuron disease caused by a deficiency of survival motor neuron. In this study, we evaluated the efficacy of intravenous administration of a recombinant adeno-associated virus (AAV1) vector encoding human insulin-like growth factor-1 (IGF-1) in a severe mouse model of spinal muscular atrophy. Measurable quantities of human IGF-1 transcripts and protein were detected in the liver (up to 3 months postinjection) and in the serum indicating that IGF-1 was secreted from the liver into systemic circulation. Spinal muscular atrophy mice administered AAV1-IGF-1 on postnatal day 1 exhibited a lower extent of motor neuron degeneration, cardiac and muscle atrophy as well as a greater extent of innervation at the neuromuscular junctions compared to untreated controls at day 8 posttreatment. Importantly, treatment with AAV1-IGF-1 prolonged the animals' lifespan, increased their body weights and improved their motor coordination. Quantitative polymerase chain reaction and western blot analyses showed that AAV1-mediated expression of IGF-1 led to an increase in survival motor neuron transcript and protein levels in the spinal cord, brain, muscles, and heart. These data indicate that systemically delivered AAV1-IGF-1 can correct several of the biochemical and behavioral deficits in spinal muscular atrophy mice through increasing tissue levels of survival motor neuron. PMID:24814151

  2. Non-transmissible Sendai virus vector encoding c-myc suppressor FBP-interacting repressor for cancer therapy

    PubMed Central

    Matsushita, Kazuyuki; Shimada, Hideaki; Ueda, Yasuji; Inoue, Makoto; Hasegawa, Mamoru; Tomonaga, Takeshi; Matsubara, Hisahiro; Nomura, Fumio

    2014-01-01

    AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR). METHODS: Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR (HA-FIR) expression vector. A fusion gene-deficient, non-transmissible, Sendai virus (SeV) vector encoding FIR cDNA, SeV/dF/FIR, was prepared. SeV/dF/FIR was examined for its gene transduction efficiency, viral dose dependency of antitumor effect and apoptosis induction in HeLa (cervical squamous cell carcinoma) cells and SW480 (colon adenocarcinoma) cells. Antitumor efficacy in a mouse xenograft model was also examined. The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A (SSA), a SAP155 inhibitor, or SAP155 siRNA which induce c-Myc by increasing FIR∆exon2 in HeLa cells. RESULTS: FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression. Thus, FIR expressing vectors are potentially applicable for cancer therapy. FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2 (FIR∆exon2), counteracting FIR for c-Myc protein expression. Furthermore, FIR forms a complex with SAP155 and inhibits mutual well-established functions. Thus, both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application. SeV/dF/FIR, a cytoplasmic RNA virus, was successfully prepared and showed highly efficient gene transduction in in vivo experiments. Furthermore, in nude mouse tumor xenograft models, SeV/dF/FIR displayed high antitumor efficiency against human cancer cells. SeV/dF/FIR suppressed SSA-activated c-Myc. SAP155 siRNA, potentially produces FIR∆exon2, and led to c-Myc overexpression with phosphorylation at Ser62. HA-FIR suppressed

  3. In vivo model of adeno-associated virus vector persistence and rescue.

    PubMed Central

    Afione, S A; Conrad, C K; Kearns, W G; Chunduru, S; Adams, R; Reynolds, T C; Guggino, W B; Cutting, G R; Carter, B J; Flotte, T R

    1996-01-01

    Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206-212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue. PMID:8627804

  4. Lentiviral vector encoding ubiquitinated hepatitis B core antigen induces potent cellular immune responses and therapeutic immunity in HBV transgenic mice.

    PubMed

    Dai, Shenglan; Zhuo, Meng; Song, Linlin; Chen, Xiaohua; Yu, Yongsheng; Zang, Guoqing; Tang, Zhenghao

    2016-07-01

    Predominant T helper cell type 1 (Th1) immune responses accompanied by boosted HBV-specific cytotoxic T lymphocyte (CTL) activity are essential for the clearance of hepatitis B virus (HBV) in chronic hepatitis B (CHB) patients. Ubiquitin (Ub) serves as a signal for the target protein to be recognized and degraded through the ubiquitin-proteasome system (UPS). Ubiquitinated hepatitis B core antigen (Ub-HBcAg) has been proved to be efficiently degraded into the peptides, which can be presented by major histocompatibility complex (MHC) class I resulting in stimulating cell-mediated responses. In the present study, lentiviral vectors encoding Ub-HBcAg (LV-Ub-HBcAg) were designed and constructed as a therapeutic vaccine for immunotherapy. HBcAg-specific cellular immune responses and anti-viral effects induced by LV-Ub-HBcAg were evaluated in HBV transgenic mice. We demonstrated that immunization with LV-Ub-HBcAg promoted the secretion of cytokines interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), generated remarkably high percentages of IFN-γ-secreting CD8(+) T cells and CD4(+) T cells, and enhanced HBcAg-specific CTL activity in HBV transgenic mice. More importantly, vaccination with LV-Ub-HBcAg could efficiently decreased the levels of serum hepatitis B surface antigen (HBsAg), HBV DNA and the expression of HBsAg and HBcAg in liver tissues of HBV transgenic mice. In addition, LV-Ub-HBcAg could upregulate the expression of T cell-specific T-box transcription factor (T-bet) and downregulate the expression of GATA-binding protein 3 (GATA-3) in spleen T lymphocytes. The therapeutic vaccine LV-Ub-HBcAg could break immune tolerance, and induce potent HBcAg specific cellular immune responses and therapeutic effects in HBV transgenic mice. PMID:26874581

  5. AAV2/8-mediated Correction of OTC Deficiency Is Robust in Adult but Not Neonatal Spfash Mice

    PubMed Central

    Cunningham, Sharon C; Spinoulas, Afroditi; Carpenter, Kevin H; Wilcken, Bridget; Kuchel, Philip W; Alexander, Ian E

    2009-01-01

    Ornithine transcarbamylase (OTC) deficiency, the most common urea cycle disorder, is associated with severe hyperammonemia accompanied by a high risk of neurological damage and death in patients presenting with the neonatal-onset form. Contemporary therapies, including liver transplantation, remain inadequate with considerable morbidity, justifying vigorous investigation of alternate therapies. Clinical evidence suggests that as little as 3% normal enzyme activity is sufficient to ameliorate the severe neonatal phenotype, making OTC deficiency an ideal model for the development of liver-targeted gene therapy. In this study, we investigated metabolic correction in neonatal and adult male OTC-deficient Spfash mice following adeno-associated virus (AAV)2/8-mediated delivery of the murine OTC complementary DNA under the transcriptional control of a liver-specific promoter. Substantially supraphysiological levels of OTC enzymatic activity were readily achieved in both adult and neonatal mice following a single intraperitoneal (i.p.) injection, with metabolic correction in adults being robust and life-long. In the neonates, however, full metabolic correction was transient, although modest levels of OTC expression persisted into adulthood. Although not directly testable in Spfash mice, these levels were theoretically sufficient to prevent hyperammonemia in a null phenotype. This loss of expression in the neonatal liver is the consequence of hepatocellular proliferation and presents an added challenge to human therapy. PMID:19384294

  6. Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors

    PubMed Central

    Zeltner, Nadja; Kohlbrenner, Erik; Clément, Nathalie; Weber, Thomas; Linden, R. Michael

    2010-01-01

    Viral vectors derived from adeno-associated viruses (AAV) are widely used for gene transfer both in vitro and in vivo. The increasing use of AAV as a gene transfer vector, as well as recently demonstrated immunological complications in clinical trials, highlight the necessity to define the specific activity of vector preparations beyond current standards. In this report, we determined the infectious, physical and genome-containing particle titers of several wild-type AAV type 2 (wtAAV2) and recombinant AAV type 2 (rAAV2) preparations that were produced and purified by standard methods. We found that the infectivity of wtAAV2 approaches a physical-to-infectious particle ratio of one. This near-perfect physical-to-infectious particle ratio defines a “ceiling” for the theoretically achievable quality of recombinant AAV vectors. In comparison, for rAAV2, only approximately 50 out of 100 viral particles contained a genome and more strikingly only approximately one of the 100 viral particles was infectious. Our findings suggest that current strategies for rAAV vector design, production and/or purification should be amenable to improvements. Ultimately, this could result in the generation of near-perfect vector particles, a prospect with significant implications for gene therapy. PMID:20336156

  7. A novel chimeric adenoassociated virus 2/human bocavirus 1 parvovirus vector efficiently transduces human airway epithelia.

    PubMed

    Yan, Ziying; Keiser, Nicholas W; Song, Yi; Deng, Xuefeng; Cheng, Fang; Qiu, Jianming; Engelhardt, John F

    2013-12-01

    Human bocavirus virus-1 (HBoV1), a newly discovered autonomous parvovirus with a 5,500 nt genome, efficiently infects human-polarized airway epithelia (HAE) from the apical membrane. We hypothesized that the larger genome and high airway tropism of HBoV1 would be ideal for creating a viral vector for lung gene therapy. To this end, we successfully generated recombinant HBoV1 (rHBoV1) from an open reading frames-disrupted rHBoV1 genome that efficiently transduces HAE from the apical surface. We next evaluated whether HBoV1 capsids could package oversized rAAV2 genomes. These studies created a rAAV2/HBoV1 chimeric virus (5.5 kb genome) capable of apically transducing HAE at 5.6- and 70-fold greater efficiency than rAAV1 or rAAV2 (4.7-kb genomes), respectively. Molecular studies demonstrated that viral uptake from the apical surface was significantly greater for rAAV2/HBoV1 than for rAAV2 or rAAV1, and that polarization of airway epithelial cells was required for HBoV1 capsid-mediated gene transfer. Furthermore, rAAV2/HBoV1-CFTR virus containing the full-length cystic fibrosis transmembrane conductance regulator (CFTR) gene coding sequence and the strong CBA promoter efficiently corrected CFTR-dependent chloride transport in cystic fibrosis (CF) HAE. In summary, using the combined advantages of AAV and HBoV1, we have developed a novel and promising viral vector for CF lung gene therapy and also potentially HBoV1 vaccine development. PMID:23896725

  8. Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach

    PubMed Central

    Mowat, FM; Gornik, KR; Dinculescu, A; Boye, SL; Hauswirth, WW; Petersen-Jones, SM; Bartoe, JT

    2013-01-01

    Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected, 4 dogs received intravitreal injections, 8 dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal), and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency, and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach. PMID:24225638

  9. Replication-competent retroviral vectors encoding alkaline phosphatase reveal spatial restriction of viral gene expression/transduction in the chick embryo.

    PubMed Central

    Fekete, D M; Cepko, C L

    1993-01-01

    Replication-competent avian retroviruses, capable of transducing and expressing up to 2 kb of nonviral sequences, are now available to effect widespread gene transfer in chicken (chick) embryos (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We have constructed novel avian retroviral vectors that encode human placental alkaline phosphatase as a marker whose expression can be histochemically monitored. These vectors have been tested for expression by introducing them into the embryonic chick nervous system. They have revealed that the expression of retrovirally transduced genes can be spatially and temporally limited without the need for tissue-specific promoters. By varying the site and time of infection, targeted gene transfer can be confined to selected populations of neural cells over the course of several days, a time window that is sufficient for many key developmental processes. The capability of differentially infecting specific target populations may avoid confounding variables such as detrimental effects of a transduced gene on processes unrelated to the cells or tissue of interest. These vectors and methods thus should be useful in studies of the effect of transduced genes on the development of various organs and tissues during avian embryogenesis. In addition, the vectors will facilitate studies aimed at an understanding of viral infection and expression patterns. Images PMID:8455633

  10. Characterization and expression analysis of gene encoding heme peroxidase HPX15 in major Indian malaria vector Anopheles stephensi (Diptera: Culicidae).

    PubMed

    Kajla, Mithilesh; Kakani, Parik; Choudhury, Tania Pal; Gupta, Kuldeep; Gupta, Lalita; Kumar, Sanjeev

    2016-06-01

    The interaction of mosquito immune system with Plasmodium is critical in determining the vector competence. Thus, blocking the crucial mosquito molecules that regulate parasite development might be effective in controlling the disease transmission. In this study, we characterized a full-length AsHPX15 gene from the major Indian malaria vector Anopheles stephensi. This gene is true ortholog of Anopheles gambiae heme peroxidase AgHPX15 (AGAP013327), which modulates midgut immunity and regulates Plasmodium falciparum development. We found that AsHPX15 is highly induced in mosquito developmental stages and blood fed midguts. In addition, this is a lineage-specific gene that has identical features and 65-99% amino acids identity with other HPX15 genes present in eighteen worldwide-distributed anophelines. We discuss that the conserved HPX15 gene might serve as a common target to manipulate mosquito immunity and arresting Plasmodium development inside the vector host. PMID:26943999

  11. Safety and efficacy of subretinal readministration of a viral vector in large animals to treat congenital blindness.

    PubMed

    Amado, Defne; Mingozzi, Federico; Hui, Daniel; Bennicelli, Jeannette L; Wei, Zhangyong; Chen, Yifeng; Bote, Erin; Grant, Rebecca L; Golden, Jeffrey A; Narfstrom, Kristina; Syed, Nasreen A; Orlin, Stephen E; High, Katherine A; Maguire, Albert M; Bennett, Jean

    2010-03-01

    Leber's congenital amaurosis (LCA) is a group of severe inherited retinal degenerations that are symptomatic in infancy and lead to total blindness in adulthood. Recent clinical trials using recombinant adeno-associated virus serotype 2 (rAAV2) successfully reversed blindness in patients with LCA caused by RPE65 mutations after one subretinal injection. However, it was unclear whether treatment of the second eye in the same manner would be safe and efficacious, given the potential for a complicating immune response after the first injection. Here, we evaluated the immunological and functional consequences of readministration of rAAV2-hRPE65v2 to the contralateral eye using large animal models. Neither RPE65-mutant (affected; RPE65(-/-)) nor unaffected animals developed antibodies against the transgene product, but all developed neutralizing antibodies against the AAV2 capsid in sera and intraocular fluid after subretinal injection. Cell-mediated immune responses were benign, with only 1 of 10 animals in the study developing a persistent T cell immune response to AAV2, a response that was mediated by CD4(+) T cells. Sequential bilateral injection caused minimal inflammation and improved visual function in affected animals. Thus, subretinal readministration of rAAV2 in animals is safe and effective, even in the setting of preexisting immunity to the vector, a parameter that has been used to exclude patients from gene therapy trials. PMID:20374996

  12. Cancer Screening by Systemic Administration of a Gene Delivery Vector Encoding Tumor-Selective Secretable Biomarker Expression

    PubMed Central

    Browne, Andrew W.; Leddon, Jennifer L.; Currier, Mark A.; Williams, Jon P.; Frischer, Jason S.; Collins, Margaret H.; Ahn, Chong H.; Cripe, Timothy P.

    2011-01-01

    Cancer biomarkers facilitate screening and early detection but are known for only a few cancer types. We demonstrated the principle of inducing tumors to secrete a serum biomarker using a systemically administered gene delivery vector that targets tumors for selective expression of an engineered cassette. We exploited tumor-selective replication of a conditionally replicative Herpes simplex virus (HSV) combined with a replication-dependent late viral promoter to achieve tumor-selective biomarker expression as an example gene delivery vector. Virus replication, cytotoxicity and biomarker production were low in quiescent normal human foreskin keratinocytes and high in cancer cells in vitro. Following intravenous injection of virus >90% of tumor-bearing mice exhibited higher levels of biomarker than non-tumor-bearing mice and upon necropsy, we detected virus exclusively in tumors. Our strategy of forcing tumors to secrete a serum biomarker could be useful for cancer screening in high-risk patients, and possibly for monitoring response to therapy. In addition, because oncolytic vectors for tumor specific gene delivery are cytotoxic, they may supplement our screening strategy as a “theragnostic” agent. The cancer screening approach presented in this work introduces a paradigm shift in the utility of gene delivery which we foresee being improved by alternative vectors targeting gene delivery and expression to tumors. Refining this approach will usher a new era for clinical cancer screening that may be implemented in the developed and undeveloped world. PMID:21589655

  13. Deficiency in MyD88 Signaling Results in Decreased Antibody Responses to an Adeno-Associated Virus Vector in Murine Pompe Disease

    PubMed Central

    Zhang, Ping; Luo, Xiaoyan; Bird, Andrew; Li, Songtao

    2012-01-01

    Abstract We have previously shown that antibody and T cell responses limit the efficacy of an adeno-associated virus (AAV) pseudotype 8 (2/8) vector containing the universally active cytomegalovirus enhancer/chicken β-actin regulatory cassette (AAV2/8-CBhGAA) in treating murine Pompe disease. However, the innate immune responses to AAV2/8-CBhGAA are largely unknown. In this study, we investigated acute immune responses to AAV2/8-CBhGAA and the role of MyD88/TRIF signaling pathway in shaping adaptive immune responses to this vector. We showed here that a small and transient increase in CXCL-1 and IL-1β expression in livers of acid-α-glucosidase knockout (GAAKO) mice 6 h following injection with AAV2/8-CBhGAA. There was a robust antibody response to GAA in wild-type mice injected with this vector. In contrast, the anti-GAA IgG1 response was diminished in MyD88KO mice, and showed a trend toward a decrease in TRIFKO mice. In addition, the vector genome and GAA activity were significantly higher in MyD88KO livers compared with wild-type livers, suggesting reduced cytotoxic T cell responses. Importantly, elevated CD4+ T cells were detected by immunohistochemistry in MyD88KO livers. When adoptively transferred to wild-type mice, these CD4+ T cells have an ability to suppress antibody responses against AAV2/8-CBhGAA and to prevent further immunization against rhGAA. Our study suggests that the MyD88 deficiency leads to the suppression of deleterious immune responses to AAV2/8-CBhGAA, which has implications for gene therapy in Pompe disease. PMID:23514839

  14. Deficiency in MyD88 Signaling Results in Decreased Antibody Responses to an Adeno-Associated Virus Vector in Murine Pompe Disease.

    PubMed

    Zhang, Ping; Luo, Xiaoyan; Bird, Andrew; Li, Songtao; Koeberl, Dwight D

    2012-06-01

    We have previously shown that antibody and T cell responses limit the efficacy of an adeno-associated virus (AAV) pseudotype 8 (2/8) vector containing the universally active cytomegalovirus enhancer/chicken β-actin regulatory cassette (AAV2/8-CBhGAA) in treating murine Pompe disease. However, the innate immune responses to AAV2/8-CBhGAA are largely unknown. In this study, we investigated acute immune responses to AAV2/8-CBhGAA and the role of MyD88/TRIF signaling pathway in shaping adaptive immune responses to this vector. We showed here that a small and transient increase in CXCL-1 and IL-1β expression in livers of acid-α-glucosidase knockout (GAAKO) mice 6 h following injection with AAV2/8-CBhGAA. There was a robust antibody response to GAA in wild-type mice injected with this vector. In contrast, the anti-GAA IgG1 response was diminished in MyD88KO mice, and showed a trend toward a decrease in TRIFKO mice. In addition, the vector genome and GAA activity were significantly higher in MyD88KO livers compared with wild-type livers, suggesting reduced cytotoxic T cell responses. Importantly, elevated CD4(+) T cells were detected by immunohistochemistry in MyD88KO livers. When adoptively transferred to wild-type mice, these CD4(+) T cells have an ability to suppress antibody responses against AAV2/8-CBhGAA and to prevent further immunization against rhGAA. Our study suggests that the MyD88 deficiency leads to the suppression of deleterious immune responses to AAV2/8-CBhGAA, which has implications for gene therapy in Pompe disease. PMID:23514839

  15. Construction of a new shuttle vector and its use for cloning and expression of two plasmid-encoded bacteriocins from Lactobacillus paracasei subsp. paracasei BGSJ2-8.

    PubMed

    Kojic, Milan; Lozo, Jelena; Jovcic, Branko; Strahinic, Ivana; Fira, Djordje; Topisirovic, Ljubisa

    2010-06-15

    A new shuttle-cloning vector, pA13, was constructed and successfully introduced into Escherichia coli, Lactobacillus and Lactococcus strains. It showed high segregational and structural stability in all three hosts. The natural plasmid pSJ2-8 from L. paracasei subsp. paracasei BGSJ2-8 was cloned into pA13 using BamHI to obtain the construct, pB5. Sequencing and in silico analysis of pB5 revealed fifteen open reading frames (ORF). Plasmid pSJ2-8 harbours genes encoding the production of two bacteriocins, BacSJ and acidocin 8912. Combined N-terminal amino acid sequencing of BacSJ in combination with DNA sequencing of the bacSJ2-8 gene enabled determination of the primary structure of bacteriocin BacSJ. The bacSJ2-8 gene encodes 68-amino-acid peptide with a double-glycine leader peptide consisting of 18 amino acids, followed by the orf2 (bacSJ2-8i) which encodes the immunity protein of BacSJ. The production and functional expression of BacSJ in homologous and heterologous hosts suggest that bacSJ2-8 and bacSJ2-8i together with the genes encoding the ABC transporter and accessory protein are the minimal requirements for production of BacSJ. Biochemical and genetic analyses showed that BacSJ belongs to class II bacteriocins. PMID:20439125

  16. Cocaine hydrolase encoded in viral vector blocks the reinstatement of cocaine seeking in rats for 6 months

    PubMed Central

    Anker, Justin J.; Brimijoin, Stephen; Gao, Yang; Geng, Liyi; Zlebnik, Natalie E.; Parks, Robin J.; Carroll, Marilyn E.

    2011-01-01

    Background Cocaine dependence is a pervasive disorder with high rates of relapse. In a previous study, direct administration of a quadruple mutant albumin-fused butyrylcholinesterase (BChE) that efficiently catalyzes hydrolysis of cocaine to benzoic acid and ecgonine methyl ester acutely blocked cocaine seeking in an animal model of relapse. In the present experiments these results were extended to achieve a long duration blockade of cocaine seeking with a gene transfer paradigm using a related BChE-based cocaine hydrolase, termed “CocH”. Methods Male and female rats were allowed to self-administer cocaine under a fixed-ratio 1 schedule of reinforcement for approximately 14 days. Following the final self-administration session, rats were injected with CocH vector or a control injection (empty vector or saline), and their cocaine solutions were replaced with saline for 14 days to allow for extinction of lever pressing. Subsequently, they were tested for drug-primed reinstatement by administering i.p. injections of saline (S), cocaine (5, 10, and 15 mg/kg, C), and d-amphetamine (A) according to the following sequence: S, C, S, C, S, C, S, A. Rats then received cocaine-priming injections once weekly for 4 weeks, and subsequently, once monthly for up to 6 months. Results Administration of CocH vector produced substantial and sustained CocH activity in plasma that corresponded with diminished cocaine- (but not amphetamine-) induced reinstatement responding for up to 6 months following treatment (compared to high responding controls). Conclusion These results demonstrate that viral transfer of CocH may be useful in promoting long-term resistance to relapse to cocaine addiction. PMID:22209637

  17. Intra-amniotic Transient Transduction of the Periderm With a Viral Vector Encoding TGFβ3 Prevents Cleft Palate in Tgfβ3−/− Mouse Embryos

    PubMed Central

    Wu, Chadwick; Endo, Masa; Yang, Byung H; Radecki, Melissa A; Davis, Patrick F; Zoltick, Philip W; Spivak, Ryan M; Flake, Alan W; Kirschner, Richard E; Nah, Hyun-Duck

    2013-01-01

    Cleft palate is a developmental defect resulting from the failure of embryonic palatal shelves to fuse with each other at a critical time. Immediately before and during palatal fusion (E13–E15 in mice), transforming growth factor β3 (TGFβ3) is expressed in the palatal shelf medial edge epithelium (MEE) and plays a pivotal role in palatal fusion. Using Tgfβ3−/− mice, which display complete penetrance of the cleft palate phenotype, we tested the hypothesis that intra-amniotic gene transfer could be used to prevent cleft palate formation by restoring palatal midline epithelial function. An adenoviral vector encoding Tgfβ3 was microinjected into the amniotic sacs of mouse embryos at successive developmental stages. Transduced Tgfβ3−/− fetuses showed efficient recovery of palatal fusion with mesenchymal confluence following injection at E12.5 (100%), E13.5 (100%), E14.5 (82%), and E15.5 (75%). Viral vectors injected into the amniotic sac transduced the most superficial and transient peridermal cell layer but not underlying basal epithelial cells. TGFβ3 transduction of the peridermdal cell layer was sufficient to induce adhesion, fusion, and disappearance of the palatal shelf MEE in a cell nonautonomous manner. We propose that intra-amniotic gene transfer approaches have therapeutic potential to prevent cleft palate in utero, especially those resulting from palatal midline epithelial dysfunction. PMID:23089732

  18. Cationic Lipid-Formulated DNA Vaccine against Hepatitis B Virus: Immunogenicity of MIDGE-Th1 Vectors Encoding Small and Large Surface Antigen in Comparison to a Licensed Protein Vaccine

    PubMed Central

    Endmann, Anne; Klünder, Katharina; Kapp, Kerstin; Riede, Oliver; Oswald, Detlef; Talman, Eduard G.; Schroff, Matthias; Kleuss, Christiane; Ruiters, Marcel H. J.; Juhls, Christiane

    2014-01-01

    Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals. PMID:24992038

  19. Schwann cells transduced with a lentiviral vector encoding Fgf-2 promote motor neuron regeneration following sciatic nerve injury.

    PubMed

    Allodi, Ilary; Mecollari, Vasil; González-Pérez, Francisco; Eggers, Ruben; Hoyng, Stefan; Verhaagen, Joost; Navarro, Xavier; Udina, Esther

    2014-10-01

    Fibroblast growth factor 2 (FGF-2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF-2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF-2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF-2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF-2 (LV-FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF-2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV-FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV-FGF2 into the rat sciatic nerve resulted in increased expression of FGF-2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF-2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV-FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV-FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow-up. PMID:24989458

  20. AAV vector-mediated reversal of hypoglycemia in canine and murine glycogen storage disease type Ia.

    PubMed

    Koeberl, Dwight D; Pinto, Carlos; Sun, Baodong; Li, Songtao; Kozink, Daniel M; Benjamin, Daniel K; Demaster, Amanda K; Kruse, Meghan A; Vaughn, Valerie; Hillman, Steven; Bird, Andrew; Jackson, Mark; Brown, Talmage; Kishnani, Priya S; Chen, Yuan-Tsong

    2008-04-01

    Glycogen storage disease type Ia (GSD-Ia) profoundly impairs glucose release by the liver due to glucose-6-phosphatase (G6Pase) deficiency. An adeno-associated virus (AAV) containing a small human G6Pase transgene was pseudotyped with AAV8 (AAV2/8) to optimize liver tropism. Survival was prolonged in 2-week-old G6Pase (-/-) mice by 600-fold fewer AAV2/8 vector particles (vp), in comparison to previous experiments involving this model (2 x 10(9) vp; 3 x 10(11) vp/kg). When the vector was pseudotyped with AAV1, survival was prolonged only at a higher dose (3 x 10(13) vp/kg). The AAV2/8 vector uniquely prevented hypoglycemia during fasting and fully corrected liver G6Pase deficiency in GSD-Ia mice and dogs. The AAV2/8 vector has prolonged survival in three GSD-Ia dogs to >11 months, which validated this strategy in the large animal model for GSD-Ia. Urinary biomarkers, including lactate and 3-hydroxybutyrate, were corrected by G6Pase expression solely in the liver. Glycogen accumulation in the liver was reduced almost to the normal level in vector-treated GSD-Ia mice and dogs, as was the hepatocyte growth factor (HGF) in GSD-Ia mice. These preclinical data demonstrated the efficacy of correcting hepatic G6Pase deficiency, and support the further preclinical development of AAV vector-mediated gene therapy for GSD-Ia. PMID:18362924

  1. Leishmania major survival in selective Phlebotomus papatasi sand fly vector requires a specific SCG-encoded lipophosphoglycan galactosylation pattern.

    PubMed

    Dobson, Deborah E; Kamhawi, Shaden; Lawyer, Phillip; Turco, Salvatore J; Beverley, Stephen M; Sacks, David L

    2010-01-01

    Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both 'poly-scGal' and 'null-scGal' lines survived poorly relative to PpapJ-sympatric L. major FV1 and other 'mono-scGal' lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing 'null-scGal'-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a 'PpapJ-optimal' scGal-LPG PAMP. Unexpectedly, these "L. major FV1-cloaked" L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific 'mono-scGal' pattern. However, failure of 'mono-scGal' L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The

  2. Leishmania major Survival in Selective Phlebotomus papatasi Sand Fly Vector Requires a Specific SCG-Encoded Lipophosphoglycan Galactosylation Pattern

    PubMed Central

    Dobson, Deborah E.; Kamhawi, Shaden; Lawyer, Phillip; Turco, Salvatore J.; Beverley, Stephen M.; Sacks, David L.

    2010-01-01

    Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In “selective” sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the “selective” fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both ‘poly-scGal’ and ‘null-scGal’ lines survived poorly relative to PpapJ-sympatric L. major FV1 and other ‘mono-scGal’ lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing ‘null-scGal’-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a ‘PpapJ-optimal’ scGal-LPG PAMP. Unexpectedly, these “L. major FV1-cloaked” L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific ‘mono-scGal’ pattern. However, failure of ‘mono-scGal’ L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L

  3. Assessment of toxicity and biodistribution of recombinant AAV8 vector-mediated immunomodulatory gene therapy in mice with Pompe disease.

    PubMed

    Wang, Gensheng; Young, Sarah P; Bali, Deeksha; Hutt, Julie; Li, Songtao; Benson, Janet; Koeberl, Dwight D

    2014-01-01

    A preclinical safety study was conducted to evaluate the short- and long-term toxicity of a recombinant adeno-associated virus serotype 8 (AAV2/8) vector that has been developed as an immune-modulatory adjunctive therapy to recombinant human acid α-glucosidase (rhGAA, Myozyme) enzyme replacement treatment (ERT) for patients with Pompe disease (AAV2/8-LSPhGAApA). The AAV2/8-LSPhGAApA vector at 1.6 × 10(13) vector particles/kg, after intravenous injection, did not cause significant short- or long-term toxicity. Recruitment of CD4(+) (but not CD8(+)) lymphocytes to the liver was elevated in the vector-dosed male animals at study day (SD) 15, and in group 8 animals at SD 113, in comparison to their respective control animals. Administration of the vector, either prior to or after the one ERT injection, uniformly prevented the hypersensitivity induced by subsequent ERT in males, but not always in female animals. The vector genome was sustained in all tissues through 16-week postdosing, except for in blood with a similar tissue tropism between males and females. Administration of the vector alone, or combined with the ERT, was effective in producing significantly increased GAA activity and consequently decreased glycogen accumulation in multiple tissues, and the urine biomarker, Glc4, was significantly reduced. The efficacy of the vector (or with ERT) was better in males than in females, as demonstrated both by the number of tissues showing significantly effective responses and the extent of response in a given tissue. Given the lack of toxicity for AAV2/8LSPhGAApA, further consideration of clinical translation is warranted in Pompe disease. PMID:26015962

  4. Virotherapy with a Semliki Forest Virus-Based Vector Encoding IL12 Synergizes with PD-1/PD-L1 Blockade.

    PubMed

    Quetglas, José I; Labiano, Sara; Aznar, M Ángela; Bolaños, Elixabet; Azpilikueta, Arantza; Rodriguez, Inmaculada; Casales, Erkuden; Sánchez-Paulete, Alfonso R; Segura, Víctor; Smerdou, Cristian; Melero, Ignacio

    2015-05-01

    Virotherapy and checkpoint inhibitors can be combined for the treatment of cancer with complementarity and potential for synergistic effects. We have developed a cytolytic but nonreplicative viral vector system based on Semliki Forest virus that encodes IL12 (SFV-IL12). Following direct intratumoral injection, infected cells release transgenic IL12, die, and elicit an inflammatory response triggered by both abundantly copied viral RNA and IL12. In difficult-to-treat mouse cancer models, such as those derived from MC38 and bilateral B16-OVA, SFV-IL12 synergized with an anti-PD-1 monoclonal antibody (mAb) to induce tumor regression and prolong survival. Similar synergistic effects were attained upon PD-L1 blockade. Combined SFV-IL12 + anti-PD-1 mAb treatment only marginally increased the elicited cytotoxic T-lymphocyte response over SFV-IL12 as a single agent, at least when measured by in vivo killing assays. In contrast, we observed that SFV-IL12 treatment induced expression of PD-L1 on tumor cells in an IFNγ-dependent fashion. PD-L1-mediated adaptive resistance thereby provides a mechanistic explanation of the observed synergistic effects achieved by the SFV-IL12 + anti-PD-1 mAb combination. PMID:25691326

  5. Dendritic cell-based vaccination with lentiviral vectors encoding ubiquitinated hepatitis B core antigen enhances hepatitis B virus-specific immune responses in vivo.

    PubMed

    Dai, Shenglan; Zhuo, Meng; Song, Linlin; Chen, Xiaohua; Yu, Yongsheng; Tang, Zhenghao; Zang, Guoqing

    2015-11-01

    The activity of hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) plays a predominant role in the clearance of HBV. Dendritic cells (DCs) are key antigen-presenting cells and play an important role in the initiation of immune responses. We previously verified that lentiviral vector encoding ubiquitinated hepatitis B core antigen (LV-Ub-HBcAg) effectively transduced DCs to induce maturation, and the mature DCs efficiently induced T cell polarization to Th1 and generated HBcAg-specific CTLs ex vivo. In this study, HBV-specific immune responses of LV-Ub-HBcAg in BALB/c mice (H-2Kd) were evaluated. It was shown that direct injection of LV-Ub-HBcAg increased the production of cytokines IL-2 and IFN-γ, elicited strong antibody responses, and remarkably generated a high percentage of IFN-γ+CD8+ T cells with HBV-specific CTL responses in BALB/c mice. In addition, direct injection of LV-Ub-HBcAg induced potent anti-HBV immune responses, similar to those elicited by in vitro-transduced DCs. In conclusion, the DC-based therapeutic vaccine LV-Ub-HBcAg elicited specific antibody immune responses and induced robust specific CTL activity in vivo. PMID:26373843

  6. Overcoming the Cystic Fibrosis Sputum Barrier to Leading Adeno-associated Virus Gene Therapy Vectors

    PubMed Central

    Schuster, Benjamin S; Kim, Anthony J; Kays, Joshua C; Kanzawa, Mia M; Guggino, William B; Boyle, Michael P; Rowe, Steven M; Muzyczka, Nicholas; Suk, Jung Soo; Hanes, Justin

    2014-01-01

    Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes. PMID:24869933

  7. Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2)

    SciTech Connect

    Weger, Stefan . E-mail: stefan.weger@charite.de; Hammer, Eva; Goetz, Anne; Heilbronn, Regine

    2007-05-25

    Through yeast two-hybrid analysis and coimmunoprecipitation studies, we have identified a novel cellular AAV-2 Rep78/Rep68 interaction partner located predominantly in the cytoplasm. In public databases, it has been assigned as KCTD5, because of a region of high similarity to the cytoplasmic tetramerization domain of voltage-gated potassium channels. Whereas Rep/KCTD5 interaction relied on the region surrounding the Rep nuclear localization signal, nuclear accumulation of Rep was not required. Wildtype Rep78/Rep68 proteins induced the translocation of large portions of KCTD5 into the nucleus pointing to functional interactions both in the cytoplasm and the nucleus. In line with an anticipated functional interference in the cytoplasm, KCTD5 overexpression completely abrogated Rep68-mediated posttranscriptional activation of a HIV-LTR driven luciferase reporter gene. Our study expands the panel of already identified nuclear Rep interaction partners to a cytoplasmic protein, which raises the awareness that important steps in the AAV life cycle may be regulated in this compartment.

  8. Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector.

    PubMed

    Blanco, D R; Giladi, M; Champion, C I; Haake, D A; Chikami, G K; Miller, J N; Lovett, M A

    1991-10-01

    Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region. Library construction with Sau3A-digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum-AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences. Triton X-114 detergent phase partitioning of individual T. pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]-palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site. The DNA sequence of Tp75 also indicates that this is a previously unreported T. pallidum lipoprotein. T. pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified. DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant

  9. [Adeno-associated viral vectors: methods for production and purification for gene therapy applications].

    PubMed

    Mena-Enriquez, Mayra; Flores-Contreras, Lucia; Armendáriz-Borunda, Juan

    2012-01-01

    Viral vectors based on adeno-associated virus (AAV) are widely used in gene therapy protocols, because they have characteristics that make them valuable for the treatment of genetic and chronic degenerative diseases. AAV2 serotype had been the best characterized to date. However, the AAV vectors developed from other serotypes is of special interest, since they have organ-specific tropism which increases their potential for transgene delivery to target cells for performing their therapeutic effects. This article summarizes AAV generalities, methods for their production and purification. It also discusses the use of these vectors in vitro, in vivo and their application in gene therapy clinical trials. PMID:23544311

  10. Therapeutic potential of CERE-110 (AAV2-NGF): Targeted, stable, and sustained NGF delivery and trophic activity on rodent basal forebrain cholinergic neurons

    PubMed Central

    Bishop, Kathie M.; Hofer, Eva K.; Mehta, Arpesh; Ramirez, Anthony; Sun, Liangwu; Tuszynski, Mark; Bartus, Raymond T.

    2009-01-01

    Treatment of degenerating basal forebrain cholinergic neurons with nerve growth factor (NGF) in Alzheimer’s disease has long been contemplated, but an effective and safe delivery method has been lacking. Towards achieving this goal, we are currently developing CERE-110, an adeno-associated virus-based gene delivery vector that encodes for human NGF, for stereotactic surgical delivery to the human nucleus basalis of Meynert. Results indicate that NGF transgene delivery to the targeted brain region via CERE-110 is reliable and accurate, that NGF transgene distribution can be controlled by altering CERE-110 dose, and that it is possible to achieve restricted NGF expression limited to but covering the target brain region. Results from animals examined at longer time periods of 3, 6, 9 and 12 months after CERE-110 delivery indicate that NGF transgene expression is stable and sustained at all time points, with no loss or build-up of protein over the long-term. In addition, results from a series of experiments indicate that CERE-110 is neuroprotective and neurorestorative to basal forebrain cholinergic neurons in the rat fimbria-fornix lesion and aged rat models, and has bioactive effects on young rat basal forebrain cholinergic neurons. These findings, as well as those from several additional non-clinical experiments conducted in both rats and monkeys, led to the initiation of a Phase I clinical study to evaluate the safety and efficacy of CERE-110 in Alzheimer’s disease subjects, which is currently ongoing. PMID:18439998

  11. Adeno-Associated Virus Type 5 Utilizes Alternative Translation Initiation To Encode a Small Rep40-Like Protein▿

    PubMed Central

    Farris, K. David; Pintel, David J.

    2010-01-01

    Alternative splicing of adeno-associated virus type 2 (AAV2) P19-generated pre-mRNAs generates the small Rep proteins Rep52 and Rep40, which differ in their carboxyl termini. Both proteins are required for optimal packaging of AAV2 genomes. AAV5 Rep-encoding P19-generated transcripts are primarily polyadenylated within the central intron and not efficiently spliced; however, surprisingly, AAV5 was found to generate high levels of a Rep40-like protein. The AAV5 Rep40-like protein was generated by internal initiation and has the same C terminus as Rep52. Although precluded from using alternative splicing to generate multiple Rep isoforms, AAV5 ensures the production of a Rep40-like protein by utilizing a novel internal translation initiation event. PMID:19889770

  12. The Interdomain Linker of AAV-2 Rep68 Is an Integral Part of Its Oligomerization Domain: Role of a Conserved SF3 Helicase Residue in Oligomerization

    PubMed Central

    Zarate-Perez, Francisco; Bardelli, Martino; Burgner, John W.; Villamil-Jarauta, Maria; Das, Kanni; Kekilli, Demet; Mansilla-Soto, Jorge; Linden, R. Michael; Escalante, Carlos R.

    2012-01-01

    The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains. PMID:22719256

  13. Foamy virus vectors.

    PubMed Central

    Russell, D W; Miller, A D

    1996-01-01

    Human foamy virus (HFV) is a retrovirus of the spumavirus family. We have constructed vectors based on HFV that encode neomycin phosphotransferase and alkaline phosphatase. These vectors are able to transduce a wide variety of vertebrate cells by integration of the vector genome. Unlike vectors based on murine leukemia virus, HFV vectors are not inactivated by human serum, and they transduce stationary-phase cultures more efficiently than murine leukemia virus vectors. These properties, as well as their large packaging capacity, make HFV vectors promising gene transfer vehicles. PMID:8523528

  14. Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors.

    PubMed

    Van der Perren, Anke; Toelen, Jaan; Casteels, Cindy; Macchi, Francesca; Van Rompuy, Anne-Sophie; Sarre, Sophie; Casadei, Nicolas; Nuber, Silke; Himmelreich, Uwe; Osorio Garcia, Maria Isabel; Michotte, Yvette; D'Hooge, Rudi; Bormans, Guy; Van Laere, Koen; Gijsbers, Rik; Van den Haute, Chris; Debyser, Zeger; Baekelandt, Veerle

    2015-03-01

    Testing of new therapeutic strategies for Parkinson's disease (PD) is currently hampered by the lack of relevant and reproducible animal models. Here, we developed a robust rat model for PD by injection of adeno-associated viral vectors (rAAV2/7) encoding α-synuclein into the substantia nigra, resulting in reproducible nigrostriatal pathology and behavioral deficits in a 4-week time period. Progressive dopaminergic dysfunction was corroborated by histopathologic and biochemical analysis, motor behavior testing and in vivo microdialysis. L-DOPA treatment was found to reverse the behavioral phenotype. Non-invasive positron emission tomography imaging and magnetic resonance spectroscopy allowed longitudinal monitoring of neurodegeneration. In addition, insoluble α-synuclein aggregates were formed in this model. This α-synuclein rat model shows improved face and predictive validity, and therefore offers the possibility to reliably test novel therapeutics. Furthermore, it will be of great value for further research into the molecular pathogenesis of PD and the importance of α-synuclein aggregation in the disease process. PMID:25599874

  15. Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

    SciTech Connect

    Puntel, Mariana; Ghulam, Muhammad A.K.M.; Farrokhi, Catherine; VanderVeen, Nathan; Paran, Christopher; Appelhans, Ashley; Kroeger, Kurt M.; Salem, Alireza; Lacayo, Liliana; Pechnick, Robert N.; Kelson, Kyle R.; Kaur, Sukhpreet; Kennedy, Sean; Palmer, Donna; Ng, Philip; and others

    2013-05-01

    Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation study at

  16. Isolation, cloning and expression mapping of a gene encoding an anti-diuretic hormone and other CAPA-related peptides in the disease vector, Rhodnius prolixus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Following a blood meal, Rhodnius prolixus undergoes a rapid diuresis in order to eliminate excess water and salts. During the voiding of this primary urine, R. prolixus acts as a vector of Chagas’ disease, with the causative agent, Trypanosoma cruzi, infecting the human host via the urine. Diuresi...

  17. Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids

    PubMed Central

    Aydemir, Fikret; Salganik, Maxim; Resztak, Justyna; Singh, Jasbir; Bennett, Antonette; Agbandje-McKenna, Mavis

    2016-01-01

    ABSTRACT We previously reported that an amino acid substitution, Y704A, near the 2-fold interface of adeno-associated virus (AAV) was defective for transcription of the packaged genome (M. Salganik, F. Aydemir, H. J. Nam, R. McKenna, M. Agbandje-McKenna, and N. Muzyczka, J Virol 88:1071–1079, 2013, doi: http://dx.doi.org/10.1128/JVI.02093-13). In this report, we have characterized the defect in 6 additional capsid mutants located in a region ∼30 Å in diameter on the surface of the AAV type 2 (AAV2) capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3 to 6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA postinfection. In addition, wild-type (wt) capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody-bound wild-type capsids, were able to enter the cell, travel to the nucleus, uncoat, and synthesize a second strand but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in the transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2-fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome that it delivers to the nucleus. IMPORTANCE Many viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses. A number of nonenveloped viruses, including AAV, carry proteases that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to

  18. Correction of murine hemophilia A following nonmyeloablative transplantation of hematopoietic stem cells engineered to encode an enhanced human factor VIII variant using a safety-augmented retroviral vector

    PubMed Central

    Ramezani, Ali

    2009-01-01

    Insertional mutagenesis by retroviral vectors is a major impediment to the clinical application of hematopoietic stem cell gene transfer for the treatment of hematologic disorders. We recently developed an insulated self-inactivating gammaretroviral vector, RMSinOFB, which uses a novel enhancer-blocking element that significantly decreases genotoxicity of retroviral integration. In this study, we used the RMSinOFB vector to evaluate the efficacy of a newly bioengineered factor VIII (fVIII) variant (efVIII)—containing a combination of A1 domain point mutations (L303E/F309S) and an extended partial B domain for improved secretion plus A2 domain mutations (R484A/R489A/P492A) for reduced immunogenicity—toward successful treatment of murine hemophilia A. In cell lines, efVIII was secreted at up to 6-fold higher levels than an L303E/F309S A1 domain–only fVIII variant (sfVIIIΔB). Most important, when compared with a conventional gammaretroviral vector expressing sfVIIIΔB, lower doses of RMSin-efVIII-OFB–transduced hematopoietic stem cells were needed to generate comparable curative fVIII levels in hemophilia A BALB/c mice after reduced-intensity total body irradiation or nonmyeloablative chemotherapy conditioning regimens. These data suggest that the safety-augmented RMSin-efVIII-OFB platform represents an encouraging step in the development of a clinically appropriate gene addition therapy for hemophilia A. PMID:19470695

  19. Vector-encoded Helicobacter pylori neutrophil-activating protein promotes maturation of dendritic cells with Th1 polarization and improved migration.

    PubMed

    Ramachandran, Mohanraj; Jin, Chuan; Yu, Di; Eriksson, Fredrik; Essand, Magnus

    2014-09-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor involved in H. pylori infection. Both HP-NAP protein and oncolytic viruses encoding HP-NAP have been suggested as immunotherapeutic anticancer agents and adjuvants for vaccination but with little known about its mode of action to activate adaptive immunity. Dendritic cells (DCs) are key players in bridging innate and adaptive immune responses, and in this study we aim to evaluate the effect of HP-NAP on DC maturation, migration, and induction of adaptive immune response. Maturation markers CD83, CD80, CD86, HLA-DR, CD40, and CCR7 were upregulated on human DCs after treatment with supernatants from HP-NAP adenovirus-infected cells. HP-NAP-activated DCs had a Th1 cytokine secretion profile, with high IL-12 and relatively low IL-10 secretion, and migrated toward CCL19. Ag-specific T cells were efficiently expanded by Ag-presenting HP-NAP-activated DCs, which is an important property of functionally mature DCs. Furthermore, intradermal injections of HP-NAP-encoding adenovirus in C57BL/6 mice enhanced resident DC migration to draining lymph nodes, which was verified by imaging lymph nodes by two-photon microscopy and by phenotyping migrating cells by flow cytometry. In conclusion, therapeutic effects of HP-NAP are mediated by maturation of DCs and subsequent activation of Ag-specific T cells in addition to provoking innate immunity. PMID:25049358

  20. Generation of Neutralizing Monoclonal Antibodies against a Conformational Epitope of Human Adenovirus Type 7 (HAdv-7) Incorporated in Capsid Encoded in a HAdv-3-Based Vector

    PubMed Central

    Li, Xiao; Zhou, Zhichao; Li, Chenyang; Zhou, Rong

    2014-01-01

    The generation of monoclonal antibodies (MAbs) by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5) of adenovirus type 7 (HAdv-7) was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA) analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses. PMID:25054273

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

    PubMed Central

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

    2016-01-01

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

  2. Enhancement of Protective Efficacy through Adenoviral Vectored Vaccine Priming and Protein Boosting Strategy Encoding Triosephosphate Isomerase (SjTPI) against Schistosoma japonicum in Mice

    PubMed Central

    Dai, Yang; Wang, Xiaoting; Tang, Jianxia; Zhao, Song; Xing, Yuntian; Dai, Jianrong; Jin, Xiaolin; Zhu, Yinchang

    2015-01-01

    Background Schistosomiasis japonica is a zoonotic parasitic disease; developing transmission blocking veterinary vaccines are urgently needed for the prevention and control of schistosomiasis in China. Heterologous prime-boost strategy, a novel vaccination approach, is more effective in enhancing vaccine efficacy against multiple pathogens. In the present study, we established a novel heterologous prime-boost vaccination strategy, the rAdV-SjTPI.opt intramuscular priming and rSjTPI subcutaneous boosting strategy, and evaluated its protective efficacy against Schistosoma japonicum in mice. Methodology/Principal Findings Adenoviral vectored vaccine (rAdV-SjTPI.opt) and recombinant protein vaccine (rSjTPI) were prepared and used in different combinations as vaccines in a mouse model. The specific immune responses and protective efficacies were evaluated. Furthermore, the longevity of protective efficacy was also determined. Results showed that the rAdV-SjTPI.opt priming-rSjTPI boosting strategy elicited higher levels of specific IgG responses and broad-spectrum specific cellular immune responses. The protective efficacy could reach up to nearly 70% and 50% of protection could be observed at 10 weeks after the last immunization in mice. Conclusions/Significance The rAdV-SjTPI.opt intramuscular priming-rSjTPI subcutaneous boosting vaccination strategy is a novel, highly efficient, and stable approach to developing vaccines against Schistosoma japonicum infections in China. PMID:25793406

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

    PubMed

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

    2016-01-01

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

  4. Lineage analysis of the late otocyst stage mouse inner ear by transuterine microinjection of a retroviral vector encoding alkaline phosphatase and an oligonucleotide library.

    PubMed

    Jiang, Han; Wang, Lingyan; Beier, Kevin T; Cepko, Constance L; Fekete, Donna M; Brigande, John V

    2013-01-01

    The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories). The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5) mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP) and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo. PMID:23935981

  5. A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

    PubMed Central

    Wang, Jin Yuan; Carrasco, Jose A.; Lloyd, Scott A.; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D.; Nataro, James P.; Pasetti, Marcela F.

    2014-01-01

    Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. PMID:25332120

  6. Adenoviral vector encoding soluble Flt-1 engineered human endometrial mesenchymal stem cells effectively regress endometriotic lesions in NOD/SCID mice.

    PubMed

    Koippallil Gopalakrishnan, A R; Pandit, H; Metkari, S M; Warty, N; Madan, T

    2016-07-01

    This study was undertaken to study the efficiency of Adsflt-1 engineered human eutopic mesenchymal stem cells (MSCs) secreting anti-angiogenic sFlt-1 as a targeted cell-based therapy for endometriosis (EM). Eutopic MSCs were transduced with Adsflt-1/AdV0 viral vectors and were evaluated for expression and secretion of sFlt-1. EM was created in NOD/SCID mice using subcutaneous implantation techniques. Four doses of 10(6) MSC-Adsflt-1/MSC-AdV0 were administered to the model and therapeutic anti-angiogenic ability was analyzed by lesion size measurement, microvessel density, immunohistochemistry and real-time reverse transcriptase-PCR analysis. Approximately 86% of transduced MSCs expressed and secreted sFlt-1. MSC-Adsflt-1-treated animals exhibited significant reduction (52.8±1.8%) in size of endometriotic lesions. We observed a 2.3-fold decrease in the number and a 10-fold decrease in the size of endometrial glands in MSC-Adsflt-1-treated animals. A two-fold decrease in stromal cell densities was also observed in MSC-Adsflt-1-treated animals compared with the MSC-AdV0 group. Specific positive immunostaining for MSC marker, CD146 and sFlt-1 in the lesion sites of the MSC-Adsflt-1 group suggests possible homing of transduced MSCs, their survival and secretion of sFlt-1 at the target sites. A marked reduction in size of microvessels and microvessel density within endometriotic lesions and surrounding host subcutaneous layers was observed in MSC-Adsflt-1 group along with significantly downregulated expression of transcripts for vascular endothelial growth factor, fetal liver kinase 1 and matrix metalloproteinases (2 and 9). Our findings indicate the efficacy of a novel eutopic MSC-Adsflt-1 therapy in EM study models. Evaluating long-term effects of genetically modified MSCs in vivo is essential in translating MSC-Adsflt-1 therapy to the clinics. PMID:26990775

  7. A Hypoxia-Responsive Glial Cell–Specific Gene Therapy Vector for Targeting Retinal Neovascularization

    PubMed Central

    Biswal, Manas R.; Prentice, Howard M.; Dorey, C. Kathleen; Blanks, Janet C.

    2014-01-01

    Purpose. Müller cells, the major glial cell in the retina, play a significant role in retinal neovascularization in response to tissue hypoxia. We previously designed and tested a vector using a hypoxia-responsive domain and a glial fibrillary acidic protein (GFAP) promoter to drive green fluorescent protein (GFP) expression in Müller cells in the murine model of oxygen-induced retinopathy (OIR). This study compares the efficacy of regulated and unregulated Müller cell delivery of endostatin in preventing neovascularization in the OIR model. Methods. Endostatin cDNA was cloned into plasmids with hypoxia-regulated GFAP or unregulated GFAP promoters, and packaged into self-complementary adeno-associated virus serotype 2 vectors (scAAV2). Before placement in hyperoxia on postnatal day (P)7, mice were given intravitreal injections of regulated or unregulated scAAV2, capsid, or PBS. Five days after return to room air, on P17, neovascular and avascular areas, as well as expression of the transgene and vascular endothelial growth factor (VEGF), were compared in OIR animals treated with a vector, capsid, or PBS. Results. The hypoxia-regulated, glial-specific, vector-expressing endostatin reduced neovascularization by 93% and reduced the central vaso-obliteration area by 90%, matching the results with the unregulated GFAP-Endo vector. Retinas treated with the regulated endostatin vector expressed substantial amounts of endostatin protein, and significantly reduced VEGF protein. Endostatin production from the regulated vector was undetectable in retinas with undamaged vasculature. Conclusions. These findings suggest that the hypoxia-regulated, glial cell–specific vector expressing endostatin may be useful for treatment of neovascularization in proliferative diabetic retinopathy. PMID:25377223

  8. Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer

    PubMed Central

    Kwon, Inchan

    2007-01-01

    Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. However, several problems should be addressed to enhance the utility of AAV vectors, particularly those based on AAV2, the best characterized AAV serotype. First, altering viral tropism would be advantageous for broadening its utility in various tissue or cell types. In response to this need, vector pseudotyping, mosaic capsids, and targeting ligand insertion into the capsid have shown promise for altering AAV specificity. In addition, library selection and directed evolution have recently emerged as promising approaches to modulate AAV tropism despite limited knowledge of viral structure–function relationships. Second, pre-existing immunity to AAV must be addressed for successful clinical application of AAV vectors. “Shielding” polymers, site-directed mutagenesis, and alternative AAV serotypes have shown success in avoiding immune neutralization. Furthermore, directed evolution of the AAV capsid is a high throughput approach that has yielded vectors with substantial resistance to neutralizing antibodies. Molecular engineering and directed evolution of AAV vectors therefore offer promise for generating ‘designer’ gene delivery vectors with enhanced properties. PMID:17763830

  9. Encoding Dictionaries.

    ERIC Educational Resources Information Center

    Ide, Nancy

    1995-01-01

    Describes problems in devising a Text Encoding Initiative (TEI) encoding format for dictionaries. Asserts that the high degree of structuring and compression of information are among the most complex text types treated in the TEI. Concludes that the source of some TEI problems lies in the design of Standard Generalized Markup Language (SGML). (CFR)

  10. Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness.

    PubMed

    Scalabrino, Miranda L; Boye, Sanford L; Fransen, Kathryn M H; Noel, Jennifer M; Dyka, Frank M; Min, Seok Hong; Ruan, Qing; De Leeuw, Charles N; Simpson, Elizabeth M; Gregg, Ronald G; McCall, Maureen A; Peachey, Neal S; Boye, Shannon E

    2015-11-01

    Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1. PMID:26310623

  11. Introducing Vectors.

    ERIC Educational Resources Information Center

    Roche, John

    1997-01-01

    Suggests an approach to teaching vectors that promotes active learning through challenging questions addressed to the class, as opposed to subtle explanations. Promotes introducing vector graphics with concrete examples, beginning with an explanation of the displacement vector. Also discusses artificial vectors, vector algebra, and unit vectors.…

  12. Deciphering ENCODE.

    PubMed

    Diehl, Adam G; Boyle, Alan P

    2016-04-01

    The ENCODE project represents a major leap from merely describing and comparing genomic sequences to surveying them for direct indicators of function. The astounding quantity of data produced by the ENCODE consortium can serve as a map to locate specific landmarks, guide hypothesis generation, and lead us to principles and mechanisms underlying genome biology. Despite its broad appeal, the size and complexity of the repository can be intimidating to prospective users. We present here some background about the ENCODE data, survey the resources available for accessing them, and describe a few simple principles to help prospective users choose the data type(s) that best suit their needs, where to get them, and how to use them to their best advantage. PMID:26962025

  13. Human adenovirus 5-vectored Plasmodium falciparum NMRC-M3V-Ad-PfCA vaccine encoding CSP and AMA1 is safe, well-tolerated and immunogenic but does not protect against controlled human malaria infection

    PubMed Central

    Tamminga, Cindy; Sedegah, Martha; Maiolatesi, Santina; Fedders, Charlotte; Reyes, Sharina; Reyes, Anatalio; Vasquez, Carlos; Alcorta, Yolanda; Chuang, Ilin; Spring, Michele; Kavanaugh, Michael; Ganeshan, Harini; Huang, Jun; Belmonte, Maria; Abot, Esteban; Belmonte, Arnel; Banania, JoGlenna; Farooq, Fouzia; Murphy, Jittawadee; Komisar, Jack; Richie, Nancy O; Bennett, Jason; Limbach, Keith; Patterson, Noelle B; Bruder, Joseph T; Shi, Meng; Miller, Edward; Dutta, Sheetij; Diggs, Carter; Soisson, Lorraine A; Hollingdale, Michael R; Epstein, Judith E; Richie, Thomas L

    2013-01-01

    Background: In a prior study, a DNA prime / adenovirus boost vaccine (DNA/Ad) expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) (NMRC-M3V-D/Ad-PfCA Vaccine) induced 27% protection against controlled human malaria infection (CHMI). To investigate the contribution of DNA priming, we tested the efficacy of adenovirus vaccine alone (NMRC-M3V-Ad-PfCA ) in a Phase 1 clinical trial. Methodology/Principal Findings: The regimen was a single intramuscular injection with two non-replicating human serotype 5 adenovectors encoding CSP and AMA1, respectively. One x 1010 particle units of each construct were combined prior to administration. The regimen was safe and well-tolerated. Four weeks later, 18 study subjects received P. falciparum CHMI administered by mosquito bite. None were fully protected although one showed delayed onset of parasitemia. Antibody responses were low, with geometric mean CSP ELISA titer of 381 (range < 50–1626) and AMA1 ELISA of 4.95 µg/mL (range 0.2–38). Summed ex vivo IFN-γ ELISpot responses to overlapping peptides were robust, with geometric mean spot forming cells/million peripheral blood mononuclear cells [sfc/m] for CSP of 273 (range 38–2550) and for AMA1 of 1303 (range 435–4594). CD4+ and CD8+ T cell IFN-γ responses to CSP were positive by flow cytometry in 25% and 56% of the research subjects, respectively, and to AMA1 in 94% and 100%, respectively. Significance: In contrast to DNA/Ad, Ad alone did not protect against CHMI despite inducing broad, cell-mediated immunity, indicating that DNA priming is required for protection by the adenovirus-vectored vaccine. ClinicalTrials.gov Identifier: NCT00392015. PMID:23899517

  14. Adeno-associated viral vectors for clinical gene transfer studies.

    PubMed

    Snyder, Richard O; Francis, Joyce

    2005-06-01

    Recombinant adeno-associated viral (rAAV) vectors can mediate the safe and long-term correction of genetic diseases in animal models following a single administration. These pre-clinical studies are the basis of human trials that have shown rAAV vector persistence and safety in humans following delivery to lung, sinus, skeletal muscle, brain and liver. Transient disease correction has also been demonstrated in humans treated for hemophilia B and cystic fibrosis using AAV2 vectors. The physiochemical properties of rAAV vector virions are amenable to industry accepted manufacturing methodologies, long-term storage and direct in vivo administration. Recombinant adeno-associated virus vectors are manufactured in compliance with current Good Manufacturing Practices (cGMPs) as outlined in the Code of Federal Regulations (21CFR). To meet these requirements, manufacturing controls and quality systems are established, including 1) adequate facilities and equipment, 2) personnel who have relevant education or experience and are trained for specific assigned duties, 3) raw materials that are qualified for use and 4) a process (including production, purification, formulation, filling, storage and shipping) that is controlled, aseptic, reliable and consistent. Quality systems including Quality Control (QC) and Quality Assurance (QA) are also implemented. These manufacturing procedures and quality systems are designed so the product meets its release specifications to ensure that patients receive a safe, pure, potent and stable investigational drug. PMID:15975008

  15. Cellobiohydrolase variants and polynucleotides encoding same

    SciTech Connect

    Wogulis, Mark

    2014-10-14

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  16. Cellobiohydrolase variants and polynucleotides encoding same

    DOEpatents

    Wogulis, Mark

    2013-09-24

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  17. Cellobiohydrolase variants and polynucleotides encoding the same

    DOEpatents

    Wogulis, Mark

    2014-09-09

    The present invention relates to variants of a parent cellobiohydrolase. The present invention also relates to polynucleotides encoding the cellobiohydrolase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the cellobiohydrolase variants.

  18. Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

    PubMed Central

    Ling, Chen; Yin, Zifei; Li, Jun; Zhang, Daniel; Aslanidi, George; Srivastava, Arun

    2016-01-01

    Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs) from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ~10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492) were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of homologous Rep

  19. Retinal transduction profiles by high-capacity viral vectors

    PubMed Central

    Puppo, Agostina; Cesi, Giulia; Marrocco, Elena; Piccolo, Pasquale; Jacca, Sarah; Shayakhmetov, Dmitry M.; Parks, Robin J.; Davidson, Beverly L.; Colloca, Stefano; Brunetti-Pierri, Nicola; Ng, Philip; Donofrio, Gaetano; Auricchio, Alberto

    2014-01-01

    Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, the limited cargo capacity of AAV prevents their use for therapy of those inherited retinopathies (IRs) due to mutations in large (>5kb) genes. Viral vectors derived from Adenovirus (Ad), Lentivirus (LV) and Herpesvirus (HV) can package large DNA sequences but do not target efficiently retinal photoreceptors (PRs) where the majority of genes responsible for IRs are expressed. Here, we have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes, and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium (RPE). We found that LV-GP64 tends to transduce more PRs than the canonical LV-VSVG albeit this was restricted to a narrow region. We observed more extensive PR transduction with HdAd1, 2 and 5/F35++ than with LV, although none of them outperformed the canonical HdAd5 or matched the extension of PR transduction achieved with AAV2/8. PMID:24989814

  20. Prophylactic Administration of Vector-Encoded Porcine Granulocyte-Colony Stimulating Factor Reduces Salmonella Shedding, Tonsil Colonization, and Microbiota Alterations of the Gastrointestinal Tract in Salmonella-Challenged Swine

    PubMed Central

    Bearson, Shawn M. D.; Bearson, Bradley L.; Loving, Crystal L.; Allen, Heather K.; Lee, InSoo; Madson, Darin; Kehrli, Marcus E.

    2016-01-01

    Salmonella colonization of food animals is a concern for animal health and public health as a food safety risk. Various obstacles impede the effort to reduce asymptomatic Salmonella carriage in food animals, including the existence of numerous serovars and the ubiquitous nature of Salmonella. To develop an intervention strategy that is non-specific yet effective against diverse Salmonella serovars, we explored the prophylactic use of a cytokine to decrease Salmonella in swine by boosting the host’s innate immune system. Granulocyte-colony stimulating factor (G-CSF) is the major cytokine regulating the production, differentiation, function, and survival of neutrophils. Neutrophils play a critical role in the response to Salmonella; therefore, we evaluated the vectored-delivery of porcine G-CSF as a prophylactic to reduce Salmonella in pigs. Crossbred pigs, 5 weeks of age, were intramuscularly injected with a replication-defective human adenovirus (Ad5) engineered to express porcine G-CSF (Ad5-G-CSF, n = 9). Control pigs received the same Ad5 vector lacking the gene encoding G-CSF (Ad5-empty, n = 7). Four days later, all pigs (n = 16) were intranasally inoculated with 1 × 107 colony forming unit (CFU) of Salmonella enterica serovar Typhimurium UK1. At 2 and 3 days post-challenge with Salmonella, Ad5-G-CSF-treated pigs shed significantly less Salmonella (~103 CFU/g) in their feces than Ad5-empty-treated pigs (~104–105 CFU/g; P < 0.05). A significant 4-log reduction in tonsil colonization was also observed in the Ad5-G-CSF-treated pigs at 7 days post-challenge (P < 0.05). In the gastrointestinal tract, the Peyer’s patch region of the ileum exhibited a significant 0.5-log reduction in colonization in the Ad5-G-CSF-treated pigs (P < 0.05). The microbiota of all challenged pigs was assessed by sequencing and analyzing the V1–V3 region of the 16S rRNA gene from fecal DNA samples. The microbial community structure of

  1. Prophylactic Administration of Vector-Encoded Porcine Granulocyte-Colony Stimulating Factor Reduces Salmonella Shedding, Tonsil Colonization, and Microbiota Alterations of the Gastrointestinal Tract in Salmonella-Challenged Swine.

    PubMed

    Bearson, Shawn M D; Bearson, Bradley L; Loving, Crystal L; Allen, Heather K; Lee, InSoo; Madson, Darin; Kehrli, Marcus E

    2016-01-01

    Salmonella colonization of food animals is a concern for animal health and public health as a food safety risk. Various obstacles impede the effort to reduce asymptomatic Salmonella carriage in food animals, including the existence of numerous serovars and the ubiquitous nature of Salmonella. To develop an intervention strategy that is non-specific yet effective against diverse Salmonella serovars, we explored the prophylactic use of a cytokine to decrease Salmonella in swine by boosting the host's innate immune system. Granulocyte-colony stimulating factor (G-CSF) is the major cytokine regulating the production, differentiation, function, and survival of neutrophils. Neutrophils play a critical role in the response to Salmonella; therefore, we evaluated the vectored-delivery of porcine G-CSF as a prophylactic to reduce Salmonella in pigs. Crossbred pigs, 5 weeks of age, were intramuscularly injected with a replication-defective human adenovirus (Ad5) engineered to express porcine G-CSF (Ad5-G-CSF, n = 9). Control pigs received the same Ad5 vector lacking the gene encoding G-CSF (Ad5-empty, n = 7). Four days later, all pigs (n = 16) were intranasally inoculated with 1 × 10(7) colony forming unit (CFU) of Salmonella enterica serovar Typhimurium UK1. At 2 and 3 days post-challenge with Salmonella, Ad5-G-CSF-treated pigs shed significantly less Salmonella (~10(3) CFU/g) in their feces than Ad5-empty-treated pigs (~10(4)-10(5) CFU/g; P < 0.05). A significant 4-log reduction in tonsil colonization was also observed in the Ad5-G-CSF-treated pigs at 7 days post-challenge (P < 0.05). In the gastrointestinal tract, the Peyer's patch region of the ileum exhibited a significant 0.5-log reduction in colonization in the Ad5-G-CSF-treated pigs (P < 0.05). The microbiota of all challenged pigs was assessed by sequencing and analyzing the V1-V3 region of the 16S rRNA gene from fecal DNA samples. The microbial community structure of

  2. The state of the art of adeno-associated virus-based vectors in gene therapy

    PubMed Central

    Coura, Renata dos Santos; Nardi, Nance Beyer

    2007-01-01

    The adeno-associated virus (AAV) has rapidly gained popularity in gene therapy since the establishment of the first AAV2 infectious clone, in 1982, due to some of their distinguishing characteristics such as lack of pathogenicity, wide range of infectivity, and ability to establish long-term transgene expression. Notably over the past decade, this virus has attracted considerable interest as a gene therapy vector, and about 85% of the currently available 2,041 PubMed references on adeno-associated viruses have been published during this time. The exponential progress of AAV-based vectors has been made possible by the advances in the knowledge of the virology and biology of this virus, which allows great improvement in AAV vectors construction and a better comprehension of their operation. Moreover, with the recent discovery of novel AAV serotypes, there is virtually one preferred serotype for nearly every organ or tissue to target. Thus, AAV-based vectors have been successfully overcoming the main gene therapy challenges such as transgene maintenance, safety and host immune response, and meeting the desirable vector system features of high level of safety combined with clinical efficacy and versatility in terms of potential applications. Consequently, AAV is increasingly becoming the vector of choice for a wide range of gene therapy approaches. This report will highlight the state of the art of AAV-based vectors studies and the advances on the use of AAV vectors for several gene therapy approaches. PMID:17939872

  3. AAV vector encoding human VEGF165–transduced pectineus muscular flaps increase the formation of new tissue through induction of angiogenesis in an in vivo chamber for tissue engineering: A technique to enhance tissue and vessels in microsurgically engineered tissue

    PubMed Central

    Moimas, Silvia; Manasseri, Benedetto; Cuccia, Giuseppe; Stagno d’Alcontres, Francesco; Geuna, Stefano; Pattarini, Lucia; Zentilin, Lorena; Giacca, Mauro; Colonna, Michele R

    2015-01-01

    In regenerative medicine, new approaches are required for the creation of tissue substitutes, and the interplay between different research areas, such as tissue engineering, microsurgery and gene therapy, is mandatory. In this article, we report a modification of a published model of tissue engineering, based on an arterio-venous loop enveloped in a cross-linked collagen–glycosaminoglycan template, which acts as an isolated chamber for angiogenesis and new tissue formation. In order to foster tissue formation within the chamber, which entails on the development of new vessels, we wondered whether we might combine tissue engineering with a gene therapy approach. Based on the well-described tropism of adeno-associated viral vectors for post-mitotic tissues, a muscular flap was harvested from the pectineus muscle, inserted into the chamber and transduced by either AAV vector encoding human VEGF165 or AAV vector expressing the reporter gene β-galactosidase, as a control. Histological analysis of the specimens showed that muscle transduction by AAV vector encoding human VEGF165 resulted in enhanced tissue formation, with a significant increase in the number of arterioles within the chamber in comparison with the previously published model. Pectineus muscular flap, transduced by adeno-associated viral vectors, acted as a source of the proangiogenic factor vascular endothelial growth factor, thus inducing a consistent enhancement of vessel growth into the newly formed tissue within the chamber. In conclusion, our present findings combine three different research fields such as microsurgery, tissue engineering and gene therapy, suggesting and showing the feasibility of a mixed approach for regenerative medicine. PMID:26848383

  4. Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector.

    PubMed

    Grieger, Joshua C; Soltys, Stephen M; Samulski, Richard Jude

    2016-02-01

    Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 10(5) vector genome containing particles (vg)/cell or greater than 1 × 10(14) vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 10(13) vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal

  5. The structure of AAVrh32.33, a Novel Gene Delivery Vector

    PubMed Central

    Mikals, Kyle; Nam, Hyun-Joo; Vliet, Kim Van; Vandenberghe, Luk H.; Mays, Lauren E.; McKenna, Robert; Wilson, James M.; Agbandje-McKenna, Mavis

    2014-01-01

    The Adeno-Associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ~40–70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded β-barrel core and αA helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral two-fold axis, three protrusions surrounding the three-fold axis, and a depression surround a cylindrical channel at the five-fold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ~61%, ~81%, and ~63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system. PMID:24704217

  6. A recursive technique for adaptive vector quantization

    NASA Technical Reports Server (NTRS)

    Lindsay, Robert A.

    1989-01-01

    Vector Quantization (VQ) is fast becoming an accepted, if not preferred method for image compression. The VQ performs well when compressing all types of imagery including Video, Electro-Optical (EO), Infrared (IR), Synthetic Aperture Radar (SAR), Multi-Spectral (MS), and digital map data. The only requirement is to change the codebook to switch the compressor from one image sensor to another. There are several approaches for designing codebooks for a vector quantizer. Adaptive Vector Quantization is a procedure that simultaneously designs codebooks as the data is being encoded or quantized. This is done by computing the centroid as a recursive moving average where the centroids move after every vector is encoded. When computing the centroid of a fixed set of vectors the resultant centroid is identical to the previous centroid calculation. This method of centroid calculation can be easily combined with VQ encoding techniques. The defined quantizer changes after every encoded vector by recursively updating the centroid of minimum distance which is the selected by the encoder. Since the quantizer is changing definition or states after every encoded vector, the decoder must now receive updates to the codebook. This is done as side information by multiplexing bits into the compressed source data.

  7. Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

    PubMed

    Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

    2015-03-01

    Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. PMID:25486083

  8. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  9. Safety, Biodistribution, and Efficacy of an AAV-5 Vector Encoding Human Interferon-Beta (ART-I02) Delivered via Intra-Articular Injection in Rhesus Monkeys with Collagen-Induced Arthritis.

    PubMed

    Bevaart, Lisette; Aalbers, Caroline J; Vierboom, Michel P M; Broekstra, Niels; Kondova, Ivanela; Breedveld, Elia; Hauck, Bernd; Wright, J Fraser; Tak, Paul Peter; Vervoordeldonk, Margriet J

    2015-06-01

    Preclinical studies to assess biodistribution, safety, and initial efficacy of ART-I02, an adeno-associated type 5 (rAAV5) vector expressing human interferon β (hIFN-β), were performed in a total of 24 rhesus monkeys with collagen-induced arthritis. All monkeys were naïve or showed limited neutralizing antibody (Nab) titers to AAV5 at the start of the study. Animals were injected with a single intra-articular dose of ART-I02 or placebo, consisting of 3.2×10(13) vg (Dose A=maximum feasible dose), 4.58×10(12) vg (Dose B), or placebo in the first affected finger joint, the ipsilateral knee, and ankle joint at the same time point. Animals were monitored for clinical parameters and well-being with a maximum of 4 weeks, with the option that the severity of arthritis could necessitate an earlier time point of sacrifice. No adverse events were noted after injection of ART-I02. No abnormalities were observed after histological evaluation of all organs. At both dose levels, immunohistochemical staining indicated expression of hIFN-β. In animals injected with Dose A, we observed stabilization or a reduction in swelling in the finger joint in which vector was administered. The highest copy numbers of vector DNA were detected in synovial tissue of the injected joint and the draining lymph node of the injected knee. High titers of Nab to rAAV5 were observed at the end of the study. Five monkeys developed an rAAV5-specific T-cell response. Two monkeys developed Nab to hIFN-β. In conclusion, intra-articular injection of ART-I02 was well-tolerated and did not induce adverse events. After administration of Dose A of ART-I02, we observed a beneficial effect on joint swelling, substantiated by decreased histological inflammation and bone erosion scores. A GMP vector for clinical application has been manufactured and is currently being tested in GLP rodent studies, with the aim to move forward to a clinical trial. PMID:26086763

  10. Vector Video

    NASA Astrophysics Data System (ADS)

    Taylor, David P.

    2001-01-01

    Vector addition is an important skill for introductory physics students to master. For years, I have used a fun example to introduce vector addition in my introductory physics classes based on one with which my high school physics teacher piqued my interest many years ago.

  11. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Zhang, Yu; Tang, Lan; Henriksen, Svend Hostgaard Bang

    2016-05-17

    The present invention provides isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2015-07-14

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Tang, Lan

    2015-07-14

    The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  14. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-11-17

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-11-17

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-10-27

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-03-10

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-15

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  19. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-10-14

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-07-14

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-02-10

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-03-31

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  3. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-08-18

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  4. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-06-24

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  5. Polypeptides having endoglucanse activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-08

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  6. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-08

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-06-24

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  8. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-15

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

    2015-06-09

    Provided are isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having endoglucanase activity and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Duan, Junxin; Tang, Lan

    2015-09-22

    The present invention provides isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cell comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Polypeptides having cellobiohydrolase activitiy and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Tang, Lan; Duan, Junxin

    2015-12-15

    The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2016-06-28

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Cloning vector

    DOEpatents

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

    1994-12-27

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

  14. Cloning vector

    DOEpatents

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

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

  15. Equivalent Vectors

    ERIC Educational Resources Information Center

    Levine, Robert

    2004-01-01

    The cross-product is a mathematical operation that is performed between two 3-dimensional vectors. The result is a vector that is orthogonal or perpendicular to both of them. Learning about this for the first time while taking Calculus-III, the class was taught that if AxB = AxC, it does not necessarily follow that B = C. This seemed baffling. The…

  16. Vector quantization

    NASA Technical Reports Server (NTRS)

    Gray, Robert M.

    1989-01-01

    During the past ten years Vector Quantization (VQ) has developed from a theoretical possibility promised by Shannon's source coding theorems into a powerful and competitive technique for speech and image coding and compression at medium to low bit rates. In this survey, the basic ideas behind the design of vector quantizers are sketched and some comments made on the state-of-the-art and current research efforts.

  17. Nucleic acid encoding TGF-. beta. and its uses

    SciTech Connect

    Derynck, R.M.A.; Goeddel, D.V.

    1989-12-12

    This patent describes a method. It comprises: constructing a vector which includes nucleic acid encoding biologically active TGF-{beta}, transforming a host eukaryotic cell with the vector, culturing the transformed cell and recovering mature TGF-{beta} from the culture medium.

  18. DNA encoding for plant digalactosyldiacylglycerol galactosyltransferase and methods of use

    DOEpatents

    Benning, Christoph; Doermann, Peter

    2003-11-04

    The cDNA encoding digalactosyldiacylglycerol galactosyltransferase (DGD1) is provided. The deduced amino acid sequence is also provided. Methods of making and using DGD1 to screen for new herbicides and alter a plant's leaf lipid composition are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors.

  19. Method and system for efficient video compression with low-complexity encoder

    NASA Technical Reports Server (NTRS)

    Chen, Jun (Inventor); He, Dake (Inventor); Jagmohan, Ashish (Inventor); Lu, Ligang (Inventor); Sheinin, Vadim (Inventor)

    2012-01-01

    Disclosed are a method and system for video compression, wherein the video encoder has low computational complexity and high compression efficiency. The disclosed system comprises a video encoder and a video decoder, wherein the method for encoding includes the steps of converting a source frame into a space-frequency representation; estimating conditional statistics of at least one vector of space-frequency coefficients; estimating encoding rates based on the said conditional statistics; and applying Slepian-Wolf codes with the said computed encoding rates. The preferred method for decoding includes the steps of; generating a side-information vector of frequency coefficients based on previously decoded source data, encoder statistics, and previous reconstructions of the source frequency vector; and performing Slepian-Wolf decoding of at least one source frequency vector based on the generated side-information, the Slepian-Wolf code bits and the encoder statistics.

  20. Immunization with a Recombinant Expression Vector Encoding NS3/NS4A of Hepatitis C Virus Genotype 3a Elicits Cell-Mediated Immune Responses in C57BL/6 Mice.

    PubMed

    Behzadi, Mohammad Amin; Alborzi, Abdolvahab; Kalani, Mehdi; Pouladfar, Gholamreza; Dianatpour, Mehdi; Ziyaeyan, Mazyar

    2016-04-01

    Today, hepatitis C virus (HCV) infection is considered as one of the most significant international health concerns. Although novel therapeutic regimens against the infection have shown satisfactory results, no approved vaccine exists yet. This study aimed to evaluate the immunogenicity of a DNA vaccine candidate for HCV-3a, based on nonstructural proteins NS3/NS4A, in C57BL/6 mice. Immunogenicity effect of pDisplay-NS3/NS4A was analyzed through immunization with 100 and 200 μg concentrations of the construct with complete Freund's adjuvant, monophosphoryl lipid A (MPL), or without adjuvant. The frequencies of different splenic mononuclear cells were measured using the Mouse Th1/Th2/Th17 Phenotyping Kit. Moreover, the number of T-CD8(+) cells was determined using conjugated anti-CD8a and anti-CD3e antibodies by flow cytometry. As observed, the frequencies of Th1, T-CD8(+), and Th2 cells increased in all the experimental groups, compared with the controls. The highest levels of the respective cells were seen in the group immunized with 200 μg of the construct with MPL. Also, there were positive correlations between the frequency of Th1 cells and those of Th2 and T-CD8(+) cells in all the immunized groups, but were significant in those receiving adjuvants. The frequency of Th17 cells did not statistically change among the groups. Taken together, our findings revealed that the constructed DNA vaccine encoding HCV-3a NS3/NS4A gene induces the cell-mediated immune responses significantly. However, its coadministration with adjuvants exhibits more efficient results than the recombinant plasmid alone. Further study is currently underway to evaluate the specific immune responses and recognize the responsible antigenic epitopes. PMID:26909520

  1. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  2. ENCODE data at the ENCODE portal.

    PubMed

    Sloan, Cricket A; Chan, Esther T; Davidson, Jean M; Malladi, Venkat S; Strattan, J Seth; Hitz, Benjamin C; Gabdank, Idan; Narayanan, Aditi K; Ho, Marcus; Lee, Brian T; Rowe, Laurence D; Dreszer, Timothy R; Roe, Greg; Podduturi, Nikhil R; Tanaka, Forrest; Hong, Eurie L; Cherry, J Michael

    2016-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments. PMID:26527727

  3. ENCODE data at the ENCODE portal

    PubMed Central

    Sloan, Cricket A.; Chan, Esther T.; Davidson, Jean M.; Malladi, Venkat S.; Strattan, J. Seth; Hitz, Benjamin C.; Gabdank, Idan; Narayanan, Aditi K.; Ho, Marcus; Lee, Brian T.; Rowe, Laurence D.; Dreszer, Timothy R.; Roe, Greg; Podduturi, Nikhil R.; Tanaka, Forrest; Hong, Eurie L.; Cherry, J. Michael

    2016-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments. PMID:26527727

  4. Highly Efficient Delivery of Adeno-Associated Viral Vectors to the Primate Retina.

    PubMed

    Boye, Shannon E; Alexander, John J; Witherspoon, C Douglas; Boye, Sanford L; Peterson, James J; Clark, Mark E; Sandefer, Kristen J; Girkin, Chris A; Hauswirth, William W; Gamlin, Paul D

    2016-08-01

    Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and

  5. Impact of Preexisting Vector Immunity on the Efficacy of Adeno-Associated Virus-Based HIV-1 Gag Vaccines

    PubMed Central

    Lin, Jianping; Calcedo, Roberto; Vandenberghe, Luk H.; Figueredo, Joanita M.

    2008-01-01

    Abstract Vectors based on primate-derived adeno-associated virus (AAV) are being considered in the development of genetic vaccines against a number of diseases including infection with HIV-1. Preexisting immunity to the vaccine carrier as a result of natural infections could potentially compromise vaccine efficacy. This study evaluates the impact of neutralizing antibodies against AAV capsids on the ability of HIV-1 Gag-expressing vectors to elicit transgene-specific T and B cell responses. Mice were passively transferred with pooled human immunoglobulin at various doses to simulate human antivector humoral immunity. Vectors based on serotype 2, which were evaluated in the clinic, were compared with those created from the novel monkey isolates AAV7 and AAV8. Inhibition of AAV2-directed Gag responses occurred at doses of human immunoglobulin 10- to 20-fold less than was required to inhibit immunogenicity of AAV7 and AAV8 vectors. Cynomolgus macaques were screened for preexisting immunity to AAV7 and AAV8 and sera from individual animals were passively transferred into mice that were analyzed for AAV vaccine efficacy. There was a correlation between the level of preexisting capsid neutralizing titers and diminution of vaccine efficacy; sera from a number of animals with no detectable neutralizing antibodies showed partial vaccine inhibition, suggesting that the in vitro assay is less sensitive than the in vivo passive transfer assay for detecting neutralizing antibodies to AAV. PMID:18549307

  6. Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle

    PubMed Central

    Léger, Adrien; Le Guiner, Caroline; Nickerson, Michael L.; McGee Im, Kate; Ferry, Nicolas; Moullier, Philippe; Snyder, Richard O.; Penaud-Budloo, Magalie

    2011-01-01

    Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (IM) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2–3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the IM or the intravenous (IV) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model. PMID:21687632

  7. Noise level and MPEG-2 encoder statistics

    NASA Astrophysics Data System (ADS)

    Lee, Jungwoo

    1997-01-01

    Most software in the movie and broadcasting industries are still in analog film or tape format, which typically contains random noise that originated from film, CCD camera, and tape recording. The performance of the MPEG-2 encoder may be significantly degraded by the noise. It is also affected by the scene type that includes spatial and temporal activity. The statistical property of noise originating from camera and tape player is analyzed and the models for the two types of noise are developed. The relationship between the noise, the scene type, and encoder statistics of a number of MPEG-2 parameters such as motion vector magnitude, prediction error, and quant scale are discussed. This analysis is intended to be a tool for designing robust MPEG encoding algorithms such as preprocessing and rate control.

  8. Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

    PubMed

    Raissadati, Alireza; Jokinen, Janne J; Syrjälä, Simo O; Keränen, Mikko A I; Krebs, Rainer; Tuuminen, Raimo; Arnaudova, Ralica; Rouvinen, Eeva; Anisimov, Andrey; Soronen, Jarkko; Pajusola, Katri; Alitalo, Kari; Nykänen, Antti I; Lemström, Karl

    2013-11-01

    Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies. PMID:24102821

  9. Vector adaptive predictive coder for speech and audio

    NASA Technical Reports Server (NTRS)

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

    1990-01-01

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

  10. Image compression using address-vector quantization

    NASA Astrophysics Data System (ADS)

    Nasrabadi, Nasser M.; Feng, Yushu

    1990-12-01

    A novel vector quantization scheme, the address-vector quantizer (A-VQ), is proposed which exploits the interblock correlation by encoding a group of blocks together using an address-codebook (AC). The AC is a set of address-codevectors (ACVs), each representing a combination of addresses or indices. Each element of the ACV is an address of an entry in the LBG-codebook, representing a vector-quantized block. The AC consists of an active (addressable) region and an inactive (nonaddressable) region. During encoding the ACVs in the AC are reordered adaptively to bring the most probable ACVs into the active region. When encoding an ACV, the active region is checked, and if such an address combination exists, its index is transmitted to the receiver. Otherwise, the address of each block is transmitted individually. The SNR of the images encoded by the A-VQ method is the same as that of a memoryless vector quantizer, but the bit rate is by a factor of approximately two.

  11. Enhanced Efficacy from Gene Therapy in Pompe Disease Using Coreceptor Blockade

    PubMed Central

    Han, Sang-oh; Li, Songtao; Brooks, Elizabeth D.; Masat, Elisa; Leborgne, Christian; Banugaria, Suhrad; Bird, Andrew; Mingozzi, Federico; Waldmann, Herman

    2015-01-01

    Abstract Enzyme replacement therapy (ERT) is the standard-of-care treatment of Pompe disease, a lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA). One limitation of ERT with recombinant human (rh) GAA is antibody formation against GAA. Similarly, in adeno-associated virus (AAV) vector-mediated gene transfer for Pompe disease, development of antibodies against the GAA transgene product and the AAV vector prevents therapeutic efficacy and vector readministration, respectively. Here a nondepleting anti-CD4 monoclonal antibody (mAb) was administrated intravenously prior to administration of an AAV2/9 vector encoding GAA to suppress anti-GAA responses, leading to a substantial reduction of anti-GAA immunoglobulins, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3. Transduction efficiency in liver with a subsequent AAV2/8 vector was massively improved by the administration of anti-CD4 mAb with the initial AAV2/9 vector, indicating a spread of benefit derived from control of the immune response to the first AAV2/9 vector. Anti-CD4 mAb along with AAV2/9-CBhGAApA significantly increased GAA activity in heart and skeletal muscles along with a significant reduction of glycogen accumulation. Taken together, these data demonstrated that the addition of nondepleting anti-CD4 mAb with gene therapy controls humoral immune responses to both vector and transgene, resulting in clear therapeutic benefit in mice with Pompe disease. PMID:25382056

  12. Enhanced efficacy from gene therapy in Pompe disease using coreceptor blockade.

    PubMed

    Han, Sang-oh; Li, Songtao; Brooks, Elizabeth D; Masat, Elisa; Leborgne, Christian; Banugaria, Suhrad; Bird, Andrew; Mingozzi, Federico; Waldmann, Herman; Koeberl, Dwight

    2015-01-01

    Enzyme replacement therapy (ERT) is the standard-of-care treatment of Pompe disease, a lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA). One limitation of ERT with recombinant human (rh) GAA is antibody formation against GAA. Similarly, in adeno-associated virus (AAV) vector-mediated gene transfer for Pompe disease, development of antibodies against the GAA transgene product and the AAV vector prevents therapeutic efficacy and vector readministration, respectively. Here a nondepleting anti-CD4 monoclonal antibody (mAb) was administrated intravenously prior to administration of an AAV2/9 vector encoding GAA to suppress anti-GAA responses, leading to a substantial reduction of anti-GAA immunoglobulins, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3. Transduction efficiency in liver with a subsequent AAV2/8 vector was massively improved by the administration of anti-CD4 mAb with the initial AAV2/9 vector, indicating a spread of benefit derived from control of the immune response to the first AAV2/9 vector. Anti-CD4 mAb along with AAV2/9-CBhGAApA significantly increased GAA activity in heart and skeletal muscles along with a significant reduction of glycogen accumulation. Taken together, these data demonstrated that the addition of nondepleting anti-CD4 mAb with gene therapy controls humoral immune responses to both vector and transgene, resulting in clear therapeutic benefit in mice with Pompe disease. PMID:25382056

  13. Viral vector mediated expression of mutant huntingtin in the dorsal raphe produces disease-related neuropathology but not depressive-like behaviors in wildtype mice.

    PubMed

    Pitzer, Mark; Lueras, Jordan; Warden, Anna; Weber, Sydney; McBride, Jodi

    2015-05-22

    depressive-like behaviors. Wildtype mice were injected with an adeno-associated virus (AAV2/1) encoding HTT containing either a pathogenic (N171-82Q) or control (N171-16Q) CAG repeat length into the ventral DRN and depressive-like behaviors and motor behaviors were assessed for 12 weeks post-surgery. Quantitative PCR and immunohistochemistry (IHC) verified positive transduction in the ventral aspects of the DRN, including the ventral sub-nucleus (DRv) and interfascicular sub-nucleus (DRif). IHC demonstrated microgliosis in and around the injection site and mHTT-positive inclusions in serotonin-producing neurons and a small percentage of astrocytes in animals injected with N171-82Q compared to controls. Moreover, N171-82Q injected mice showed a 75% reduction in cells that stained positive for the serotonin synthesis enzyme, tryptophan hydroxylase-2 (TPH2) compared to controls (p<0.05). Despite mHTT-mediated pathology in the DRv and DRif, no significant changes in depressive-like behavior were detected. Consequently, we conclude that 12 weeks of N171-82Q expression in the ventral sub-nuclei of the DRN of wildtype mice causes characteristic disease-related cellular neuropathology but is not sufficient to elicit depressive-like behaviors. Ongoing studies are investigating whether a larger injection volume that transfects a larger percentage of the DRN and/or a longer time course of mHTT expression might elicit depressive-like behaviors. Moreover, mHTT expression in other regions of the brain, such as the hippocampal dentate gyrus and/or the frontal cortex might be necessary to elicit HD depression. Together, these results may prove helpful in addressing which therapeutic and/or pharmacological strategies might be most efficacious when treating depressive symptomology in patients suffering from HD. PMID:25732261

  14. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG

    PubMed Central

    Fuchs, Sebastian P.; Martinez-Navio, José M.; Gao, Guangping; Desrosiers, Ronald C.

    2016-01-01

    Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5–2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG. PMID:27332822

  15. Alignment system for encoders

    NASA Technical Reports Server (NTRS)

    Villani, Daniel D. (Inventor)

    1988-01-01

    An improved encoder alignment system is disclosed which provides an indication of the extent of misalignment and a measure of the rate at which the misalignment may be changing. The invention is adapted for use with a conventional encoder which provides a digital coarse word having at least significant bit and a digital fine word having a least significant bit and a most significant bit. The invention generates the exclusive or of the least significant bit of the coarse digital signal and the least significant bit of the fine digital signal to provide a first signal. The invention then generates the exclusive or of the first signal and the complement of the most significant bit of the fine digital signal to provide an output signal which represents the misalignment of the encoder.

  16. Determination of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies in Patients With Heart Failure in the Cardiovascular Foundation of Colombia (ANVIAS): Study Protocol

    PubMed Central

    Prada, Carlos E; Lopez, Marcos; Castillo, Victor; Echeverria, Luis Eduardo; Serrano, Norma

    2016-01-01

    Background Recent progress in the pathophysiology of heart failure (HF) has led to the development of new therapeutic options such as gene therapy and the use of adeno-associated viral (AAV) vectors. Despite the promising results in early clinical trials of gene therapy for HF, various obstacles have been faced, such as the presence of neutralizing antibodies (NAbs) against the capsid vectors. NAb activity limits vector transduction levels and therefore diminishes the final therapeutic response. Recent studies evaluating the prevalence of NAbs in various populations found considerable geographic variability for each AAV serotype. However, the levels of NAbs in Latin American populations are unknown, becoming a limiting factor to conducting AAV vector therapeutic trials in this population. Objective The goal of this study is to determine for the first time, the prevalence of anti-AAV NAbs for the serotypes 1, 2, and 9 in HF patients from the city of Bucaramanga, Colombia, using the in vitro transduction inhibition assay. Methods We will conduct a cross-sectional study with patients who periodically attend the HF clinic of the Cardiovascular Foundation of Colombia and healthy volunteers matched for age and sex. For all participants, we will evaluate the NAb levels against serotypes AAV1, AAV2, and AAV9. We will determine NAb levels using the in vitro transduction inhibition assay. In addition, participants will answer a survey to evaluate their epidemiological and socioeconomic variables. Participation in the study will be voluntary and all participants will sign an informed consent document before any intervention. Results The project is in the first phase: elaboration of case report forms and the informed consent form, and design of the recruitment strategy. Patient recruitment is expected to begin in the spring of 2016. We expect to have preliminary results, including the titer of the viral vectors, multiplicity of infections that we will use for each serotype

  17. Polarization encoded color camera.

    PubMed

    Schonbrun, Ethan; Möller, Guðfríður; Di Caprio, Giuseppe

    2014-03-15

    Digital cameras would be colorblind if they did not have pixelated color filters integrated into their image sensors. Integration of conventional fixed filters, however, comes at the expense of an inability to modify the camera's spectral properties. Instead, we demonstrate a micropolarizer-based camera that can reconfigure its spectral response. Color is encoded into a linear polarization state by a chiral dispersive element and then read out in a single exposure. The polarization encoded color camera is capable of capturing three-color images at wavelengths spanning the visible to the near infrared. PMID:24690806

  18. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2016-02-23

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  19. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

    2016-06-14

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2016-05-31

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Schnorr, Kirk; Kramer, Randall

    2016-04-05

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    DOEpatents

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2016-08-09

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  3. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2010-06-22

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  4. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Lopez de Leon, Alfredo; Rey, Micheal; Ding, Hanshu; Vlasenko, Elena

    2012-02-21

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  5. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Ding, Hanshu; Brown, Kimberly

    2011-10-25

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  6. EGVII endoglucanase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2014-02-25

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  7. EGVII endoglucanase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2015-04-14

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  8. EGVII endoglucanase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2013-07-16

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  9. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    SciTech Connect

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2014-09-30

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  10. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    SciTech Connect

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2012-10-16

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  11. EGVII endoglucanase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2012-02-14

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  12. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Hanshu, Ding

    2012-10-30

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Polypeptides having xylanase activity and polynucleotides encoding same

    SciTech Connect

    Lopez de Leon, Alfredo; Rey, Michael

    2015-01-27

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  14. Polypeptides having endoglucanase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj; Shagasi, Tarana

    2015-06-30

    The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

  15. Polypeptides having endoglucanase activity and polynucleotides encoding same

    SciTech Connect

    Lopez de Leon, Alfredo; Rey, Michael

    2015-03-10

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2011-06-14

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  17. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Morant, Marc

    2014-01-14

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase, or beta-glucosidase activity and isolated polynucleotides encoding polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. EGVI endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2008-04-01

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  19. Polynucleotides encoding polypeptides having beta-glucosidase activity

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2010-03-02

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  20. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Harris, Paul; Tang, Lan; Wu, Wenping

    2013-11-19

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Brown, Kimberly; Harris, Paul; Lopez De Leon, Alfredo; Merino, Sandra

    2007-05-22

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  2. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2012-09-18

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  3. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

    DOEpatents

    Duan, Junxin; Schnorr, Kirk Matthew; Wu, Wenping

    2013-11-19

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  4. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2007-07-17

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  5. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

    2009-05-19

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

  6. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2012-11-27

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  7. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2009-05-05

    The present invention provides an endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  8. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Lopez de Leon, Alfredo; Rey, Michael; Ding, Hanshu; Vlasenko, Elena

    2010-11-02

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  9. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2013-06-18

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2010-12-14

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

    2013-10-29

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

    2007-09-18

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

  13. Polypeptides having xylanase activity and polynucleotides encoding the same

    DOEpatents

    Spodsberg, Nikolaj [Bagsvaed, DK

    2014-01-07

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The inventino also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  14. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-10-21

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. EGVII endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2008-11-11

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  16. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-10-14

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Polypeptides having xylanase activity and polynucleotides encoding same

    SciTech Connect

    Tang, Lan; Liu, Ye; Duan, Junxin; Ding, Hanshu

    2013-04-30

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Tang, Lan; Harris, Paul; Wu, Wenping

    2012-10-02

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  19. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc D; Patkar, Shamkant; Ding, Hanshu

    2013-11-12

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Joergensen, Christian; Kramer, Randall

    2014-09-16

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

    2013-12-24

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

    2013-04-16

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  3. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2015-11-17

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  4. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Tang, Lan

    2015-11-20

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  5. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc D.; Harris, Paul

    2015-10-13

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  6. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

    2012-04-03

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Duan, Junxin; Tang, Lan; Liu, Ye; Wu, Wenping; Quinlan, Jason; Kramer, Randall

    2013-06-18

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  8. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

    2014-10-21

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Duan, Junxin; Liu, Ye; Tang, Lan; Wu, Wenping; Quinlan, Jason; Kramer, Randall

    2012-03-27

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. EGVI endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2010-10-05

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  11. EGVI endoglucanase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2010-10-12

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  12. Time-Encoded Imagers.

    SciTech Connect

    Marleau, Peter; Brubaker, Erik

    2014-11-01

    This report provides a short overview of the DNN R&D funded project, Time-Encoded Imagers. The project began in FY11 and concluded in FY14. The Project Description below provides the overall motivation and objectives for the project as well as a summary of programmatic direction. It is followed by a short description of each task and the resulting deliverables.

  13. Video Time Encoding Machines

    PubMed Central

    Lazar, Aurel A.; Pnevmatikakis, Eftychios A.

    2013-01-01

    We investigate architectures for time encoding and time decoding of visual stimuli such as natural and synthetic video streams (movies, animation). The architecture for time encoding is akin to models of the early visual system. It consists of a bank of filters in cascade with single-input multi-output neural circuits. Neuron firing is based on either a threshold-and-fire or an integrate-and-fire spiking mechanism with feedback. We show that analog information is represented by the neural circuits as projections on a set of band-limited functions determined by the spike sequence. Under Nyquist-type and frame conditions, the encoded signal can be recovered from these projections with arbitrary precision. For the video time encoding machine architecture, we demonstrate that band-limited video streams of finite energy can be faithfully recovered from the spike trains and provide a stable algorithm for perfect recovery. The key condition for recovery calls for the number of neurons in the population to be above a threshold value. PMID:21296708

  14. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  15. Reed-Solomon Encoder

    NASA Technical Reports Server (NTRS)

    Troung, T. K.; Reed, I. S.; Deutsch, L. J.; Hsu, I. S.; Wang, K.; Yeh, C. S.

    1985-01-01

    Report presents mathematical principles of Berlekamp bit serial multiplier algorithm and its application to design of very-large-scale integrated (VLSI) encoders for Reed-Solomon error-correcting codes. Structure made readily on single chip of negatively doped channel metal oxide semiconductor.

  16. A Flexible Turbulent Vector Field Generator

    NASA Astrophysics Data System (ADS)

    Benassi, A.; Davis, A.

    2004-12-01

    Analysis and generation of turbulent vector fields is a necessity in many areas, such as Atmospheric Science. A candidate model of vector field must be flexible enough to tune some features, such as the spacial distribution of vortices, sinks and sources, according to physical measures. To achieve that goal, we propose a model that depends upon a given matricial function called "topolet" and a law of random vectors family. This model has a hierarchical structure. Its spinal column is a tree: the encoding tree of the domain where the vector field lives. The sets of vortices, sinks and sources are driven by some Bernouilli subtrees, directly giving their fractal dimension. At each node of the tree is attached a rate of energy loose giving the spectral slope. All those quantities are independantly identifiable on the base of mathematical proofs. A primitive version of this model have been proposed for generating clouds.

  17. Vector Design Tour de Force: Integrating Combinatorial and Rational Approaches to Derive Novel Adeno-associated Virus Variants

    PubMed Central

    Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

    2014-01-01

    Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 105 complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. PMID:25048217

  18. Vector design Tour de Force: integrating combinatorial and rational approaches to derive novel adeno-associated virus variants.

    PubMed

    Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

    2014-11-01

    Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of "virtual family shuffling" to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 10(5) complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. PMID:25048217

  19. Rotations with Rodrigues' Vector

    ERIC Educational Resources Information Center

    Pina, E.

    2011-01-01

    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears…

  20. Raster and vector processing for scanned linework

    USGS Publications Warehouse

    Greenlee, David D.

    1987-01-01

    An investigation of raster editing techniques, including thinning, filling, and node detecting, was performed by using specialized software. The techniques were based on encoding the state of the 3-by-3 neighborhood surrounding each pixel into a single byte. A prototypical method for converting the edited raster linkwork into vectors was also developed. Once vector representations of the lines were formed, they were formatted as a Digital Line Graph, and further refined by deletion of nonessential vertices and by smoothing with a curve-fitting technique.

  1. The Text Encoding Initiative: Flexible and Extensible Document Encoding.

    ERIC Educational Resources Information Center

    Barnard, David T.; Ide, Nancy M.

    1997-01-01

    The Text Encoding Initiative (TEI), an international collaboration aimed at producing a common encoding scheme for complex texts, examines the requirement for generality versus the requirement to handle specialized text types. Discusses how documents and users tax the limits of fixed schemes requiring flexible extensible encoding to support…

  2. Reduced Vector Preisach Model

    NASA Technical Reports Server (NTRS)

    Patel, Umesh D.; Torre, Edward Della; Day, John H. (Technical Monitor)

    2002-01-01

    A new vector Preisach model, called the Reduced Vector Preisach model (RVPM), was developed for fast computations. This model, derived from the Simplified Vector Preisach model (SVPM), has individual components that like the SVPM are calculated independently using coupled selection rules for the state vector computation. However, the RVPM does not require the rotational correction. Therefore, it provides a practical alternative for computing the magnetic susceptibility using a differential approach. A vector version, using the framework of the DOK model, is implemented. Simulation results for the reduced vector Preisach model are also presented.

  3. VLSI Reed-Solomon Encoder

    NASA Technical Reports Server (NTRS)

    Liu, K. Y.

    1983-01-01

    Modular Reed-solomon encoder uses identical custom VLSI chips called "symbol slices." By cascading and properly interconnecting group of these chips, encoder is made for any desired error-correcting capability and interleaving level. VLSI encoder requires only one-tenth the number of chips required by conventional Reed-Solomon Circuit implemented with discrete IC's.

  4. Understanding Singular Vectors

    ERIC Educational Resources Information Center

    James, David; Botteron, Cynthia

    2013-01-01

    matrix yields a surprisingly simple, heuristical approximation to its singular vectors. There are correspondingly good approximations to the singular values. Such rules of thumb provide an intuitive interpretation of the singular vectors that helps explain why the SVD is so…

  5. The vector ruling protractor

    NASA Technical Reports Server (NTRS)

    Zahm, A F

    1924-01-01

    The theory, structure and working of a vector slide rule is presented in this report. This instrument is used for determining a vector in magnitude and position when given its components and its moment about a point in their plane.

  6. Restart 68000 vector remapping

    SciTech Connect

    Gustin, J.

    1984-05-03

    The circuit described allows power-on-reset (POR) vector fetch from ROM for a 68000 microprocessor. It offers programmability of exception vectors, including the restart vector. This method eliminates the need for high-resolution, address-decoder peripheral circuitry.

  7. Rhotrix Vector Spaces

    ERIC Educational Resources Information Center

    Aminu, Abdulhadi

    2010-01-01

    By rhotrix we understand an object that lies in some way between (n x n)-dimensional matrices and (2n - 1) x (2n - 1)-dimensional matrices. Representation of vectors in rhotrices is different from the representation of vectors in matrices. A number of vector spaces in matrices and their properties are known. On the other hand, little seems to be…

  8. MATRIX AND VECTOR SERVICES

    2001-10-18

    PETRA V2 provides matrix and vector services and the ability construct, query, and use matrix and vector objects that are used and computed by TRILINOS solvers. It provides all basic matr5ix and vector operations for solvers in TRILINOS.

  9. Insulated Foamy Viral Vectors.

    PubMed

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

    2016-03-01

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

  10. Complete shaping of optical vector beams.

    PubMed

    Chen, Zhaozhong; Zeng, Tingting; Qian, Binjie; Ding, Jianping

    2015-07-13

    We propose and experimentally demonstrate the complete and simultaneous modulation of the amplitude, phase and arbitrary state of polarization of optical beams. Based on a 4-f system including a spatial light modulator (SLM), two orthogonally polarized beams serving as the base vector components are produced by a computer generated hologram. The complex amplitude of orthogonal components is realized by a macro-pixel encoding technique purposely designed for phase-only SLMs. Vector beams can be created from the coaxial superposition of the two base beams. This enables us to design optical fields with arbitrarily structured amplitude, phase and polarization by using only one SLM, and thus provides an easy-to-implement route for exploring the novel effects and expanding the functionality of vector beams with space-variant parameters. PMID:26191832

  11. Time encoded radiation imaging

    DOEpatents

    Marleau, Peter; Brubaker, Erik; Kiff, Scott

    2014-10-21

    The various technologies presented herein relate to detecting nuclear material at a large stand-off distance. An imaging system is presented which can detect nuclear material by utilizing time encoded imaging relating to maximum and minimum radiation particle counts rates. The imaging system is integrated with a data acquisition system that can utilize variations in photon pulse shape to discriminate between neutron and gamma-ray interactions. Modulation in the detected neutron count rates as a function of the angular orientation of the detector due to attenuation of neighboring detectors is utilized to reconstruct the neutron source distribution over 360 degrees around the imaging system. Neutrons (e.g., fast neutrons) and/or gamma-rays are incident upon scintillation material in the imager, the photons generated by the scintillation material are converted to electrical energy from which the respective neutrons/gamma rays can be determined and, accordingly, a direction to, and the location of, a radiation source identified.

  12. Rotary encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A device for position encoding of a rotating shaft in which a polygonal mirror having a number of facets is mounted to the shaft and a light beam is directed towards the facets is presented. The facets of the polygonal mirror reflect the light beam such that a light spot is created on a linear array detector. An analog-to-digital converter is connected to the linear array detector for reading the position of the spot on the linear array detector. A microprocessor with memory is connected to the analog-to-digital converter to hold and manipulate the data provided by the analog-to-digital converter on the position of the spot and to compute the position of the shaft based upon the data from the analog-to-digital converter.

  13. Linear encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A Linear Motion Encoding device for measuring the linear motion of a moving object is disclosed in which a light source is mounted on the moving object and a position sensitive detector such as an array photodetector is mounted on a nearby stationary object. The light source emits a light beam directed towards the array photodetector such that a light spot is created on the array. An analog-to-digital converter, connected to the array photodetector is used for reading the position of the spot on the array photodetector. A microprocessor and memory is connected to the analog-to-digital converter to hold and manipulate data provided by the analog-to-digital converter on the position of the spot and to compute the linear displacement of the moving object based upon the data from the analog-to-digital converter.

  14. More About Vector Adaptive/Predictive Coding Of Speech

    NASA Technical Reports Server (NTRS)

    Jedrey, Thomas C.; Gersho, Allen

    1992-01-01

    Report presents additional information about digital speech-encoding and -decoding system described in "Vector Adaptive/Predictive Encoding of Speech" (NPO-17230). Summarizes development of vector adaptive/predictive coding (VAPC) system and describes basic functions of algorithm. Describes refinements introduced enabling receiver to cope with errors. VAPC algorithm implemented in integrated-circuit coding/decoding processors (codecs). VAPC and other codecs tested under variety of operating conditions. Tests designed to reveal effects of various background quiet and noisy environments and of poor telephone equipment. VAPC found competitive with and, in some respects, superior to other 4.8-kb/s codecs and other codecs of similar complexity.

  15. Covariantized vector Galileons

    NASA Astrophysics Data System (ADS)

    Hull, Matthew; Koyama, Kazuya; Tasinato, Gianmassimo

    2016-03-01

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

  16. Space vehicle onboard command encoder

    NASA Technical Reports Server (NTRS)

    1975-01-01

    A flexible onboard encoder system was designed for the space shuttle. The following areas were covered: (1) implementation of the encoder design into hardware to demonstrate the various encoding algorithms/code formats, (2) modulation techniques in a single hardware package to maintain comparable reliability and link integrity of the existing link systems and to integrate the various techniques into a single design using current technology. The primary function of the command encoder is to accept input commands, generated either locally onboard the space shuttle or remotely from the ground, format and encode the commands in accordance with the payload input requirements and appropriately modulate a subcarrier for transmission by the baseband RF modulator. The following information was provided: command encoder system design, brassboard hardware design, test set hardware and system packaging, and software.

  17. Security authentication using phase-encoded nanoparticle structures and polarized light.

    PubMed

    Carnicer, Artur; Hassanfiroozi, Amir; Latorre-Carmona, Pedro; Huang, Yi-Pai; Javidi, Bahram

    2015-01-15

    Phase-encoded nanostructures such as quick response (QR) codes made of metallic nanoparticles are suggested to be used in security and authentication applications. We present a polarimetric optical method able to authenticate random phase-encoded QR codes. The system is illuminated using polarized light, and the QR code is encoded using a phase-only random mask. Using classification algorithms, it is possible to validate the QR code from the examination of the polarimetric signature of the speckle pattern. We used Kolmogorov-Smirnov statistical test and Support Vector Machine algorithms to authenticate the phase-encoded QR codes using polarimetric signatures. PMID:25679827

  18. N-Consecutive-Phase Encoder

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Lee, Ho-Kyoung; Weber, Charles

    1995-01-01

    N-consecutive-phase encoder (NCPE) is conceptual encoder for generating alphabet of N consecutive full-response continuous-phase-modulation (CPM) signals. Enables use of binary preencoder of higher rate than used with simple continuous-phase encoder (CPE). NCPE makes possible to achieve power efficiencies and bandwidth efficiencies greater than conventional trellis coders with continuous-phase frequency-shift keying (CPFSK).

  19. Contour-map encoding of shape for early vision

    NASA Technical Reports Server (NTRS)

    Kanerva, Pentti

    1990-01-01

    Contour maps provide a general method for recognizing 2-D shapes. All but blank images give rise to such maps, and people are good at recognizing objects and shapes from them. The maps are encoded easily in long feature vectors that are suitable for recognition by an associative memory. These properties of contour maps suggest a role for them in early visual perception. The prevalence of direction sensitive neurons in the visual cortex of mammals supports this view.

  20. Index Sets and Vectorization

    SciTech Connect

    Keasler, J A

    2012-03-27

    Vectorization is data parallelism (SIMD, SIMT, etc.) - extension of ISA enabling the same instruction to be performed on multiple data items simultaeously. Many/most CPUs support vectorization in some form. Vectorization is difficult to enable, but can yield large efficiency gains. Extra programmer effort is required because: (1) not all algorithms can be vectorized (regular algorithm structure and fine-grain parallelism must be used); (2) most CPUs have data alignment restrictions for load/store operations (obey or risk incorrect code); (3) special directives are often needed to enable vectorization; and (4) vector instructions are architecture-specific. Vectorization is the best way to optimize for power and performance due to reduced clock cycles. When data is organized properly, a vector load instruction (i.e. movaps) can replace 'normal' load instructions (i.e. movsd). Vector operations can potentially have a smaller footprint in the instruction cache when fewer instructions need to be executed. Hybrid index sets insulate users from architecture specific details. We have applied hybrid index sets to achieve optimal vectorization. We can extend this concept to handle other programming models.

  1. Viral vectors for cancer immunotherapy.

    PubMed

    Harrop, Richard; Carroll, Miles W

    2006-01-01

    Over the last decade, immunotherapy approaches for the treatment of cancer have been investigated with renewed vigour, perhaps catalyzed by the clinical successes seen with monoclonal antibody and cytokine based therapies. The identification of tumor-associated antigens (TAAs) in multiple cancer types has enabled the development of targeted immunotherapies and allayed some of the safety concerns associated with the induction of deleterious autoimmune reactions. In addition to the TAA or therapeutic gene, the antigen delivery system is equally as important for the development of a successful cancer vaccine. One approach to induce a potent and targeted antitumor response is to use viruses to deliver the TAA to cells of the immune system. A diverse array of oncolytic viruses and recombinant viral vectors encoding numerous therapeutic genes or TAAs have been tested in pre-clinical studies and produced results which, in some cases, justify their clinical development as potential cancer immunotherapies. Within the last 5-10 years, many such recombinant vectors have made the transition from pre-clinical research to clinical development and it is these, which are given most weight in this review. PMID:16146772

  2. A sparse embedding and least variance encoding approach to hashing.

    PubMed

    Zhu, Xiaofeng; Zhang, Lei; Huang, Zi

    2014-09-01

    Hashing is becoming increasingly important in large-scale image retrieval for fast approximate similarity search and efficient data storage. Many popular hashing methods aim to preserve the kNN graph of high dimensional data points in the low dimensional manifold space, which is, however, difficult to achieve when the number of samples is big. In this paper, we propose an effective and efficient hashing approach by sparsely embedding a sample in the training sample space and encoding the sparse embedding vector over a learned dictionary. To this end, we partition the sample space into clusters via a linear spectral clustering method, and then represent each sample as a sparse vector of normalized probabilities that it falls into its several closest clusters. This actually embeds each sample sparsely in the sample space. The sparse embedding vector is employed as the feature of each sample for hashing. We then propose a least variance encoding model, which learns a dictionary to encode the sparse embedding feature, and consequently binarize the coding coefficients as the hash codes. The dictionary and the binarization threshold are jointly optimized in our model. Experimental results on benchmark data sets demonstrated the effectiveness of the proposed approach in comparison with state-of-the-art methods. PMID:24968174

  3. The decoding method based on wavelet image En vector quantization

    NASA Astrophysics Data System (ADS)

    Liu, Chun-yang; Li, Hui; Wang, Tao

    2013-12-01

    With the rapidly progress of internet technology, large scale integrated circuit and computer technology, digital image processing technology has been greatly developed. Vector quantization technique plays a very important role in digital image compression. It has the advantages other than scalar quantization, which possesses the characteristics of higher compression ratio, simple algorithm of image decoding. Vector quantization, therefore, has been widely used in many practical fields. This paper will combine the wavelet analysis method and vector quantization En encoder efficiently, make a testing in standard image. The experiment result in PSNR will have a great improvement compared with the LBG algorithm.

  4. Prosodic Encoding in Silent Reading.

    ERIC Educational Resources Information Center

    Wilkenfeld, Deborah

    In silent reading, short-memory tasks, such as semantic and syntactic processing, require a stage of phonetic encoding between visual representation and the actual extraction of meaning, and this encoding includes prosodic as well as segmental features. To test for this suprasegmental coding, an experiment was conducted in which subjects were…

  5. Symbolic computer vector analysis

    NASA Technical Reports Server (NTRS)

    Stoutemyer, D. R.

    1977-01-01

    A MACSYMA program is described which performs symbolic vector algebra and vector calculus. The program can combine and simplify symbolic expressions including dot products and cross products, together with the gradient, divergence, curl, and Laplacian operators. The distribution of these operators over sums or products is under user control, as are various other expansions, including expansion into components in any specific orthogonal coordinate system. There is also a capability for deriving the scalar or vector potential of a vector field. Examples include derivation of the partial differential equations describing fluid flow and magnetohydrodynamics, for 12 different classic orthogonal curvilinear coordinate systems.

  6. Peri-encoding predictors of memory encoding and consolidation.

    PubMed

    Cohen, Noga; Pell, Liat; Edelson, Micah G; Ben-Yakov, Aya; Pine, Alex; Dudai, Yadin

    2015-03-01

    We review reports of brain activations that occur immediately prior to the onset or following the offset of to-be-remembered information and can predict subsequent mnemonic success. Memory-predictive pre-encoding processes, occurring from fractions of a second to minutes prior to event onset, are mainly associated with activations in the medial temporal lobe (MTL), amygdala and midbrain, and with enhanced theta oscillations. These activations may be considered as the neural correlates of one or more cognitive operations, including contextual processing, attention, and the engagement of distinct computational modes associated with prior encoding or retrieval. Post-encoding activations that correlate with subsequent memory performance are mainly observed in the MTL, sensory cortices and frontal regions. These activations may reflect binding of elements of the encoded information and initiation of memory consolidation. In all, the findings reviewed here illustrate the importance of brain states in the immediate peri-encoding time windows in determining encoding success. Understanding these brain states and their specific effects on memory may lead to optimization of the encoding of desired memories and mitigation of undesired ones. PMID:25446944

  7. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, Samuel Lee; Miller, William Michael; McWhorter, Paul Jackson

    1997-01-01

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals.

  8. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, S.L.; Miller, W.M.; McWhorter, P.J.

    1997-10-21

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals. 32 figs.

  9. Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same

    DOEpatents

    Tsien, Roger Y.; Wang, Lei

    2008-07-01

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  10. Vector theories in cosmology

    SciTech Connect

    Esposito-Farese, Gilles; Pitrou, Cyril; Uzan, Jean-Philippe

    2010-03-15

    This article provides a general study of the Hamiltonian stability and the hyperbolicity of vector field models involving both a general function of the Faraday tensor and its dual, f(F{sup 2},FF-tilde), as well as a Proca potential for the vector field, V(A{sup 2}). In particular it is demonstrated that theories involving only f(F{sup 2}) do not satisfy the hyperbolicity conditions. It is then shown that in this class of models, the cosmological dynamics always dilutes the vector field. In the case of a nonminimal coupling to gravity, it is established that theories involving Rf(A{sup 2}) or Rf(F{sup 2}) are generically pathologic. To finish, we exhibit a model where the vector field is not diluted during the cosmological evolution, because of a nonminimal vector field-curvature coupling which maintains second-order field equations. The relevance of such models for cosmology is discussed.

  11. Vector generator scan converter

    DOEpatents

    Moore, J.M.; Leighton, J.F.

    1988-02-05

    High printing speeds for graphics data are achieved with a laser printer by transmitting compressed graphics data from a main processor over an I/O channel to a vector generator scan converter which reconstructs a full graphics image for input to the laser printer through a raster data input port. The vector generator scan converter includes a microprocessor with associated microcode memory containing a microcode instruction set, a working memory for storing compressed data, vector generator hardware for drawing a full graphic image from vector parameters calculated by the microprocessor, image buffer memory for storing the reconstructed graphics image and an output scanner for reading the graphics image data and inputting the data to the printer. The vector generator scan converter eliminates the bottleneck created by the I/O channel for transmitting graphics data from the main processor to the laser printer, and increases printer speed up to thirty fold. 7 figs.

  12. Vector generator scan converter

    DOEpatents

    Moore, James M.; Leighton, James F.

    1990-01-01

    High printing speeds for graphics data are achieved with a laser printer by transmitting compressed graphics data from a main processor over an I/O (input/output) channel to a vector generator scan converter which reconstructs a full graphics image for input to the laser printer through a raster data input port. The vector generator scan converter includes a microprocessor with associated microcode memory containing a microcode instruction set, a working memory for storing compressed data, vector generator hardward for drawing a full graphic image from vector parameters calculated by the microprocessor, image buffer memory for storing the reconstructed graphics image and an output scanner for reading the graphics image data and inputting the data to the printer. The vector generator scan converter eliminates the bottleneck created by the I/O channel for transmitting graphics data from the main processor to the laser printer, and increases printer speed up to thirty fold.

  13. Nonviral Vectors for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Baoum, Abdulgader Ahmed

    2011-12-01

    The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,L-lactide- co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (˜200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium. On the other hand, a more simple method to synthesize 50-200 nm complexes capable of high transfection efficiency or high gene knockdown was

  14. Two digital video encoder circuits

    NASA Astrophysics Data System (ADS)

    Eldon, John A.

    1992-11-01

    Central to `multimedia' image processing is the desire to encode computer graphics data into a standard television signal, complete with line, field, and color subcarrier synchronizing information. The numerous incompatibilities between television and computer display standards render this operation far less trivial than it sounds to anyone who hasn't worked with both types of signals. To simplify the task of encoding computer graphics signals into standard NTSC (North America and Japan) or PAL (most of Europe) television format for display, broadcast, or recording, TRW LSI Products Inc. has introduced the two newest members of it multimedia integrated circuit family, the TMC22090 and TMC22190 digital video encoders.

  15. Line Integral of a Vector.

    ERIC Educational Resources Information Center

    Balabanian, Norman

    This programed booklet is designed for the engineering student who understands and can use vector and unit vector notation, components of a vector, parallel law of vector addition, and the dot product of two vectors. Content begins with work done by a force in moving a body a certain distance along some path. For each of the examples and problem…

  16. Serial position encoding of signs.

    PubMed

    Miozzo, Michele; Petrova, Anna; Fischer-Baum, Simon; Peressotti, Francesca

    2016-09-01

    Reduced short-term memory (STM) capacity has been reported for sign as compared to speech when items have to be recalled in a specific order. This difference has been attributed to a more precise and efficient serial position encoding in verbal STM (used for speech) than visuo-spatial STM (used for sign). We tested in the present investigation whether the reduced STM capacity with signs stems from a lack of positional encoding available in verbal STM. Error analyses reported in prior studies have revealed that positions are defined in verbal STM by distance from both the start and the end of the sequence (both-edges positional encoding scheme). Our analyses of the errors made by deaf participants with finger-spelled letters revealed that the both-edges positional encoding scheme underlies the STM representation of signs. These results indicate that the cause of the STM disadvantage is not the type of positional encoding but rather the difficulties in binding an item in visuo-spatial STM to its specific position in the sequence. Both-edges positional encoding scheme could be specific of sign, since it has not been found in visuo-spatial STM tasks conducted with hearing participants. PMID:27244095

  17. Fractal vector optical fields.

    PubMed

    Pan, Yue; Gao, Xu-Zhen; Cai, Meng-Qiang; Zhang, Guan-Lin; Li, Yongnan; Tu, Chenghou; Wang, Hui-Tian

    2016-07-15

    We introduce the concept of a fractal, which provides an alternative approach for flexibly engineering the optical fields and their focal fields. We propose, design, and create a new family of optical fields-fractal vector optical fields, which build a bridge between the fractal and vector optical fields. The fractal vector optical fields have polarization states exhibiting fractal geometry, and may also involve the phase and/or amplitude simultaneously. The results reveal that the focal fields exhibit self-similarity, and the hierarchy of the fractal has the "weeding" role. The fractal can be used to engineer the focal field. PMID:27420485

  18. Fast Packet Classification Using Multi-Dimensional Encoding

    NASA Astrophysics Data System (ADS)

    Huang, Chi Jia; Chen, Chien

    Internet routers need to classify incoming packets quickly into flows in order to support features such as Internet security, virtual private networks and Quality of Service (QoS). Packet classification uses information contained in the packet header, and a predefined rule table in the routers. Packet classification of multiple fields is generally a difficult problem. Hence, researchers have proposed various algorithms. This study proposes a multi-dimensional encoding method in which parameters such as the source IP address, destination IP address, source port, destination port and protocol type are placed in a multi-dimensional space. Similar to the previously best known algorithm, i.e., bitmap intersection, multi-dimensional encoding is based on the multi-dimensional range lookup approach, in which rules are divided into several multi-dimensional collision-free rule sets. These sets are then used to form the new coding vector to replace the bit vector of the bitmap intersection algorithm. The average memory storage of this encoding is Θ (L · N · log N) for each dimension, where L denotes the number of collision-free rule sets, and N represents the number of rules. The multi-dimensional encoding practically requires much less memory than bitmap intersection algorithm. Additionally, the computation needed for this encoding is as simple as bitmap intersection algorithm. The low memory requirement of the proposed scheme means that it not only decreases the cost of packet classification engine, but also increases the classification performance, since memory represents the performance bottleneck in the packet classification engine implementation using a network processor.

  19. Vector quantization of 3-D point clouds

    NASA Astrophysics Data System (ADS)

    Sim, Jae-Young; Kim, Chang-Su; Lee, Sang-Uk

    2005-10-01

    A geometry compression algorithm for 3-D QSplat data using vector quantization (VQ) is proposed in this work. The positions of child spheres are transformed to the local coordinate system, which is determined by the parent children relationship. The coordinate transform makes child positions more compactly distributed in 3-D space, facilitating effective quantization. Moreover, we develop a constrained encoding method for sphere radii, which guarantees hole-free surface rendering at the decoder side. Simulation results show that the proposed algorithm provides a faithful rendering quality even at low bitrates.

  20. Analysis and study of the interlaced encoding pixels in Hadamard transform spectral imager based on DMD

    NASA Astrophysics Data System (ADS)

    Xu, Jun; Hu, Bingliang; Feng, Dazheng; Fan, Xiaohui; Qian, Qingming

    2012-03-01

    The key innovation in Hadamard transform spectral imager (HTSI) introduced recently is the use of digital micro-mirror device (DMD) to encode spectral information. However, because the size of individual micro-mirrors does not match the detector pixel size or for other unavoidable errors in the optical design and the system assembling, an interlaced encoding phenomenon appears on some pixels of the encoded images obtained from the detector. These interlaced encoding pixels are not encoded based on Hadamard transform, so they should be processed specially in spectrum recovery. This paper analyzes the interlaced encoding phenomenon and proposes a positioning method and a decoding method for the interlaced encoding pixels on the encoded images. In our experiment, we direct a beam of laser into our HTSI and fill the entire field of view; by observing the column vector, which is made up of the gray values of a pixel on the encoded images from the detector in sequence, the interlaced encoding pixels can be distinguished easily and a coefficient is obtained simultaneously, which denotes the ratio of the area between the left part and the right part of the interlaced encoding pixel. By substituting the coefficient and the encoded gray values of the interlaced pixel into its encoding equation, we can recover the spectral elements of the interlaced pixel with ease. By comparing the spectral curve of the interlaced encoding pixels recovered by the method mentioned in this paper and the spectral curves of its two adjacent pixels, we find the decoding results are quite effective.

  1. Understanding Vector Fields.

    ERIC Educational Resources Information Center

    Curjel, C. R.

    1990-01-01

    Presented are activities that help students understand the idea of a vector field. Included are definitions, flow lines, tangential and normal components along curves, flux and work, field conservation, and differential equations. (KR)

  2. AAV Vectorization of DSB-mediated Gene Editing Technologies.

    PubMed

    Moser, Rachel J; Hirsch, Matthew L

    2016-01-01

    Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases. PMID:27280971

  3. Bloch vector projection noise

    NASA Technical Reports Server (NTRS)

    Wang, Li-Jun; Bacon, A. M.; Zhao, H.-Z.; Thomas, J. E.

    1994-01-01

    In the optical measurement of the Bloch vector components describing a system of N two-level atoms, the quantum fluctuations in these components are coupled into the measuring optical field. This paper develops the quantum theory of optical measurement of Bloch vector projection noise. The preparation and probing of coherence in an effective two-level system consisting of the two ground states in an atomic three-level lambda-scheme are analyzed.

  4. Poynting-vector filter

    SciTech Connect

    Carrigan, Charles R.

    2011-08-02

    A determination is made of frequency components associated with a particular bearing or location resulting from sources emitting electromagnetic-wave energy for which a Poynting-Vector can be defined. The broadband frequency components associated with a specific direction or location of interest are isolated from other components in the power spectrum that are not associated with the direction or location of interest. The collection of pointing vectors can be used to characterize the source.

  5. Null Space Imaging: Nonlinear Magnetic Encoding Fields Designed Complementary to Receiver Coil Sensitivities for Improved Acceleration in Parallel Imaging

    PubMed Central

    Tam, Leo K.; Galiana, Gigi; Stockmann, Jason P.; Constable, R. Todd

    2012-01-01

    To increase image acquisition efficiency, we develop alternative gradient encoding strategies designed to provide spatial encoding complementary to the spatial encoding provided by the multiple receiver coil elements in parallel image acquisitions. Intuitively, complementary encoding is achieved when the magnetic field encoding gradients are designed to encode spatial information where receiver spatial encoding is ambiguous, for example, along sensitivity isocontours. Specifically, the method generates a basis set for the null space of the coil sensitivities with the singular value decomposition (SVD) and calculates encoding fields from the null space vectors. A set of nonlinear gradients is used as projection imaging readout magnetic fields, replacing the conventional linear readout field and phase encoding. Multiple encoding fields are used as projections to capture the null space information, hence the term Null Space Imaging (NSI). The method is compared to conventional Cartesian SENSitivity Encoding (SENSE) as evaluated by mean squared error and robustness to noise. Strategies for developments in the area of nonlinear encoding schemes are discussed. The NSI approach yields a parallel imaging method that provides high acceleration factors with a limited number of receiver coil array elements through increased time efficiency in spatial encoding. PMID:22190380

  6. Gravity referenced elevation encoder development

    NASA Astrophysics Data System (ADS)

    Goddard, R. E.

    1993-05-01

    Recent progress in the development of a gravity-sensor-based instrument for determining the elevation angle of DSN antennas is described. The benefits of such a system include the capability to locate the Gravity Referenced Elevation Encoder (GREE) directly on the primary reflector (thus bypassing structural flexure and deformation error sources), anticipated lower maintenance costs compared to the present gimbal encoders, direct replaceability, or supplementation of the present gimbal encoders and the utilization of off-the-shelf components to construct the GREE. This article includes a description of the nominal GREE design. Test results on a laboratory breadboard model are given. Rigid-body dynamics of the GREE are derived and the simulated performance in response to measured antenna vibrations is given.

  7. Gravity referenced elevation encoder development

    NASA Technical Reports Server (NTRS)

    Goddard, R. E.

    1993-01-01

    Recent progress in the development of a gravity-sensor-based instrument for determining the elevation angle of DSN antennas is described. The benefits of such a system include the capability to locate the Gravity Referenced Elevation Encoder (GREE) directly on the primary reflector (thus bypassing structural flexure and deformation error sources), anticipated lower maintenance costs compared to the present gimbal encoders, direct replaceability, or supplementation of the present gimbal encoders and the utilization of off-the-shelf components to construct the GREE. This article includes a description of the nominal GREE design. Test results on a laboratory breadboard model are given. Rigid-body dynamics of the GREE are derived and the simulated performance in response to measured antenna vibrations is given.

  8. Fly Photoreceptors Encode Phase Congruency

    PubMed Central

    Friederich, Uwe; Billings, Stephen A.; Hardie, Roger C.; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  9. Fly Photoreceptors Encode Phase Congruency.

    PubMed

    Friederich, Uwe; Billings, Stephen A; Hardie, Roger C; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  10. Vector-Borne Bacterial Plant Pathogens: Interactions with Hemipteran Insects and Plants.

    PubMed

    Perilla-Henao, Laura M; Casteel, Clare L

    2016-01-01

    Hemipteran insects are devastating pests of crops due to their wide host range, rapid reproduction, and ability to transmit numerous plant-infecting pathogens as vectors. While the field of plant-virus-vector interactions has flourished in recent years, plant-bacteria-vector interactions remain poorly understood. Leafhoppers and psyllids are by far the most important vectors of bacterial pathogens, yet there are still significant gaps in our understanding of their feeding behavior, salivary secretions, and plant responses as compared to important viral vectors, such as whiteflies and aphids. Even with an incomplete understanding of plant-bacteria-vector interactions, some common themes have emerged: (1) all known vector-borne bacteria share the ability to propagate in the plant and insect host; (2) particular hemipteran families appear to be incapable of transmitting vector-borne bacteria; (3) all known vector-borne bacteria have highly reduced genomes and coding capacity, resulting in host-dependence; and (4) vector-borne bacteria encode proteins that are essential for colonization of specific hosts, though only a few types of proteins have been investigated. Here, we review the current knowledge on important vector-borne bacterial pathogens, including Xylella fastidiosa, Spiroplasma spp., Liberibacter spp., and 'Candidatus Phytoplasma spp.'. We then highlight recent approaches used in the study of vector-borne bacteria. Finally, we discuss the application of this knowledge for control and future directions that will need to be addressed in the field of vector-plant-bacteria interactions. PMID:27555855

  11. Vector-Mediated In Vivo Antibody Expression.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. PMID:26104192

  12. BGL7 beta-glucosidase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Ward, Michael

    2015-04-14

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  13. BGL6 beta-glucosidase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Ward, Michael

    2015-08-11

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  14. BGL7 beta-glucosidase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Ward, Michael

    2014-03-25

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  15. BGL6 beta-glucosidase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Ward, Michael

    2014-03-04

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  16. BGL7 beta-glucosidase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Ward, Michael

    2013-01-29

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  17. BGL6 .beta.-glucosidase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Ward, Michael

    2012-10-02

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  18. BGL3 beta-glucosidase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2012-10-30

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  19. BGL3 beta-glucosidase and nucleic acids encoding the same

    SciTech Connect

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2011-06-14

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  20. BGL3 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2008-04-01

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  1. BGL5 .beta.-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2008-03-18

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl5, and the corresponding BGL5 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL5, recombinant BGL5 proteins and methods for producing the same.

  2. BGL6 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Ward, Michael

    2009-09-01

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

  3. Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

    DOEpatents

    Brown, Kimberly; Harris, Paul

    2013-12-17

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  4. BGL7 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Ward, Michael

    2008-08-05

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

  5. Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-04-29

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  6. Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

    DOEpatents

    Morant, Marc Dominique

    2014-05-06

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. Polypeptides having beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

    DOEpatents

    Morant, Marc Dominique

    2014-05-06

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  8. Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

    DOEpatents

    Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

    2015-04-21

    The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  9. The cloning and expression of a gene encoding haemolytic activity from the fish pathogen Renibacterium salmoninarum.

    PubMed

    Evenden, A J; Gilpin, M L; Munn, C B

    1990-09-01

    A gene encoding haemolytic activity from Renibacterium salmoninarum (strain PPD) was cloned into Escherichia coli using the cosmid vector pHC79, and subsequently subcloned on a 1.6 kbp SAlI fragment into pBR328. Southern blot hybridisation revealed that a homologous sequence is found in other strains of R. salmoninarum. PMID:2276613

  10. BGL4 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2008-01-22

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

  11. BGL3 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2007-09-25

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

  12. BGL4 beta-glucosidase and nucleic acids encoding the same

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

    2011-12-06

    The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

  13. Breast ultrasound computed tomography using waveform inversion with source encoding

    NASA Astrophysics Data System (ADS)

    Wang, Kun; Matthews, Thomas; Anis, Fatima; Li, Cuiping; Duric, Neb; Anastasio, Mark A.

    2015-03-01

    Ultrasound computed tomography (USCT) holds great promise for improving the detection and management of breast cancer. Because they are based on the acoustic wave equation, waveform inversion-based reconstruction methods can produce images that possess improved spatial resolution properties over those produced by ray-based methods. However, waveform inversion methods are computationally demanding and have not been applied widely in USCT breast imaging. In this work, source encoding concepts are employed to develop an accelerated USCT reconstruction method that circumvents the large computational burden of conventional waveform inversion methods. This method, referred to as the waveform inversion with source encoding (WISE) method, encodes the measurement data using a random encoding vector and determines an estimate of the speed-of-sound distribution by solving a stochastic optimization problem by use of a stochastic gradient descent algorithm. Computer-simulation studies are conducted to demonstrate the use of the WISE method. Using a single graphics processing unit card, each iteration can be completed within 25 seconds for a 128 × 128 mm2 reconstruction region. The results suggest that the WISE method maintains the high spatial resolution of waveform inversion methods while significantly reducing the computational burden.

  14. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 1 2012-10-01 2012-10-01 false EAS Encoder. 11.32 Section 11.32 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMERGENCY ALERT SYSTEM (EAS) Equipment Requirements § 11.32 EAS Encoder. (a) EAS Encoders must at a minimum be capable of encoding the EAS protocol described in § 11.31 and providing the EAS...

  15. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 1 2014-10-01 2014-10-01 false EAS Encoder. 11.32 Section 11.32 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMERGENCY ALERT SYSTEM (EAS) Equipment Requirements § 11.32 EAS Encoder. (a) EAS Encoders must at a minimum be capable of encoding the EAS protocol described in § 11.31 and providing the EAS...

  16. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 1 2011-10-01 2011-10-01 false EAS Encoder. 11.32 Section 11.32 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL EMERGENCY ALERT SYSTEM (EAS) Equipment Requirements § 11.32 EAS Encoder. (a) EAS Encoders must at a minimum be capable of encoding the EAS protocol described in § 11.31 and providing the EAS...

  17. Vector WIMP miracle

    NASA Astrophysics Data System (ADS)

    Abe, Tomohiro; Kakizaki, Mitsuru; Matsumoto, Shigeki; Seto, Osamu

    2012-07-01

    Weakly interacting massive particle (WIMP) is well known to be a good candidate for dark matter, and it is also predicted by many new physics models beyond the standard model at the TeV scale. We found that, if the WIMP is a vector particle (spin-one particle) which is associated with some gauge symmetry broken at the TeV scale, the Higgs mass is often predicted to be 120-125 GeV, which is very consistent with the result of Higgs searches recently reported by ATLAS and CMS Collaborations at the Large Hadron Collider experiment. In this Letter, we consider the vector WIMP using a non-linear sigma model in order to confirm this result as general as possible in a bottom-up approach. Near-future prospects to detect the vector WIMP at both direct and indirect detection experiments of dark matter are also discussed.

  18. Vector financial rogue waves

    NASA Astrophysics Data System (ADS)

    Yan, Zhenya

    2011-11-01

    The coupled nonlinear volatility and option pricing model presented recently by Ivancevic is investigated, which generates a leverage effect, i.e., stock volatility is (negatively) correlated to stock returns, and can be regarded as a coupled nonlinear wave alternative of the Black-Scholes option pricing model. In this Letter, we analytically propose vector financial rogue waves of the coupled nonlinear volatility and option pricing model without an embedded w-learning. Moreover, we exhibit their dynamical behaviors for chosen different parameters. The vector financial rogue wave (rogon) solutions may be used to describe the possible physical mechanisms for the rogue wave phenomena and to further excite the possibility of relative researches and potential applications of vector rogue waves in the financial markets and other related fields.

  19. Vectorized garbage collection

    SciTech Connect

    Appel, A.W.; Bendiksen, A.

    1988-01-01

    Garbage collection can be done in vector mode on supercomputers like the Cray-2 and the Cyber 205. Both copying collection and mark-and-sweep can be expressed as breadth-first searches in which the queue can be processed in parallel. The authors have designed a copying garbage collector whose inner loop works entirely in vector mode. The only significant limitation of the algorithm is that if the size of the records is not constant, the implementation becomes much more complicated. The authors give performance measurements of the algorithm as implemented for Lisp CONS cells on the Cyber 205. Vector-mode garbage collection performs up to 9 times faster than scalar-mode collection.

  20. How Infants Encode Spatial Extent

    ERIC Educational Resources Information Center

    Duffy, Sean; Huttenlocher, Janellen; Levine, Susan; Duffy, Renee

    2005-01-01

    This study explores how infants encode an object's spatial extent. We habituated 6.5-month-old infants to a dowel inside a container and then tested whether they dishabituate to a change in absolute size when the relation between dowel and container is held constant (by altering the size of both container and dowel) and when the relation changes…

  1. Shaft encoder presents digital output

    NASA Technical Reports Server (NTRS)

    Hillis, D. A.

    1966-01-01

    Circuits that include compensation circuitry time a capacitance relative to a reference voltage so that a digital presentation occurs that is representative of the positional condition of the mechanical shaft being monitored. This circuitry may be employed in multiples to furnish binary encoding of a number of rotating devices simultaneously.

  2. Encoding Standards for Linguistic Corpora.

    ERIC Educational Resources Information Center

    Ide, Nancy

    The demand for extensive reusability of large language text collections for natural languages processing research requires development of standardized encoding formats. Such formats must be capable of representing different kinds of information across the spectrum of text types and languages, capable of representing different levels of…

  3. Bunyavirus-Vector Interactions

    PubMed Central

    Horne, Kate McElroy; Vanlandingham, Dana L.

    2014-01-01

    The Bunyaviridae family is comprised of more than 350 viruses, of which many within the Hantavirus, Orthobunyavirus, Nairovirus, Tospovirus, and Phlebovirus genera are significant human or agricultural pathogens. The viruses within the Orthobunyavirus, Nairovirus, and Phlebovirus genera are transmitted by hematophagous arthropods, such as mosquitoes, midges, flies, and ticks, and their associated arthropods not only serve as vectors but also as virus reservoirs in many cases. This review presents an overview of several important emerging or re-emerging bunyaviruses and describes what is known about bunyavirus-vector interactions based on epidemiological, ultrastructural, and genetic studies of members of this virus family. PMID:25402172

  4. Scalar-vector bootstrap

    NASA Astrophysics Data System (ADS)

    Rejon-Barrera, Fernando; Robbins, Daniel

    2016-01-01

    We work out all of the details required for implementation of the conformal bootstrap program applied to the four-point function of two scalars and two vectors in an abstract conformal field theory in arbitrary dimension. This includes a review of which tensor structures make appearances, a construction of the projectors onto the required mixed symmetry representations, and a computation of the conformal blocks for all possible operators which can be exchanged. These blocks are presented as differential operators acting upon the previously known scalar conformal blocks. Finally, we set up the bootstrap equations which implement crossing symmetry. Special attention is given to the case of conserved vectors, where several simplifications occur.

  5. Vector insert-targeted integrative antisense expression system for plasmid stabilization.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2011-01-01

    Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides. PMID:20607625

  6. Vector-Borne Bacterial Plant Pathogens: Interactions with Hemipteran Insects and Plants

    PubMed Central

    Perilla-Henao, Laura M.; Casteel, Clare L.

    2016-01-01

    Hemipteran insects are devastating pests of crops due to their wide host range, rapid reproduction, and ability to transmit numerous plant-infecting pathogens as vectors. While the field of plant–virus–vector interactions has flourished in recent years, plant–bacteria–vector interactions remain poorly understood. Leafhoppers and psyllids are by far the most important vectors of bacterial pathogens, yet there are still significant gaps in our understanding of their feeding behavior, salivary secretions, and plant responses as compared to important viral vectors, such as whiteflies and aphids. Even with an incomplete understanding of plant–bacteria–vector interactions, some common themes have emerged: (1) all known vector-borne bacteria share the ability to propagate in the plant and insect host; (2) particular hemipteran families appear to be incapable of transmitting vector-borne bacteria; (3) all known vector-borne bacteria have highly reduced genomes and coding capacity, resulting in host-dependence; and (4) vector-borne bacteria encode proteins that are essential for colonization of specific hosts, though only a few types of proteins have been investigated. Here, we review the current knowledge on important vector-borne bacterial pathogens, including Xylella fastidiosa, Spiroplasma spp., Liberibacter spp., and ‘Candidatus Phytoplasma spp.’. We then highlight recent approaches used in the study of vector-borne bacteria. Finally, we discuss the application of this knowledge for control and future directions that will need to be addressed in the field of vector–plant–bacteria interactions. PMID:27555855

  7. Pattern vectors from algebraic graph theory.

    PubMed

    Wilson, Richard C; Hancock, Edwin R; Luo, Bin

    2005-07-01

    Graph structures have proven computationally cumbersome for pattern analysis. The reason for this is that, before graphs can be converted to pattern vectors, correspondences must be established between the nodes of structures which are potentially of different size. To overcome this problem, in this paper, we turn to the spectral decomposition of the Laplacian matrix. We show how the elements of the spectral matrix for the Laplacian can be used to construct symmetric polynomials that are permutation invariants. The coefficients of these polynomials can be used as graph features which can be encoded in a vectorial manner. We extend this representation to graphs in which there are unary attributes on the nodes and binary attributes on the edges by using the spectral decomposition of a Hermitian property matrix that can be viewed as a complex analogue of the Laplacian. To embed the graphs in a pattern space, we explore whether the vectors of invariants can be embedded in a low-dimensional space using a number of alternative strategies, including principal components analysis (PCA), multidimensional scaling (MDS), and locality preserving projection (LPP). Experimentally, we demonstrate that the embeddings result in well-defined graph clusters. Our experiments with the spectral representation involve both synthetic and real-world data. The experiments with synthetic data demonstrate that the distances between spectral feature vectors can be used to discriminate between graphs on the basis of their structure. The real-world experiments show that the method can be used to locate clusters of graphs. PMID:16013758

  8. Production of lentiviral vectors

    PubMed Central

    Merten, Otto-Wilhelm; Hebben, Matthias; Bovolenta, Chiara

    2016-01-01

    Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented. PMID:27110581

  9. Support vector machines

    NASA Technical Reports Server (NTRS)

    Garay, Michael J.; Mazzoni, Dominic; Davies, Roger; Wagstaff, Kiri

    2004-01-01

    Support Vector Machines (SVMs) are a type of supervised learning algorith,, other examples of which are Artificial Neural Networks (ANNs), Decision Trees, and Naive Bayesian Classifiers. Supervised learning algorithms are used to classify objects labled by a 'supervisor' - typically a human 'expert.'.

  10. Singular Vectors' Subtle Secrets

    ERIC Educational Resources Information Center

    James, David; Lachance, Michael; Remski, Joan

    2011-01-01

    Social scientists use adjacency tables to discover influence networks within and among groups. Building on work by Moler and Morrison, we use ordered pairs from the components of the first and second singular vectors of adjacency matrices as tools to distinguish these groups and to identify particularly strong or weak individuals.

  11. Killing vectors and anisotropy

    SciTech Connect

    Krisch, J. P.; Glass, E. N.

    2009-08-15

    We consider an action that can generate fluids with three unequal stresses for metrics with a spacelike Killing vector. The parameters in the action are directly related to the stress anisotropies. The field equations following from the action are applied to an anisotropic cosmological expansion and an extension of the Gott-Hiscock cosmic string.

  12. Vector-borne diseases.

    PubMed

    Gubler, D J

    2009-08-01

    Vector-borne diseases have been the scourge of man and animals since the beginning of time. Historically, these are the diseases that caused the great plagues such as the 'Black Death' in Europe in the 14th Century and the epidemics of yellow fever that plagued the development of the New World. Others, such as Nagana, contributed to the lack of development in Africa for many years. At the turn of the 20th Century, vector-borne diseases were among the most serious public and animal health problems in the world. For the most part, these diseases were controlled by the middle of the 20th Century through the application of knowledge about their natural history along with the judicious use of DDT (dichlorodiphenyltrichloroethane) and other residual insecticides to interrupt the transmission cycle between arthropod and vertebrate host. However, this success initiated a period of complacency in the 1960s and 1970s, which resulted in the redirection of resources away from prevention and control of vector-borne diseases. The 1970s was also a time in which there were major changes to public health policy. Global trends, combined with changes in animal husbandry, urbanisation, modern transportation and globalisation, have resulted in a global re-emergence of epidemic vector-borne diseases affecting both humans and animals over the past 30 years. PMID:20128467

  13. Research in vector control

    PubMed Central

    Quarterman, K. D.

    1963-01-01

    Current research on vector control is directed mainly at finding answers to the problem of resistance. Despite considerable advances in knowledge of the genetics, biochemistry, physiology, and ecology of resistant vectors, the only practical answer found so far has been the development of new, substitute insecticides. At present the operational needs of existing large-scale control or eradication programmes swallow up much of the funds, personnel and facilities that might otherwise be devoted to basic research. Moreover, to back up these programmes, there is a continuing need for applied research on such questions as the packaging of pesticides, improvements in equipment and the development of new formulations. The author gives examples of applied research already carried out or in progress and indicates some areas of both basic and applied research demanding urgent attention. Like other participants in the seminar, he stresses the fundamental importance of ecological studies. He also examines the concept of integrated vector control and points out that the realization of this concept presupposes close co-ordination between basic and applied research, laboratory and field studies, and investigations on chemical and non-chemical vector control measures. PMID:20604177

  14. Monolithic-integrated microlaser encoder.

    PubMed

    Sawada, R; Higurashi, E; Ito, T; Ohguchi, O; Tsubamoto, M

    1999-11-20

    We have developed an extremely small integrated microencoder whose sides are less than 1 mm long. It is 1/100 the size of conventional encoders. This microencoder consists of a laser diode, monolithic photodiodes, and fluorinated polyimide waveguides with total internal reflection mirrors. The instrument can measure the relative displacement between a grating scale and the encoder with a resolution of the order of 0.01 microm; it can also determine the direction in which the scale is moving. By using the two beams that were emitted from the two etched mirrors of the laser diode, by monolithic integration of the waveguide and photodiodes, and by fabrication of a step at the edge of the waveguide, we were able to eliminate conventional bulky optical components such as the beam splitter, the quarter-wavelength plate, bulky mirrors, and bulky photodetectors. PMID:18324228

  15. The PsychENCODE project

    PubMed Central

    Akbarian, Schahram; Liu, Chunyu; Knowles, James A; Vaccarino, Flora M; Farnham, Peggy J; Crawford, Gregory E; Jaffe, Andrew E; Pinto, Dalila; Dracheva, Stella; Geschwind, Daniel H; Mill, Jonathan; Nairn, Angus C; Abyzov, Alexej; Pochareddy, Sirisha; Prabhakar, Shyam; Weissman, Sherman; Sullivan, Patrick F; State, Matthew W; Weng, Zhiping; Peters, Mette A; White, Kevin P; Gerstein, Mark B; Senthil, Geetha; Lehner, Thomas; Sklar, Pamela; Sestan, Nenad

    2015-01-01

    Recent research on disparate psychiatric disorders has implicated rare variants in genes involved in global gene regulation and chromatin modification, as well as many common variants located primarily in regulatory regions of the genome. Understanding precisely how these variants contribute to disease will require a deeper appreciation for the mechanisms of gene regulation in the developing and adult human brain. The PsychENCODE project aims to produce a public resource of multidimensional genomic data using tissue- and cell type–specific samples from approximately 1,000 phenotypically well-characterized, high-quality healthy and disease-affected human post-mortem brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. We are beginning with a focus on autism spectrum disorder, bipolar disorder and schizophrenia, and expect that this knowledge will apply to a wide variety of psychiatric disorders. This paper outlines the motivation and design of PsychENCODE. PMID:26605881

  16. Nonconjugative Plasmids Encoding Sulfanilamide Resistance

    PubMed Central

    Mitsuhashi, Susumu; Inoue, Kunio; Inoue, Matsuhisa

    1977-01-01

    Nonconjugative plasmids encoding sulfanilamide (Sa) resistance were demonstrated at a high frequency in Shigella and Escherichia coli strains resistant to sulfanilamide. These Sa plasmids were all compatible with the standard plasmids used in compatibility testing. The sizes of seven Sa plasmids were measured by electron microscopy and ranged from 1.79 to 2.08 μm, corresponding to 3.5 to 3.9 megadaltons. Images PMID:334067

  17. Lessons from modENCODE.

    PubMed

    Brown, James B; Celniker, Susan E

    2015-01-01

    The modENCODE (Model Organism Encyclopedia of DNA Elements) Consortium aimed to map functional elements-including transcripts, chromatin marks, regulatory factor binding sites, and origins of DNA replication-in the model organisms Drosophila melanogaster and Caenorhabditis elegans. During its five-year span, the consortium conducted more than 2,000 genome-wide assays in developmentally staged animals, dissected tissues, and homogeneous cell lines. Analysis of these data sets provided foundational insights into genome, epigenome, and transcriptome structure and the evolutionary turnover of regulatory pathways. These studies facilitated a comparative analysis with similar data types produced by the ENCODE Consortium for human cells. Genome organization differs drastically in these distant species, and yet quantitative relationships among chromatin state, transcription, and cotranscriptional RNA processing are deeply conserved. Of the many biological discoveries of the modENCODE Consortium, we highlight insights that emerged from integrative studies. We focus on operational and scientific lessons that may aid future projects of similar scale or aims in other, emerging model systems. PMID:26133010

  18. Methods of treating Parkinson's disease using viral vectors

    DOEpatents

    Bankiewicz, Krys; Cunningham, Janet

    2012-11-13

    Methods of delivering viral vectors, particularly recombinant AAV virions, to the central nervous system (CNS) are provided for the treatment of CNS disorders, particularly those disorders which involve the neurotransmitter dopamine. The methods entail providing rAAV virions that comprise a transgene encoding aromatic amino acid decarboxylase (AADC) and administering the virions to the brain of a mammal using a non-manual pump.

  19. Efficient morse decompositions of vector fields.

    PubMed

    Chen, Guoning; Mischaikow, Konstantin; Laramee, Robert S; Zhang, Eugene

    2008-01-01

    Existing topology-based vector field analysis techniques rely on the ability to extract the individual trajectories such as fixed points, periodic orbits, and separatrices that are sensitive to noise and errors introduced by simulation and interpolation. This can make such vector field analysis unsuitable for rigorous interpretations. We advocate the use of Morse decompositions, which are robust with respect to perturbations, to encode the topological structures of a vector field in the form of a directed graph, called a Morse connection graph (MCG). While an MCG exists for every vector field, it need not be unique. Previous techniques for computing MCG's, while fast, are overly conservative and usually results in MCG's that are too coarse to be useful for the applications. To address this issue, we present a new technique for performing Morse decomposition based on the concept of tau-maps, which typically provides finer MCG's than existing techniques. Furthermore, the choice of tau provides a natural tradeoff between the fineness of the MCG's and the computational costs. We provide efficient implementations of Morse decomposition based on tau-maps, which include the use of forward and backward mapping techniques and an adaptive approach in constructing better approximations of the images of the triangles in the meshes used for simulation.. Furthermore, we propose the use of spatial tau-maps in addition to the original temporal tau-maps. These techniques provide additional trade-offs between the quality of the MCGs and the speed of computation. We demonstrate the utility of our technique with various examples in the plane and on surfaces including engine simulation data sets. PMID:18467759

  20. Preloadable vector sensitive latch

    NASA Technical Reports Server (NTRS)

    Acres, William R. (Inventor)

    1987-01-01

    A preloadable vector-sensitive latch which automatically releases when the force vector from a latch memebr reaches a specified release angle is presented. In addition, it contains means to remove clearance between the latched members and to preload the latch to prevent separation at angles less than the specified release angle. The latch comprises a triangular main link, a free link connected between a first corner of the main link and a yoke member, a housing, and an actuator connected between the yoke member and the housing. A return spring bias means connects the main link to a portion of the housing. A second corner of the main link is slidably and pivotally connected to the housing via a slot in a web portion of the housing. The latch housing has a rigid docking ring alignable with a mating locking ring which is engageable by a locking roller journalled on the third corner of the triangular main link.

  1. Vector Magnetograph Design

    NASA Technical Reports Server (NTRS)

    Chipman, Russell A.

    1996-01-01

    This report covers work performed during the period of November 1994 through March 1996 on the design of a Space-borne Solar Vector Magnetograph. This work has been performed as part of a design team under the supervision of Dr. Mona Hagyard and Dr. Alan Gary of the Space Science Laboratory. Many tasks were performed and this report documents the results from some of those tasks, each contained in the corresponding appendix. Appendices are organized in chronological order.

  2. Some experiences with Krylov vectors and Lanczos vectors

    NASA Technical Reports Server (NTRS)

    Craig, Roy R., Jr.; Su, Tzu-Jeng; Kim, Hyoung M.

    1993-01-01

    This paper illustrates the use of Krylov vectors and Lanczos vectors for reduced-order modeling in structural dynamics and for control of flexible structures. Krylov vectors and Lanczos vectors are defined and illustrated, and several applications that have been under study at The University of Texas at Austin are reviewed: model reduction for undamped structural dynamics systems, component mode synthesis using Krylov vectors, model reduction of damped structural dynamics systems, and one-sided and two-sided unsymmetric block-Lanczos model-reduction algorithms.

  3. Permissive growth of human adenovirus type 4 vaccine strain-based vector in porcine cell lines.

    PubMed

    Gao, Dong-Sheng; Li, Xiao-Jing; Wan, Wen-Yan; Li, Hong-Jie; Wang, Xiao-Xue; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-02-01

    In recent years, there has been considerable interest in using adenoviruses as live vectors to develop recombinant vaccines. Previous studies have demonstrated the safety and effectiveness of HIV/SIV and influenza vaccine candidates based on human adenovirus type 4 (Ad4) replication-competent vectors in rhesus macaque and human model. To explore the possibility of human Ad4 vaccine strain used as a vector in developing porcine vaccines, the growth properties of replication-competent human Ad4 vaccine strain recombinant encoding EGFP in different porcine cell lines were investigated. All tested cell lines are permissive for Ad4 vaccine strain vector with varied replication efficiency. Thus, human Ad4 based vectors would be promising supplement to adenovirus vectors as a delivery vehicle for recombinant vaccines in swine industry. PMID:26850542

  4. RSF1010-based shuttle vectors for cloning and expression in Pasteurella multocida.

    PubMed

    Lee, M D; Henk, A D

    1997-03-01

    The broad host-range cloning vectors, pJRD215 and pMMB67EH, were evaluated for stability and cloning efficiency in Pasteurella multocida. Transformation of P. multocida by electroporation was unreliable and poorly efficient regardless of whether the transforming DNA was isolated from E. coli or P. multocida. Both vectors contain a mob site that enabled transfer by conjugation from E. coli to P. multocida with high efficiency. Kanamycin, streptomycin, and ampicillin resistance encoded by the vectors were expressed in P. multocida. LacZ was cloned in pMMB67EH, an expression vector, and was transferred to P. multocida by conjugation. The transconjugants expressed a functional beta-galactosidase as determined by o-nitrophenyl-beta-D-galactopyranoside (ONPG) test. We propose the use of these cosmid and expression vectors as a shuttle vectors for cloning in P. multocida. PMID:9100336

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

    PubMed

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

    2008-02-01

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

  6. Isomap based supporting vector machine

    NASA Astrophysics Data System (ADS)

    Liang, W. N.

    2015-12-01

    This research presents a new isomap based supporting vector machine method. Isomap is a dimension reduction method which is able to analyze nonlinear relationship of data on manifolds. Accordingly, support vector machine is established on the isomap manifold to classify given and predict unknown data. A case study of the isomap based supporting vector machine for environmental planning problems is conducted.

  7. Hall effect encoding of brushless dc motors

    NASA Technical Reports Server (NTRS)

    Berard, C. A.; Furia, T. J.; Goldberg, E. A.; Greene, R. C.

    1970-01-01

    Encoding mechanism integral to the motor and using the permanent magnets embedded in the rotor eliminates the need for external devices to encode information relating the position and velocity of the rotating member.

  8. Novel optical encoder for harsh environments

    NASA Astrophysics Data System (ADS)

    Kress, Bernard; Mueller, Ulrich; Brac-de-la-Perriere, Vincent

    2014-09-01

    We are presenting a new optical encoder architecture for shaft encoding, both in incremental and absolute modes. This encoder is based on a diffractive optics technology platform. We have developed various disk based rotary diffractive encoders previously. This encoder is different in the way it is not a disk composed of successive gratings or computer generated holograms, but rather composed of a single element placed on the shaft. It is thus best suited for hollow shaft or end of shaft applications such as in encoder controlled electrical motors. This new architecture aims at solving some of the problems encountered with previous implementations of diffractive encoders such as disk wobble, disk to shaft centering and also encoding in harsh environments.

  9. Vector lifting scheme for phase-shifting holographic data compression

    NASA Astrophysics Data System (ADS)

    Xing, Yafei; Kaaniche, Mounir; Pesquet-Popescu, Béatrice; Dufaux, Frédéric

    2014-11-01

    With the increasing interest in holography in three-dimensional imaging applications, the use of hologram compression techniques is mandatory for storage and transmission purposes. The state-of-the-art approach aims at encoding separately each interference pattern by resorting to common still-image compression techniques. Contrary to such an independent scheme, a joint hologram coding scheme is investigated in this paper. More precisely, instead of encoding all the interference patterns, it is proposed that only two sets of data be compressed by taking into account the redundancies existing among them. The resulting data are encoded by applying a joint multiscale decomposition based on the vector lifting concept. Experimental results show the benefits that can be drawn from the proposed hologram compression approach.

  10. Image Compression Using Vector Quantization with Variable Block Size Division

    NASA Astrophysics Data System (ADS)

    Matsumoto, Hiroki; Kichikawa, Fumito; Sasazaki, Kazuya; Maeda, Junji; Suzuki, Yukinori

    In this paper, we propose a method for compressing a still image using vector quantization (VQ). Local fractal dimension (LFD) is computed to divided an image into variable block size. The LFD shows the complexity of local regions of an image, so that a region of an image that shows higher LFD values than those of other regions is partitioned into small blocks of pixels, while a region of an image that shows lower LFD values than those of other regions is partitioned into large blocks. Furthermore, we developed a division and merging algorithm to decrease the number of blocks to encode. This results in improvement of compression rate. We construct code books for respective blocks sizes. To encode an image, a block of pixels is transformed by discrete cosine transform (DCT) and the closest vector is chosen from the code book (CB). In decoding, the code vector corresponding to the index is selected from the CB and then the code vector is transformed by inverse DCT to reconstruct a block of pixels. Computational experiments were carried out to show the effectiveness of the proposed method. Performance of the proposed method is slightly better than that of JPEG. In the case of learning images to construct a CB being different from test images, the compression rate is comparable to compression rates of methods proposed so far, while image quality evaluated by NPIQM (normalized perceptual image quality measure) is almost the highest step. The results show that the proposed method is effective for still image compression.

  11. Persistent, circulative transmission of begomoviruses by whitefly vectors.

    PubMed

    Rosen, Ran; Kanakala, Surapathrudu; Kliot, Adi; Cathrin Pakkianathan, Britto; Farich, Basheer Abu; Santana-Magal, Nadine; Elimelech, Meytar; Kontsedalov, Svetlana; Lebedev, Galina; Cilia, Michelle; Ghanim, Murad

    2015-12-01

    Begomoviruses comprise an emerging and economically important group of plant viruses exclusively transmitted by the sweetpotato whitefly Bemisia tabaci in many regions of the world. The past twenty years have witnessed significant progress in studying the molecular interactions between members of this virus group and B. tabaci. Mechanisms and proteins encoded by the insect vector and its bacterial symbionts, which have been shown to be important for virus transmission, have been identified and thoroughly studied. Despite the economic importance of this group of viruses and their impact on the global agriculture, progress in investigating the virus-vector interactions is moving slowly when compared with similar virus-vector systems in plants and animals. Major advances in this field and future perspectives will be discussed in this review. PMID:26196230

  12. Encoding Cortical Dynamics in Sparse Features

    PubMed Central

    Khan, Sheraz; Lefèvre, Julien; Baillet, Sylvain; Michmizos, Konstantinos P.; Ganesan, Santosh; Kitzbichler, Manfred G.; Zetino, Manuel; Hämäläinen, Matti S.; Papadelis, Christos; Kenet, Tal

    2014-01-01

    Distributed cortical solutions of magnetoencephalography (MEG) and electroencephalography (EEG) exhibit complex spatial and temporal dynamics. The extraction of patterns of interest and dynamic features from these cortical signals has so far relied on the expertise of investigators. There is a definite need in both clinical and neuroscience research for a method that will extract critical features from high-dimensional neuroimaging data in an automatic fashion. We have previously demonstrated the use of optical flow techniques for evaluating the kinematic properties of motion field projected on non-flat manifolds like in a cortical surface. We have further extended this framework to automatically detect features in the optical flow vector field by using the modified and extended 2-Riemannian Helmholtz–Hodge decomposition (HHD). Here, we applied these mathematical models on simulation and MEG data recorded from a healthy individual during a somatosensory experiment and an epilepsy pediatric patient during sleep. We tested whether our technique can automatically extract salient dynamical features of cortical activity. Simulation results indicated that we can precisely reproduce the simulated cortical dynamics with HHD; encode them in sparse features and represent the propagation of brain activity between distinct cortical areas. Using HHD, we decoded the somatosensory N20 component into two HHD features and represented the dynamics of brain activity as a traveling source between two primary somatosensory regions. In the epilepsy patient, we displayed the propagation of the epileptic activity around the margins of a brain lesion. Our findings indicate that HHD measures computed from cortical dynamics can: (i) quantitatively access the cortical dynamics in both healthy and disease brain in terms of sparse features and dynamic brain activity propagation between distinct cortical areas, and (ii) facilitate a reproducible, automated analysis of experimental and clinical

  13. Encoding cortical dynamics in sparse features.

    PubMed

    Khan, Sheraz; Lefèvre, Julien; Baillet, Sylvain; Michmizos, Konstantinos P; Ganesan, Santosh; Kitzbichler, Manfred G; Zetino, Manuel; Hämäläinen, Matti S; Papadelis, Christos; Kenet, Tal

    2014-01-01

    Distributed cortical solutions of magnetoencephalography (MEG) and electroencephalography (EEG) exhibit complex spatial and temporal dynamics. The extraction of patterns of interest and dynamic features from these cortical signals has so far relied on the expertise of investigators. There is a definite need in both clinical and neuroscience research for a method that will extract critical features from high-dimensional neuroimaging data in an automatic fashion. We have previously demonstrated the use of optical flow techniques for evaluating the kinematic properties of motion field projected on non-flat manifolds like in a cortical surface. We have further extended this framework to automatically detect features in the optical flow vector field by using the modified and extended 2-Riemannian Helmholtz-Hodge decomposition (HHD). Here, we applied these mathematical models on simulation and MEG data recorded from a healthy individual during a somatosensory experiment and an epilepsy pediatric patient during sleep. We tested whether our technique can automatically extract salient dynamical features of cortical activity. Simulation results indicated that we can precisely reproduce the simulated cortical dynamics with HHD; encode them in sparse features and represent the propagation of brain activity between distinct cortical areas. Using HHD, we decoded the somatosensory N20 component into two HHD features and represented the dynamics of brain activity as a traveling source between two primary somatosensory regions. In the epilepsy patient, we displayed the propagation of the epileptic activity around the margins of a brain lesion. Our findings indicate that HHD measures computed from cortical dynamics can: (i) quantitatively access the cortical dynamics in both healthy and disease brain in terms of sparse features and dynamic brain activity propagation between distinct cortical areas, and (ii) facilitate a reproducible, automated analysis of experimental and clinical

  14. Jam-resistant speech encoding

    NASA Astrophysics Data System (ADS)

    Poole, M. A.; Rifkin, R.

    1983-06-01

    This report describes techniques that provide increased jam resistance for digitized speech. Methods for increasing the jam resistance of pulse code modulated data are analyzed and evaluated in listener tests. Special emphasis is placed on new voice encoding approaches that take advantage of a spread spectrum system with a variable (or multiple)-data-rate/variable (or multiple)-AJ capability. Methods for matching a source to a channel in a jamming environment are investigated. Several techniques that provide about a 4 dB increase in jam resistance have been identified.

  15. Cistrons encoding Escherichia coli heat-labile toxin.

    PubMed Central

    Dallas, W S; Gill, D M; Falkow, S

    1979-01-01

    The structure and products of the two cistrons encoding the Escherichia coli heat-labile toxin (LT) were studied. The LT deoxyribonucleic acid (DNA) region had been isolated as part of a DNA fragment from the plasmid P307, and this fragment was joined to the cloning vector pBR313. Deletion mutations of various lengths were introduced into the LT DNA region and into the adjacent DNA sequences. Analysis of the deletions indicated that the maximum size of the LT DNA region was 1.2 x 10(6) daltons. Two proteins of 11,500 daltons and 25,500 daltons had been shown to be encoded by the LT DNA region. The functions of these LT gene products were investigated. The 11,500-dalton protein had an adsorption activity for Y-1 adrenal cells, and this protein was shown to form aggregates of four or five monomers. The 25,500-dalton protein was shown to have an adenylate cyclase-activating activity. The two cistrons encoding for each of the LT proteins have been located on a genetic map of the LT DNA region. Both cistrons are probably transcribed from the same promoter. Images PMID:383697

  16. Towards Encoding Background Knowledge with Temporal Extent into Neural Networks

    NASA Astrophysics Data System (ADS)

    Anh, Han; Marques, Nuno C.

    Neuro-symbolic integration merges background knowledge and neural networks to provide a more effective learning system. It uses the Core Method as a means to encode rules. However, this method has several drawbacks in dealing with rules that have temporal extent. First, it demands some interface with the world which buffers the input patterns so they can be represented all at once. This imposes a rigid limit on the duration of patterns and further suggests that all input vectors be the same length. These are troublesome in domains where one would like comparable representations for patterns that are of variable length (e.g. language). Second, it does not allow dynamic insertion of rules conveniently. Finally and also most seriously, it cannot encode rules having preconditions satisfied at non-deterministic time points - an important class of rules. This paper presents novel methods for encoding such rules, thereby improves and extends the power of the state-of-the-art neuro-symbolic integration.

  17. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

    PubMed

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær; Rank, Julie; Hansen, Bjarne Gram; Andersen, Mikael Rørdam; Mortensen, Uffe Hasbro

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors. PMID:24879460

  18. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    PubMed Central

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær; Rank, Julie; Hansen, Bjarne Gram; Andersen, Mikael Rørdam; Mortensen, Uffe Hasbro

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors. PMID:24879460

  19. Time Course of Grammatical Encoding in Agrammatism

    ERIC Educational Resources Information Center

    Lee, Jiyeon

    2011-01-01

    Producing a sentence involves encoding a preverbal message into a grammatical structure by retrieving lexical items and integrating them into a functional (semantic-to-grammatical) structure. Individuals with agrammatism are impaired in this grammatical encoding process. However, it is unclear what aspect of grammatical encoding is impaired and…

  20. Schematic driven layout of Reed Solomon encoders

    NASA Technical Reports Server (NTRS)

    Arave, Kari; Canaris, John; Miles, Lowell; Whitaker, Sterling

    1992-01-01

    Two Reed Solomon error correcting encoders are presented. Schematic driven layout tools were used to create the encoder layouts. Special consideration had to be given to the architecture and logic to provide scalability of the encoder designs. Knowledge gained from these projects was used to create a more flexible schematic driven layout system.

  1. Vector representation of tourmaline compositions

    NASA Technical Reports Server (NTRS)

    Burt, Donald M.

    1989-01-01

    The vector method for representing mineral compositions of amphibole and mica groups is applied to the tourmaline group. Consideration is given to the methods for drawing the relevant vector diagrams, relating the exchange vectors to one another, and contouring the diagrams for constant values of Na, Ca, Li, Fe, Mg, Al, Si, and OH. The method is used to depict a wide range of possible tourmaline end-member compositions and solid solutions, starting from a single point. In addition to vector depictions of multicomponent natural tourmalines, vectors are presented for simpler systems such as (Na,Al)-tourmalines, alkali-free tourmalines, and elbaites.

  2. Vector Theory of Ultrasonic Imaging

    NASA Astrophysics Data System (ADS)

    Gan, W. S.

    So far, works on ultrasonic diffraction imaging are based on scalar theory of sound wave. This is not correct as sound has vector nature. But when sound propagates in solids, its vector nature has to be considered as polarization occurs and transverse wave as well as longitudinal wave will appear. Vector theory is especially needed when the obstacle size is smaller than the wavelength. We use the Smythe-Kirchhoff approach for the vector theory of diffraction. We derive the image formation theory based on the vector diffraction theory. The effect of polarization on acoustical imaging is discussed.

  3. Distributed Signal Decorrelation and Detection in Multi View Camera Networks Using the Vector Sparse Matrix Transform.

    PubMed

    Bachega, Leonardo R; Hariharan, Srikanth; Bouman, Charles A; Shroff, Ness B

    2015-12-01

    This paper introduces the vector sparse matrix transform (vector SMT), a new decorrelating transform suitable for performing distributed processing of high-dimensional signals in sensor networks. We assume that each sensor in the network encodes its measurements into vector outputs instead of scalar ones. The proposed transform decorrelates a sequence of pairs of vector outputs, until these vectors are decorrelated. In our experiments, we simulate distributed anomaly detection by a network of cameras, monitoring a spatial region. Each camera records an image of the monitored environment from its particular viewpoint and outputs a vector encoding the image. Our results, with both artificial and real data, show that the proposed vector SMT transform effectively decorrelates image measurements from the multiple cameras in the network while maintaining low overall communication energy consumption. Since it enables joint processing of the multiple vector outputs, our method provides significant improvements to anomaly detection accuracy when compared with the baseline case when the images are processed independently. PMID:26415179

  4. Semi-fixed-length motion vector coding for H.263-based low bit rate video compression.

    PubMed

    Côté, G; Gallant, M; Kossentini, F

    1999-01-01

    We present a semi-fixed-length motion vector coding method for H.263-based low bit rate video compression. The method exploits structural constraints within the motion field. The motion vectors are encoded using semi-fixed-length codes, yielding essentially the same levels of rate-distortion performance and subjective quality achieved by H.263's Huffman-based variable length codes in a noiseless environment. However, such codes provide substantially higher error resilience in a noisy environment. PMID:18267417

  5. Vector ecology of equine piroplasmosis.

    PubMed

    Scoles, Glen A; Ueti, Massaro W

    2015-01-01

    Equine piroplasmosis is a disease of Equidae, including horses, donkeys, mules, and zebras, caused by either of two protozoan parasites, Theileria equi or Babesia caballi. These parasites are biologically transmitted between hosts via tick vectors, and although they have inherent differences they are categorized together because they cause similar pathology and have similar morphologies, life cycles, and vector relationships. To complete their life cycle, these parasites must undergo a complex series of developmental events, including sexual-stage development in their tick vectors. Consequently, ticks are the definitive hosts as well as vectors for these parasites, and the vector relationship is restricted to a few competent tick species. Because the vector relationship is critical to the epidemiology of these parasites, we highlight current knowledge of the vector ecology of these tick-borne equine pathogens, emphasizing tick transmissibility and potential control strategies to prevent their spread. PMID:25564746

  6. Angular encoding in attosecond recollision

    NASA Astrophysics Data System (ADS)

    Kitzler, Markus; Xie, Xinhua; Roither, Stefan; Scrinzi, Armin; Baltuska, Andrius

    2008-02-01

    We describe a general concept of using the spatial information encoded in the time-dependent polarization of high harmonic radiation generated by orthogonally polarized two-color laser fields. The main properties of recolliding electron wave packets driven by such fields are reviewed. It is shown that in addition to the recollision energy the angle of recollision of such wave packets, which is directly mapped onto the polarization direction of the emitted high harmonic radiation, varies on a sub-laser-cycle time-scale. Thus, a mapping between the polarization angle and the frequency of the emitted radiation is established on an attosecond time scale. While the polarization angle encodes the spatial properties of the recollision process, the frequency is linked to time via the well-known dispersion relations of high harmonic generation. Based on these principles, we show that in combination with polarization selective detection the use of orthogonally polarized drive pulses for high harmonic generation permit one to construct spatially resolved attosecond measurements. Here, we present two examples of possible applications: (i) a method for isolating a single attosecond pulse from an attosecond pulse train which is more efficient than the cut-off selection method, and (ii) a technique for orbital tomography of molecules with attosecond resolution.

  7. Reconstruction of turbo-code encoders

    NASA Astrophysics Data System (ADS)

    Barbier, Johann

    2005-06-01

    Turbo-code encoders are one of the spreadest family of error correcting codes used in the communication's world, especially in space transmissions. This paper presents an efficient technique to reconstruct turbo-code encoders which allows a passive adversary, with only few bits of an intercepted message encoded by the target turbocode encoder, to determine the parameters of the turbo-code encoder used, and therefore to decode online the communications. Thereby, our results confirm that keeping secret the parameters of turbo-code encoders can not be considered as a cryptographically way to ensure confidentiality. The starting point of our work is algorithms due to Filiol which enable to find the parameters of each convolutional encoder in the turbo-code encoder. Then, we recover the interleaver with two new algorithms, the first one based on the dynamic trie structure and the second one on a first order statistical test. The first algorithm is dedicated to noiseless channels. The asymptotic complexity of the complete process is O(n4) when a n2-bit message is available to attack a n-bit turbo-code encoder. The second algorithm works for every kind of channel and the noise does not matter much. Additionally, we present experimental results which underline the right detection threshold to use to recover the interleaver with a high probability. Furthermore, this method also works for turbo-code encoders composed of punctured convolutional encoders.

  8. Aerodynamics of thrust vectoring

    NASA Technical Reports Server (NTRS)

    Tseng, J. B.; Lan, C. Edward

    1989-01-01

    Thrust vectoring as a means to enhance maneuverability and aerodynamic performane of a tactical aircraft is discussed. This concept usually involves the installation of a multifunction nozzle. With the nozzle, the engine thrust can be changed in direction without changing the attitude of the aircraft. Change in the direction of thrust induces a significant change in the aerodynamic forces on the aircraft. Therefore, this device can be used for lift-augmenting as well as stability and control purposes. When the thrust is deflected in the longitudinal direction, the lift force and the pitching stability can be manipulated, while the yawing stability can be controlled by directing the thrust in the lateral direction.

  9. Molecular mechanisms for protein-encoded inheritance

    SciTech Connect

    Wiltzius, Jed J.W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

    2009-12-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of {beta}-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct {beta}-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  10. Horse cDNA clones encoding two MHC class I genes

    SciTech Connect

    Barbis, D.P.; Maher, J.K.; Stanek, J.; Klaunberg, B.A.; Antczak, D.F.

    1994-12-31

    Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

  11. Vector-vector production in photon-photon interactions

    NASA Astrophysics Data System (ADS)

    Ronan, Micheal T.

    1989-04-01

    Measurements of exclusive untagged ρ0ρ0,ρφ,K*K¯*, and ρω production and tagged ρ0ρ0 production in photon-photon interactions by the TPC/Two-Gamma experiment are reviewed. Comparisons to the results of other experiments and to models of vector-vector production are made. Fits to the data following a four quark model prescription for vector meson pair production are also presented.

  12. Scalar-vector quantization of medical images.

    PubMed

    Mohsenian, N; Shahri, H; Nasrabadi, N M

    1996-01-01

    A new coding scheme based on the scalar-vector quantizer (SVQ) is developed for compression of medical images. The SVQ is a fixed rate encoder and its rate-distortion performance is close to that of optimal entropy-constrained scalar quantizers (ECSQs) for memoryless sources. The use of a fixed-rate quantizer is expected to eliminate some of the complexity of using variable-length scalar quantizers. When transmission of images over noisy channels is considered, our coding scheme does not suffer from error propagation that is typical of coding schemes using variable-length codes. For a set of magnetic resonance (MR) images, coding results obtained from SVQ and ECSQ at low bit rates are indistinguishable. Furthermore, our encoded images are perceptually indistinguishable from the original when displayed on a monitor. This makes our SVQ-based coder an attractive compression scheme for picture archiving and communication systems (PACS). PACS are currently under study for use in an all-digital radiology environment in hospitals, where reliable transmission, storage, and high fidelity reconstruction of images are desired. PMID:18285124

  13. Novelty's effect on memory encoding.

    PubMed

    Rangel-Gomez, Mauricio; Janenaite, Sigita; Meeter, Martijn

    2015-07-01

    It is often thought that novelty benefits memory formation. However, support for this idea mostly comes from paradigms that are open to alternative explanations. In the present study we manipulated novelty in a word-learning task through task-irrelevant background images. These background images were either standard (presented repeatedly), or novel (presented only once). Two types of background images were used: Landscape pictures and fractals. EEG was also recorded during encoding. Contrary to the idea that novelty aids memory formation, memory performance was not affected by the novelty of the background. In the evoked response potentials, we found evidence of distracting effects of novelty: both the N1 and P3b components were smaller to words studied with novel backgrounds, and the amplitude of the N2b component correlated negatively with subsequent retrieval. We conclude that although evidence from other studies does suggest benefits on a longer time scale, novelty has no instantaneous benefits for learning. PMID:26005196

  14. Digital plus analog output encoder

    NASA Technical Reports Server (NTRS)

    Hafle, R. S. (Inventor)

    1976-01-01

    The disclosed encoder is adapted to produce both digital and analog output signals corresponding to the angular position of a rotary shaft, or the position of any other movable member. The digital signals comprise a series of binary signals constituting a multidigit code word which defines the angular position of the shaft with a degree of resolution which depends upon the number of digits in the code word. The basic binary signals are produced by photocells actuated by a series of binary tracks on a code disc or member. The analog signals are in the form of a series of ramp signals which are related in length to the least significant bit of the digital code word. The analog signals are derived from sine and cosine tracks on the code disc.

  15. Engineering Genetically Encoded FRET Sensors

    PubMed Central

    Lindenburg, Laurens; Merkx, Maarten

    2014-01-01

    Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening. PMID:24991940

  16. Attenuated Measles Virus as a Vaccine Vector

    PubMed Central

    Zuniga, Armando; Wang, ZiLi; Liniger, Matthias; Hangartner, Lars; Caballero, Michael; Pavlovic, Jovan; Wild, Peter; Viret, Jean Francois; Glueck, Reinhard; Billeter, Martin A.; Naim, Hussein Y.

    2013-01-01

    Live attenuated measles virus (MV) vaccines have an impressive record of safety, efficacy and ability to induce life-long immunity against measles infection. Using reverse genetics technology, such negative-strand RNA viruses can now be rescued from cloned DNA. This technology allows the insertion of exogenous genes encoding foreign antigens into the MV genome in such a way that they can be expressed by the MV vaccine strain, without affecting virus structure, propagation and cell targeting. Recombinant viruses rescued from cloned cDNA induce immune responses against both measles virus and the cloned antigens. The tolerability of MV to gene(s) insertion makes it an attractive flexible vector system, especially if broad immune responses are required. The fact that measles replication strictly occurs in the cytoplasm of infected cells without DNA intermediate has important biosafety implications and adds to the attractiveness of MV as a vector. In this article we report the characteristics of reporter gene expression (GFP, LacZ and CAT) and the biochemical, biophysical and immunological properties of recombinant MV expressing heterologous antigens of simian immunogeficiency virus (SIV). PMID:17303293

  17. Gene encoding plant asparagine synthetase

    DOEpatents

    Coruzzi, Gloria M.; Tsai, Fong-Ying

    1993-10-26

    The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

  18. A novel Streptomyces spp. integration vector derived from the S. venezuelae phage, SV1

    PubMed Central

    2014-01-01

    Background Integrating vectors based on the int/attP loci of temperate phages are convenient and used widely, particularly for cloning genes in Streptomyces spp. Results We have constructed and tested a novel integrating vector based on g27, encoding integrase, and attP site from the phage, SV1. This plasmid, pBF3 integrates efficiently in S. coelicolor and S. lividans but surprisingly fails to generate stable integrants in S. venezuelae, the natural host for phage SV1. Conclusion pBF3 promises to be a useful addition to the range of integrating vectors currently available for Streptomyces molecular genetics. PMID:24885867

  19. Multiscale hierarchical support vector clustering

    NASA Astrophysics Data System (ADS)

    Hansen, Michael Saas; Holm, David Alberg; Sjöstrand, Karl; Ley, Carsten Dan; Rowland, Ian John; Larsen, Rasmus

    2008-03-01

    Clustering is the preferred choice of method in many applications, and support vector clustering (SVC) has proven efficient for clustering noisy and high-dimensional data sets. A method for multiscale support vector clustering is demonstrated, using the recently emerged method for fast calculation of the entire regularization path of the support vector domain description. The method is illustrated on artificially generated examples, and applied for detecting blood vessels from high resolution time series of magnetic resonance imaging data. The obtained results are robust while the need for parameter estimation is reduced, compared to support vector clustering.

  20. VLSI Processor For Vector Quantization

    NASA Technical Reports Server (NTRS)

    Tawel, Raoul

    1995-01-01

    Pixel intensities in each kernel compared simultaneously with all code vectors. Prototype high-performance, low-power, very-large-scale integrated (VLSI) circuit designed to perform compression of image data by vector-quantization method. Contains relatively simple analog computational cells operating on direct or buffered outputs of photodetectors grouped into blocks in imaging array, yielding vector-quantization code word for each such block in sequence. Scheme exploits parallel-processing nature of vector-quantization architecture, with consequent increase in speed.

  1. Localization and vector spherical harmonics

    NASA Astrophysics Data System (ADS)

    von Brecht, James H.

    2016-01-01

    This paper establishes the following localization property for vector spherical harmonics: a wide class of non-local, vector-valued operators reduce to local, multiplication-type operations when applied to a vector spherical harmonic. As localization occurs in a very precise, quantifiable and explicitly computable fashion, the localization property provides a set of useful formulae for analyzing vector-valued fractional diffusion and non-local differential equations defined on S d - 1. As such analyses require a detailed understanding of operators for which localization occurs, we provide several applications of the result in the context of non-local differential equations.

  2. The MSFC Vector Magnetograph

    NASA Astrophysics Data System (ADS)

    Hagyard, M. J.; Cumings, N. P.; West, E. A.; Smith, J. E.

    1982-09-01

    The NASA/Marshall Space Flight Center's solar vector magnetograph system is described; this system allows measurements of all components of the Sun's photospheric magnetic field over a 5 × 5 or 2.0 × 2.0 arc min square field-of-view with an optimum time resolution of ˜ 100 s and an optimum signal-to-noise of ˜1600. The basic system components are described, including the optics, detector, digital system and associated electronics. Automatic sequencing and control functions are outlined as well as manual selections of system parameters which afford unique system flexibility. Results of system calibration and performance are presented, including linearity, dynamic range, uniformity, spatial and spectral resolutions, signal-to-noise, electro-optical retardation and polarization calibration. Scientific investigations which utilize the unique characteristics of the instrument are described and typical results are presented.

  3. The MSFC vector magnetograph

    NASA Astrophysics Data System (ADS)

    Hagyard, M. J.; Cumings, N. P.; West, E. A.

    1981-02-01

    The NASA/Marshall Space Flight Center's solar vector magnetograph system allows measurements of all components of the Sun's photospheric magnetic field over a 5 x 5 or 2.5 x 2.5 arc min square field of view with an optimum time resolution of approximately 100 sec and an optimum signal-to-noise of approximately 1000. The basic system components are described, including the optics, detector, digital system, and associated electronics. Automatic sequencing and control functions are outlined as well as manual selections of system parameters which afford unique system flexibility. Results of system calibration and performance are presented, including linearity, dynamic range, uniformity, spatial and spectral resolutions, signal-to-noise, electro-optical retardation and polarization calibration.

  4. Multistage vector (MSV) therapeutics.

    PubMed

    Wolfram, Joy; Shen, Haifa; Ferrari, Mauro

    2015-12-10

    One of the greatest challenges in the field of medicine is obtaining controlled distribution of systemically administered therapeutic agents within the body. Indeed, biological barriers such as physical compartmentalization, pressure gradients, and excretion pathways adversely affect localized delivery of drugs to pathological tissue. The diverse nature of these barriers requires the use of multifunctional drug delivery vehicles that can overcome a wide range of sequential obstacles. In this review, we explore the role of multifunctionality in nanomedicine by primarily focusing on multistage vectors (MSVs). The MSV is an example of a promising therapeutic platform that incorporates several components, including a microparticle, nanoparticles, and small molecules. In particular, these components are activated in a sequential manner in order to successively address transport barriers. PMID:26264836

  5. Solar imaging vector magnetograph

    NASA Technical Reports Server (NTRS)

    Canfield, Richard C.

    1993-01-01

    This report describes an instrument which has been constructed at the University of Hawaii to make observations of the magnetic field in solar active regions. Detailed knowledge of active region magnetic structures is crucial to understanding many solar phenomena, because the magnetic field both defines the morphology of structures seen in the solar atmosphere and is the apparent energy source for solar flares. The new vector magnetograph was conceived in response to a perceived discrepancy between the capabilities of X ray imaging telescopes to be operating during the current solar maximum and those of existing magnetographs. There were no space-based magnetographs planned for this period; the existing ground-based instruments variously suffered from lack of sensitivity, poor time resolution, inadequate spatial resolution or unreliable sites. Yet the studies of flares and their relationship to the solar corona planned for the 1991-1994 maximum absolutely required high quality vector magnetic field measurements. By 'vector' measurements we mean that the observation attempts to deduce the complete strength and direction of the field at the measurement site, rather than just the line of sight component as obtained by a traditional longitudinal magnetograph. Knowledge of the vector field permits one to calculate photospheric electric currents, which might play a part in heating the corona, and to calculate energy stored in coronal magnetic fields as the result of such currents. Information about the strength and direction of magnetic fields in the solar atmosphere can be obtained in a number of ways, but quantitative data is best obtained by observing Zeeman-effect polarization in solar spectral lines. The technique requires measuring the complete state of polarization at one or more wavelengths within a magnetically sensitive line of the solar spectrum. This measurement must be done for each independent spatial point for which one wants magnetic field data. All the

  6. Enhanced Transgene Expression from Recombinant Single-Stranded D-Sequence-Substituted Adeno-Associated Virus Vectors in Human Cell Lines In Vitro and in Murine Hepatocytes In Vivo

    PubMed Central

    Wang, Yuan; Lu, Yuan; Wang, Lina; Jayandharan, Giridhara R.; Aslanidi, George V.; Li, Baozheng; Cheng, Binbin; Ma, Wenqin; Lentz, Thomas; Ling, Changquan; Xiao, Xiao; Samulski, R. Jude; Muzyczka, Nicholas

    2014-01-01

    ABSTRACT We have previously reported that the removal of a 20-nucleotide sequence, termed the D sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome significantly impairs rescue, replication, and encapsidation of the viral genomes (X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Mol Biol 250:573–580, 1995; X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Virol 70:1668–1677, 1996). Here we describe that replacement of only one D sequence in either ITR restores each of these functions, but DNA strands of only single polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded DNA (ssDNA), which is transcriptionally inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription suppressor sequence in one of the D sequences, which shares homology with the binding site for the cellular NF-κB-repressing factor (NRF). The removal of this D sequence from, and replacement with a sequence containing putative binding sites for transcription factors in, single-stranded AAV (ssAAV) vectors significantly augments transgene expression both in human cell lines in vitro and in murine hepatocytes in vivo. The development of these genome-modified ssAAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy. IMPORTANCE The results of the studies described here not only have provided novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV), that causes no known disease but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased levels of gene expression. Thus, these studies have significant implications for the potential use of these novel AAV vectors in human gene therapy

  7. An efficient method for recovering Lyapunov vectors from singular vectors

    NASA Astrophysics Data System (ADS)

    Wolfe, Christopher L.; Samelson, Roger M.

    2007-05-01

    Lyapunov vectors are natural generalizations of normal modes for linear disturbances to aperiodic deterministic flows and offer insights into the physical mechanisms of aperiodic flow and the maintenance of chaos. Most standard techniques for computing Lyapunov vectors produce results which are norm-dependent and lack invariance under the linearized flow (except for the leading Lyapunov vector) and these features can make computation and physical interpretation problematic. An efficient, norm-independent method for constructing the n most rapidly growing Lyapunov vectors from n - 1 leading forward and n leading backward asymptotic singular vectors is proposed. The Lyapunov vectors so constructed are invariant under the linearized flow in the sense that, once computed at one time, they are defined, in principle, for all time through the tangent linear propagator. An analogous method allows the construction of the n most rapidly decaying Lyapunov vectors from n decaying forward and n - 1 decaying backward singular vectors. This method is demonstrated using two low-order geophysical models.

  8. Control Circuit For Reed-Solomon Encoder

    NASA Technical Reports Server (NTRS)

    Ross, Douglas

    1992-01-01

    Control circuit designed for use with commercially available AHA4610 Reed-Solomon encoder. Needed to select depth of interleaving and to synchronize input and output blocks of data and parity bits with suitable clock signals. Circuit provides synchronizing and control signals for Reed-Solomon encoder. Encoder can operate with asynchronous input and output data streams at rates up to 80 Mb/s. Interleaving depth selectable, and accommodation to input data rate automatic.

  9. NMDA receptors and memory encoding.

    PubMed

    Morris, Richard G M

    2013-11-01

    It is humbling to think that 30 years have passed since the paper by Collingridge, Kehl and McLennan showing that one of Jeff Watkins most interesting compounds, R-2-amino-5-phosphonopentanoate (d-AP5), blocked the induction of long-term potentiation in vitro at synapses from area CA3 of the hippocampus to CA1 without apparent effect on baseline synaptic transmission (Collingridge et al., 1983). This dissociation was one of the key triggers for an explosion of interest in glutamate receptors, and much has been discovered since that collectively contributes to our contemporary understanding of glutamatergic synapses - their biophysics and subunit composition, of the agonists and antagonists acting on them, and their diverse functions in different networks of the brain and spinal cord. It can be fairly said that Collingridge et al.'s (1983) observation was the stimulus that has led, on the one hand, to structural biological work at the atomic scale describing the key features of NMDA receptors that enables their coincidence function to happen; and, on the other, to work with whole animals investigating the contributions that calcium signalling via this receptor can have on rhythmical activities controlled by spinal circuits, memory encoding in the hippocampus (the topic of this article), visual cortical plasticity, sensitization in pain, and other functions. In this article, I lay out how my then interest in long-term potentiation (LTP) as a model of memory enabled me to recognise the importance of Collingridge et al.'s discovery - and how I and my colleagues endeavoured to take things forward in the area of learning and memory. This is in some respects a personal story, and I tell it as such. The idea that NMDA receptor activation is essential for memory encoding, though not for storage, took time to develop and to be accepted. Along the way, there have been confusions, challenges, and surprises surrounding the idea that activation of NMDA receptors can

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

    PubMed

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

    1999-11-01

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

  11. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of streptococcus pneumontae

    DOEpatents

    Lacks, Sanford A.

    1990-01-01

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252.

  12. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of Streptococcus pneumontae

    DOEpatents

    Lacks, S.A.

    1990-10-02

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252. 9 figs.

  13. Encoders for block-circulant LDPC codes

    NASA Technical Reports Server (NTRS)

    Andrews, Kenneth; Dolinar, Sam; Thorpe, Jeremy

    2005-01-01

    In this paper, we present two encoding methods for block-circulant LDPC codes. The first is an iterative encoding method based on the erasure decoding algorithm, and the computations required are well organized due to the block-circulant structure of the parity check matrix. The second method uses block-circulant generator matrices, and the encoders are very similar to those for recursive convolutional codes. Some encoders of the second type have been implemented in a small Field Programmable Gate Array (FPGA) and operate at 100 Msymbols/second.

  14. Unconscious relational encoding depends on hippocampus

    PubMed Central

    Duss, Simone B.; Reber, Thomas P.; Hänggi, Jürgen; Schwab, Simon; Wiest, Roland; Müri, René M.; Brugger, Peter; Gutbrod, Klemens

    2014-01-01

    Textbooks divide between human memory systems based on consciousness. Hippocampus is thought to support only conscious encoding, while neocortex supports both conscious and unconscious encoding. We tested whether processing modes, not consciousness, divide between memory systems in three neuroimaging experiments with 11 amnesic patients (mean age = 45.55 years, standard deviation = 8.74, range = 23–60) and 11 matched healthy control subjects. Examined processing modes were single item versus relational encoding with only relational encoding hypothesized to depend on hippocampus. Participants encoded and later retrieved either single words or new relations between words. Consciousness of encoding was excluded by subliminal (invisible) word presentation. Amnesic patients and controls performed equally well on the single item task activating prefrontal cortex. But only the controls succeeded on the relational task activating the hippocampus, while amnesic patients failed as a group. Hence, unconscious relational encoding, but not unconscious single item encoding, depended on hippocampus. Yet, three patients performed normally on unconscious relational encoding in spite of amnesia capitalizing on spared hippocampal tissue and connections to language cortex. This pattern of results suggests that processing modes divide between memory systems, while consciousness divides between levels of function within a memory system. PMID:25273998

  15. Perceptually adapted MPEG video encoding

    NASA Astrophysics Data System (ADS)

    Bordes, Philippe; Guillotel, Philippe

    2000-06-01

    In picture quality assessment, the amount of distortion perceived by a human observer differs from one region to another according to its particular local content. This subjective perception can be explained/predicted by considering some simple psychovisual properties (masking) of the Human Visual System (HVS). We have implemented a HVS model based on a pyramid decomposition for extracting the spatial frequencies, associated with a multi-resolution motion representation. Then the visibility of the decoded errors is computed by exploiting the Kelly's contrast sensitivity spatio-velocity model. The resulting data is called a 'Quality-map.' Special attention has been paid to temporal/moving effects since, in the case of video sequences, motion strongly influences the subjective quality assessment. The quality of the motion information is thus preponderant. In the second part, two possible uses of these psychovisual properties for improving MPEG video encoding performances are depicted: (1) The pre-processing of the pictures to remove non-visible information using a motion adapted filtering. This process is efficient in term of bits saved and degradation is not significant especially on consumer electronic TV sets. (2) A perceptual quantizer based on a local adaptation scheme in order to obtain Quality-maps as uniform as possible (homogeneous perceived distortion), at constant bit-rate. Further improvements have been considered, especially when the viewer is tracking a moving object in the scene.

  16. Evaluation of GOES encoder lamps

    NASA Technical Reports Server (NTRS)

    Viehmann, W.; Helmold, N.

    1983-01-01

    Aging characteristics and life expectancies of flight quality, tungsten filament, encoder lamps are similar to those of 'commercial' grade gas filled lamps of similar construction, filament material and filament temperature. The aging and final failure by filament burnout are caused by single crystal growth over large portions of the filament with the concomitant development of facets and notches resulting in reduction of cross section and mechanical weakening of the filament. The life expectancy of presently produced lamps is about one year at their nominal operating voltage of five volts dc. At 4.5 volts, it is about two years. These life times are considerably shorter, and the degradation rates of lamp current and light flux are considerably higher, than were observed in the laboratory and in orbit on lamps of the same type manufactured more than a decade ago. It is speculated that the filaments of these earlier lamps contained a crystallization retarding dopant, possibly thorium oxide. To obtain the desired life expectancy of or = to four years in present lamps, operating voltages of or = to four volts dc would be required.

  17. Protection of chickens against avian influenza with non-replicating adenovirus-vectored vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA) -free human adenovirus (Ad) vector encoding a H7 hemagglutinin gene from a low pathogenic North American isolate (AdChNY94.H7). Chickens vaccinate...

  18. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The integrative vector pINTRS was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128, thus allowing for the production of '-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. ...

  19. One Hidden Object, Two Spatial Codes: Young Children's Use of Relational and Vector Coding

    ERIC Educational Resources Information Center

    Uttal, David H.; Sandstrom, Lisa B.; Newcombe, Nora S.

    2006-01-01

    An important characteristic of mature spatial cognition is the ability to encode spatial locations in terms of relations among landmarks as well as in terms of vectors that include distance and direction. In this study, we examined children's use of the relation "middle" to code the location of a hidden toy, using a procedure adapted from prior…

  20. Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protective immunity against avian influenza (AI) virus was elicited in chickens by single dose in ovo vaccination with a replication competent adenovirus (RCA) -free human adenovirus vector (Ad5) encoding an avian AI virus H5 hemagglutinin. Vaccinated chickens were protected against both H5N1 and H5...

  1. Vectors on the Basketball Court

    ERIC Educational Resources Information Center

    Bergman, Daniel

    2010-01-01

    An Idea Bank published in the April/May 2009 issue of "The Science Teacher" describes an experiential physics lesson on vectors and vector addition (Brown 2009). Like its football predecessor, the basketball-based investigation presented in this Idea Bank addresses National Science Education Standards Content B, Physical Science, 9-12 (NRC 1996)…

  2. Bubble vector in automatic merging

    NASA Technical Reports Server (NTRS)

    Pamidi, P. R.; Butler, T. G.

    1987-01-01

    It is shown that it is within the capability of the DMAP language to build a set of vectors that can grow incrementally to be applied automatically and economically within a DMAP loop that serves to append sub-matrices that are generated within a loop to a core matrix. The method of constructing such vectors is explained.

  3. GPU Accelerated Vector Median Filter

    NASA Technical Reports Server (NTRS)

    Aras, Rifat; Shen, Yuzhong

    2011-01-01

    Noise reduction is an important step for most image processing tasks. For three channel color images, a widely used technique is vector median filter in which color values of pixels are treated as 3-component vectors. Vector median filters are computationally expensive; for a window size of n x n, each of the n(sup 2) vectors has to be compared with other n(sup 2) - 1 vectors in distances. General purpose computation on graphics processing units (GPUs) is the paradigm of utilizing high-performance many-core GPU architectures for computation tasks that are normally handled by CPUs. In this work. NVIDIA's Compute Unified Device Architecture (CUDA) paradigm is used to accelerate vector median filtering. which has to the best of our knowledge never been done before. The performance of GPU accelerated vector median filter is compared to that of the CPU and MPI-based versions for different image and window sizes, Initial findings of the study showed 100x improvement of performance of vector median filter implementation on GPUs over CPU implementations and further speed-up is expected after more extensive optimizations of the GPU algorithm .

  4. Encoding and decoding in fMRI

    PubMed Central

    Naselaris, Thomas; Kay, Kendrick N.; Nishimoto, Shinji; Gallant, Jack L.

    2010-01-01

    Over the past decade fMRI researchers have developed increasingly sensitive techniques for analyzing the information represented in BOLD activity. The most popular of these techniques is linear classification, a simple technique for decoding information about experimental stimuli or tasks from patterns of activity across an array of voxels. A more recent development is the voxel-based encoding model, which describes the information about the stimulus or task that is represented in the activity of single voxels. Encoding and decoding are complementary operations: encoding uses stimuli to predict activity while decoding uses activity to predict information about stimuli. However, in practice these two operations are often confused, and their respective strengths and weaknesses have not been made clear. Here we use the concept of a linearizing feature space to clarify the relationship between encoding and decoding. We show that encoding and decoding operations can both be used to investigate some of the most common questions about how information is represented in the brain. However, focusing on encoding models offers two important advantages over decoding. First, an encoding model can in principle provide a complete functional description of a region of interest, while a decoding model can provide only a partial description. Second, while it is straightforward to derive an optimal decoding model from an encoding model it is much more difficult to derive an encoding model from a decoding model. We propose a systematic modeling approach that begins by estimating an encoding model for every voxel in a scan and ends by using the estimated encoding models to perform decoding. PMID:20691790

  5. Divergence-based vector quantization.

    PubMed

    Villmann, Thomas; Haase, Sven

    2011-05-01

    Supervised and unsupervised vector quantization methods for classification and clustering traditionally use dissimilarities, frequently taken as Euclidean distances. In this article, we investigate the applicability of divergences instead, focusing on online learning. We deduce the mathematical fundamentals for its utilization in gradient-based online vector quantization algorithms. It bears on the generalized derivatives of the divergences known as Fréchet derivatives in functional analysis, which reduces in finite-dimensional problems to partial derivatives in a natural way. We demonstrate the application of this methodology for widely applied supervised and unsupervised online vector quantization schemes, including self-organizing maps, neural gas, and learning vector quantization. Additionally, principles for hyperparameter optimization and relevance learning for parameterized divergences in the case of supervised vector quantization are given to achieve improved classification accuracy. PMID:21299418

  6. Rice Reoviruses in Insect Vectors.

    PubMed

    Wei, Taiyun; Li, Yi

    2016-08-01

    Rice reoviruses, transmitted by leafhopper or planthopper vectors in a persistent propagative manner, seriously threaten the stability of rice production in Asia. Understanding the mechanisms that enable viral transmission by insect vectors is a key to controlling these viral diseases. This review describes current understanding of replication cycles of rice reoviruses in vector cell lines, transmission barriers, and molecular determinants of vector competence and persistent infection. Despite recent breakthroughs, such as the discoveries of actin-based tubule motility exploited by viruses to overcome transmission barriers and mutually beneficial relationships between viruses and bacterial symbionts, there are still many gaps in our knowledge of transmission mechanisms. Advances in genome sequencing, reverse genetics systems, and molecular technologies will help to address these problems. Investigating the multiple interaction systems among the virus, insect vector, insect symbiont, and plant during natural infection in the field is a central topic for future research on rice reoviruses. PMID:27296147

  7. A neural support vector machine.

    PubMed

    Jändel, Magnus

    2010-06-01

    Support vector machines are state-of-the-art pattern recognition algorithms that are well founded in optimization and generalization theory but not obviously applicable to the brain. This paper presents Bio-SVM, a biologically feasible support vector machine. An unstable associative memory oscillates between support vectors and interacts with a feed-forward classification pathway. Kernel neurons blend support vectors and sensory input. Downstream temporal integration generates the classification. Instant learning of surprising events and off-line tuning of support vector weights trains the system. Emotion-based learning, forgetting trivia, sleep and brain oscillations are phenomena that agree with the Bio-SVM model. A mapping to the olfactory system is suggested. PMID:20092978

  8. Spatial-Spectral Classification Based on the Unsupervised Convolutional Sparse Auto-Encoder for Hyperspectral Remote Sensing Imagery

    NASA Astrophysics Data System (ADS)

    Han, Xiaobing; Zhong, Yanfei; Zhang, Liangpei

    2016-06-01

    Current hyperspectral remote sensing imagery spatial-spectral classification methods mainly consider concatenating the spectral information vectors and spatial information vectors together. However, the combined spatial-spectral information vectors may cause information loss and concatenation deficiency for the classification task. To efficiently represent the spatial-spectral feature information around the central pixel within a neighbourhood window, the unsupervised convolutional sparse auto-encoder (UCSAE) with window-in-window selection strategy is proposed in this paper. Window-in-window selection strategy selects the sub-window spatial-spectral information for the spatial-spectral feature learning and extraction with the sparse auto-encoder (SAE). Convolution mechanism is applied after the SAE feature extraction stage with the SAE features upon the larger outer window. The UCSAE algorithm was validated by two common hyperspectral imagery (HSI) datasets - Pavia University dataset and the Kennedy Space Centre (KSC) dataset, which shows an improvement over the traditional hyperspectral spatial-spectral classification methods.

  9. Strategies for targeting lentiviral vectors.

    PubMed

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

    2008-12-01

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

  10. Encoding Sequential Information in Semantic Space Models: Comparing Holographic Reduced Representation and Random Permutation

    PubMed Central

    Recchia, Gabriel; Sahlgren, Magnus; Kanerva, Pentti; Jones, Michael N.

    2015-01-01

    Circular convolution and random permutation have each been proposed as neurally plausible binding operators capable of encoding sequential information in semantic memory. We perform several controlled comparisons of circular convolution and random permutation as means of encoding paired associates as well as encoding sequential information. Random permutations outperformed convolution with respect to the number of paired associates that can be reliably stored in a single memory trace. Performance was equal on semantic tasks when using a small corpus, but random permutations were ultimately capable of achieving superior performance due to their higher scalability to large corpora. Finally, “noisy” permutations in which units are mapped to other units arbitrarily (no one-to-one mapping) perform nearly as well as true permutations. These findings increase the neurological plausibility of random permutations and highlight their utility in vector space models of semantics. PMID:25954306

  11. Congruity of Encoding in Children's Redintegrative Memory.

    ERIC Educational Resources Information Center

    Hall, Donald M.; Geis, Mary Fulcher

    The mnemonic consequences of semantic, acoustic, and orthographic encoding and the relationships between encoding and retrieval cues were investigated in an incidental-learning experiment involving 24 first-, third-, and fifth-grade pupils. Each child was asked one orienting question for each of 18 words; the questions differed in the type of…

  12. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... for either manual or automatic operation. (2) Inputs. The encoder shall have two inputs, one for audio... encoder shall have two outputs, one audio port and one data port (RS-232C with standard protocol and 1200... frequency components outside 200 to 4000 Hz shall be attenuated by 40 dB or more with respect to the...

  13. Experiments in encoding multilevel images as quadtrees

    NASA Technical Reports Server (NTRS)

    Lansing, Donald L.

    1987-01-01

    Image storage requirements for several encoding methods are investigated and the use of quadtrees with multigray level or multicolor images are explored. The results of encoding a variety of images having up to 256 gray levels using three schemes (full raster, runlength and quadtree) are presented. Although there is considerable literature on the use of quadtrees to store and manipulate binary images, their application to multilevel images is relatively undeveloped. The potential advantage of quadtree encoding is that an entire area with a uniform gray level may be encoded as a unit. A pointerless quadtree encoding scheme is described. Data are presented on the size of the quadtree required to encode selected images and on the relative storage requirements of the three encoding schemes. A segmentation scheme based on the statistical variation of gray levels within a quadtree quadrant is described. This parametric scheme may be used to control the storage required by an encoded image and to preprocess a scene for feature identification. Several sets of black and white and pseudocolor images obtained by varying the segmentation parameter are shown.

  14. Encoding information using Laguerre Gaussian modes

    NASA Astrophysics Data System (ADS)

    Trichili, Abderrahmen; Dudley, Angela; Ben Salem, Amine; Ndagano, Bienvenu; Zghal, Mourad; Forbes, Andrew

    2015-08-01

    We experimentally demonstrate an information encoding protocol using the two degrees of freedom of Laguerre Gaussian modes having different radial and azimuthal components. A novel method, based on digital holography, for information encoding and decoding using different data transmission scenarios is presented. The effects of the atmospheric turbulence introduced in free space communication is discussed as well.

  15. The Acquisition of Syntactically Encoded Evidentiality

    ERIC Educational Resources Information Center

    Rett, Jessica; Hyams, Nina

    2014-01-01

    This article presents several empirical studies of syntactically encoded evidentiality in English. The first part of our study consists of an adult online experiment that confirms claims in Asudeh & Toivonen (2012) that raised Perception Verb Similatives (PVSs; e.g. "John looks like he is sick") encode direct evidentiality. We then…

  16. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  17. Vector Network Analysis

    1997-10-20

    Vector network analyzers are a convenient way to measure scattering parameters of a variety of microwave devices. However, these instruments, unlike oscilloscopes for example, require a relatively high degree of user knowledge and expertise. Due to the complexity of the instrument and of the calibration process, there are many ways in which an incorrect measurement may be produced. The Microwave Project, which is part of Sandia National Laboratories Primary Standards Laboratory, routinely uses check standardsmore » to verify that the network analyzer is operating properly. In the past, these measurements were recorded manually and, sometimes, interpretation of the results was problematic. To aid our measurement assurance process, a software program was developed to automatically measure a check standard and compare the new measurements with an historical database of measurements of the same device. The program acquires new measurement data from selected check standards, plots the new data against the mean and standard deviation of prior data for the same check standard, and updates the database files for the check standard. The program is entirely menu-driven requiring little additional work by the user.« less

  18. Vector-vector production in photon-photon interactions

    SciTech Connect

    Ronan, M.T.

    1988-12-09

    Measurements of exclusive untagged /rho//sup 0//rho//sup 0/, /rho//phi/, K/sup *//bar K//sup */, and /rho/..omega.. production and tagged /rho//sup 0//rho//sup 0/ production in photon-photon interactions by the TPC/Two-Gamma experiment are reviewed. Comparisons to the results of other experiments and to models of vector-vector production are made. Fits to the data following a four quark model prescription for vector meson pair production are also presented. 10 refs., 9 figs.

  19. Vector-vector production in photon-photon interactions

    SciTech Connect

    Ronan, M. T.

    1989-04-25

    Measurements of exclusive untagged /rho//sup 0//rho0/,/rho//phi/,/ital K//sup *//ital K/bar /*/, and /rho/..omega.. production and tagged /rho//sup 0//rho0/ production in photon-photon interactions by the TPC/Two-Gamma experiment are reviewed. Comparisons to the results of other experiments and to models of vector-vector production are made. Fits to the data following a four quark model prescription for vector meson pair production are also presented.

  20. Industrial Applications Of Optical Shaft Encoders

    NASA Astrophysics Data System (ADS)

    Edmister, Brian W.

    1980-11-01

    The development of the microprocessor and mini-computer for industrial process control has made the optical shaft angle encoder a natural choice for a position feedback transducer. Many of these applications, however, require the encoder to operate reliably in extremely hostile environments. In response to this, the encoder manufacturer has been faced with reliability problems which fall into the following general categories: 1. Exposure to weather 2. Wide operating and storage temperature range 3. Exposure to corrosive chemicals 4. Severe shock and vibration 5. High electrical noise levels 6. Severe blows to encoder housing 7. Operation in explosive atmospheres Three of these applications expose the encoder to most of these environmental conditions: 1. A jack-up control position feedback for an offshore oil well drilling rig 2. A depth measurement system for oil well logging instrumentation 3. Elevation and azimuth feedback for a solar power plant heliostat

  1. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

    PubMed

    Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

    2014-11-01

    Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates

  2. Chikungunya Virus–Vector Interactions

    PubMed Central

    Coffey, Lark L.; Failloux, Anna-Bella; Weaver, Scott C.

    2014-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever, a severe, debilitating disease that often produces chronic arthralgia. Since 2004, CHIKV has emerged in Africa, Indian Ocean islands, Asia, Europe, and the Americas, causing millions of human infections. Central to understanding CHIKV emergence is knowledge of the natural ecology of transmission and vector infection dynamics. This review presents current understanding of CHIKV infection dynamics in mosquito vectors and its relationship to human disease emergence. The following topics are reviewed: CHIKV infection and vector life history traits including transmission cycles, genetic origins, distribution, emergence and spread, dispersal, vector competence, vector immunity and microbial interactions, and co-infection by CHIKV and other arboviruses. The genetics of vector susceptibility and host range changes, population heterogeneity and selection for the fittest viral genomes, dual host cycling and its impact on CHIKV adaptation, viral bottlenecks and intrahost diversity, and adaptive constraints on CHIKV evolution are also discussed. The potential for CHIKV re-emergence and expansion into new areas and prospects for prevention via vector control are also briefly reviewed. PMID:25421891

  3. Enhancing poxvirus vectors vaccine immunogenicity

    PubMed Central

    García-Arriaza, Juan; Esteban, Mariano

    2014-01-01

    Attenuated recombinant poxvirus vectors expressing heterologous antigens from pathogens are currently at various stages in clinical trials with the aim to establish their efficacy. This is because these vectors have shown excellent safety profiles, significant immunogenicity against foreign expressed antigens and are able to induce protective immune responses. In view of the limited efficacy triggered by some poxvirus strains used in clinical trials (i.e, ALVAC in the RV144 phase III clinical trial for HIV), and of the restrictive replication capacity of the highly attenuated vectors like MVA and NYVAC, there is a consensus that further improvements of these vectors should be pursuit. In this review we considered several strategies that are currently being implemented, as well as new approaches, to improve the immunogenicity of the poxvirus vectors. This includes heterologous prime/boost protocols, use of co-stimulatory molecules, deletion of viral immunomodulatory genes still present in the poxvirus genome, enhancing virus promoter strength, enhancing vector replication capacity, optimizing expression of foreign heterologous sequences, and the combined use of adjuvants. An optimized poxvirus vector triggering long-lasting immunity with a high protective efficacy against a selective disease should be sought. PMID:25424927

  4. AAV8-mediated expression of N-acetylglucosamine-1-phosphate transferase attenuates bone loss in a mouse model of mucolipidosis II.

    PubMed

    Ko, Ah-Ra; Jin, Dong-Kyu; Cho, Sung Yoon; Park, Sung Won; Przybylska, Malgorzata; Yew, Nelson S; Cheng, Seng H; Kim, Jung-Sun; Kwak, Min Jung; Kim, Su Jin; Sohn, Young Bae

    2016-04-01

    Mucolipidoses II and III (ML II and ML III) are lysosomal disorders in which the mannose 6-phosphate recognition marker is absent from lysosomal hydrolases and other glycoproteins due to mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase. Both disorders are caused by the same gene, but ML II represents the more severe phenotype. Bone manifestations of ML II include hip dysplasia, scoliosis, rickets and osteogenesis imperfecta. In this study, we sought to determine whether a recombinant adeno-associated viral vector (AAV2/8-GNPTAB) could confer high and prolonged gene expression of GNPTAB and thereby influence the pathology in the cartilage and bone tissue of a GNPTAB knock out (KO) mouse model. The results demonstrated significant increases in bone mineral density and content in AAV2/8-GNPTAB-treated as compared to non-treated KO mice. We also showed that IL-6 (interleukin-6) expression in articular cartilage was reduced in AAV2/8-GNPTAB treated ML II mice. Together, these data suggest that AAV-mediated expression of GNPTAB in ML II mice can attenuate bone loss via inhibition of IL-6 production. This study emphasizes the value of the MLII KO mouse to recapitulate the clinical manifestations of the disease and highlights its amenability to therapy. PMID:26857995

  5. Genome sequence of Aedes aegypti, a major arbovirus vector.

    PubMed

    Nene, Vishvanath; Wortman, Jennifer R; Lawson, Daniel; Haas, Brian; Kodira, Chinnappa; Tu, Zhijian Jake; Loftus, Brendan; Xi, Zhiyong; Megy, Karyn; Grabherr, Manfred; Ren, Quinghu; Zdobnov, Evgeny M; Lobo, Neil F; Campbell, Kathryn S; Brown, Susan E; Bonaldo, Maria F; Zhu, Jingsong; Sinkins, Steven P; Hogenkamp, David G; Amedeo, Paolo; Arensburger, Peter; Atkinson, Peter W; Bidwell, Shelby; Biedler, Jim; Birney, Ewan; Bruggner, Robert V; Costas, Javier; Coy, Monique R; Crabtree, Jonathan; Crawford, Matt; Debruyn, Becky; Decaprio, David; Eiglmeier, Karin; Eisenstadt, Eric; El-Dorry, Hamza; Gelbart, William M; Gomes, Suely L; Hammond, Martin; Hannick, Linda I; Hogan, James R; Holmes, Michael H; Jaffe, David; Johnston, J Spencer; Kennedy, Ryan C; Koo, Hean; Kravitz, Saul; Kriventseva, Evgenia V; Kulp, David; Labutti, Kurt; Lee, Eduardo; Li, Song; Lovin, Diane D; Mao, Chunhong; Mauceli, Evan; Menck, Carlos F M; Miller, Jason R; Montgomery, Philip; Mori, Akio; Nascimento, Ana L; Naveira, Horacio F; Nusbaum, Chad; O'leary, Sinéad; Orvis, Joshua; Pertea, Mihaela; Quesneville, Hadi; Reidenbach, Kyanne R; Rogers, Yu-Hui; Roth, Charles W; Schneider, Jennifer R; Schatz, Michael; Shumway, Martin; Stanke, Mario; Stinson, Eric O; Tubio, Jose M C; Vanzee, Janice P; Verjovski-Almeida, Sergio; Werner, Doreen; White, Owen; Wyder, Stefan; Zeng, Qiandong; Zhao, Qi; Zhao, Yongmei; Hill, Catherine A; Raikhel, Alexander S; Soares, Marcelo B; Knudson, Dennis L; Lee, Norman H; Galagan, James; Salzberg, Steven L; Paulsen, Ian T; Dimopoulos, George; Collins, Frank H; Birren, Bruce; Fraser-Liggett, Claire M; Severson, David W

    2007-06-22

    We present a draft sequence of the genome of Aedes aegypti, the primary vector for yellow fever and dengue fever, which at approximately 1376 million base pairs is about 5 times the size of the genome of the malaria vector Anopheles gambiae. Nearly 50% of the Ae. aegypti genome consists of transposable elements. These contribute to a factor of approximately 4 to 6 increase in average gene length and in sizes of intergenic regions relative to An. gambiae and Drosophila melanogaster. Nonetheless, chromosomal synteny is generally maintained among all three insects, although conservation of orthologous gene order is higher (by a factor of approximately 2) between the mosquito species than between either of them and the fruit fly. An increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to An. gambiae suggests that members of these protein families underpin some of the biological differences between the two mosquito species. PMID:17510324

  6. Improved vector quantization scheme for grayscale image compression

    NASA Astrophysics Data System (ADS)

    Hu, Y.-C.; Chen, W.-L.; Lo, C.-C.; Chuang, J.-C.

    2012-06-01

    This paper proposes an improved image coding scheme based on vector quantization. It is well known that the image quality of a VQ-compressed image is poor when a small-sized codebook is used. In order to solve this problem, the mean value of the image block is taken as an alternative block encoding rule to improve the image quality in the proposed scheme. To cut down the storage cost of compressed codes, a two-stage lossless coding approach including the linear prediction technique and the Huffman coding technique is employed in the proposed scheme. The results show that the proposed scheme achieves better image qualities than vector quantization while keeping low bit rates.

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2015-12-16

    Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development. PMID:26514419

  9. Decoding the ubiquitin-mediated pathway of arthropod disease vectors.

    PubMed

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

    2013-01-01

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

  10. Vector statistics of LANDSAT imagery

    NASA Technical Reports Server (NTRS)

    Jayroe, R. R., Jr.; Underwood, D.

    1977-01-01

    A digitized multispectral image, such as LANDSAT data, is composed of numerous four dimensional vectors, which quantitatively describe the ground scene from which the data are acquired. The statistics of unique vectors that occur in LANDSAT imagery are studied to determine if that information can provide some guidance on reducing image processing costs. A second purpose of this report is to investigate how the vector statistics are changed by various types of image processing techniques and determine if that information can be useful in choosing one processing approach over another.

  11. Baculovirus as a vaccine vector

    PubMed Central

    Lu, Hsin-Yu; Chen, Yi-Hsuan; Liu, Hung-Jen

    2012-01-01

    Baculovirus is extensively utilized as an excellent tool for production of recombinant protein in insect cells. Baculovirus infects insects in nature and is non-pathogenic to humans. In addition to insect cells, baculovirus is capable of transducing a broad range of animal cells. Due to its biosafety, large cloning capacity, low cytotoxicity, and non-replication nature in the transduced cells as well as the ease of manipulation and production, baculovirus has been utilized as RNA interference mediators, gene delivery vectors, and vaccine vectors for a wide variety of applications. This article focuses on the utilization of baculoviruses as vaccine vectors to prepare antigen or subunit vaccines. PMID:22705893

  12. Relativistic Gamow vectors: State vectors for unstable particles

    NASA Astrophysics Data System (ADS)

    Kaldas, Hany Kamel Halim

    The relativistic Gamow vectors are derived from the analytic continuation of the angular momentum velocity kets to the resonance pole of the S- matrix. This construction is justifiable within a Rigged Hilbert Space of Hardy class functions. The kets obtained | p j3[ sRjR ]-> are characterized by a spin jR and a complex mass square sR = (MR - iΓ R/2)2. Our use of the velocity kets renders the Gamow vectors | p j3[ sRjR ]-> ``minimally complex'', as the 4-velocities p̂μ = p μ/ s are taken real and they remain real under Lorentz transformations. When the symmetry transformations of the Gamow vectors are considered, it is found that they obey a semigroup time evolution in the forward light cone for the subgroup of P with causal space- time translations, i.e., for space-time translations with 4-vectors x such that x2 >= 0. This semigroup evolution, which is a consequence of the characterization obtained for the Gamow vectors as functionals in a Rigged Hilbert Space, is in conformity with the time directedness associated with decay phenomena. The Gamow vectors, with a Breit-Wigner distribution and exponential decay law, provide a description of decaying particles with a wide range of Γ/ M. Moreover, the Gamow vectors, being members of a complex basis vector expansion, allow the Wigner-Weisskopf's based effective theories, such as the Lee-Oehme-Yang theory for the neutral K-mesons, to be obtained as an approximation in an exact formalism.

  13. Are Bred Vectors The Same As Lyapunov Vectors?

    NASA Astrophysics Data System (ADS)

    Kalnay, E.; Corazza, M.; Cai, M.

    Regional loss of predictability is an indication of the instability of the underlying flow, where small errors in the initial conditions (or imperfections in the model) grow to large amplitudes in finite times. The stability properties of evolving flows have been studied using Lyapunov vectors (e.g., Alligood et al, 1996, Ott, 1993, Kalnay, 2002), singular vectors (e.g., Lorenz, 1965, Farrell, 1988, Molteni and Palmer, 1993), and, more recently, with bred vectors (e.g., Szunyogh et al, 1997, Cai et al, 2001). Bred vectors (BVs) are, by construction, closely related to Lyapunov vectors (LVs). In fact, after an infinitely long breeding time, and with the use of infinitesimal ampli- tudes, bred vectors are identical to leading Lyapunov vectors. In practical applications, however, bred vectors are different from Lyapunov vectors in two important ways: a) bred vectors are never globally orthogonalized and are intrinsically local in space and time, and b) they are finite-amplitude, finite-time vectors. These two differences are very significant in a dynamical system whose size is very large. For example, the at- mosphere is large enough to have "room" for several synoptic scale instabilities (e.g., storms) to develop independently in different regions (say, North America and Aus- tralia), and it is complex enough to have several different possible types of instabilities (such as barotropic, baroclinic, convective, and even Brownian motion). Bred vectors share some of their properties with leading LVs (Corazza et al, 2001a, 2001b, Toth and Kalnay, 1993, 1997, Cai et al, 2001). For example, 1) Bred vectors are independent of the norm used to define the size of the perturba- tion. Corazza et al. (2001) showed that bred vectors obtained using a potential enstro- phy norm were indistinguishable from bred vectors obtained using a streamfunction squared norm, in contrast with singular vectors. 2) Bred vectors are independent of the length of the rescaling period as long as the

  14. Double Virus Vector Infection to the Prefrontal Network of the Macaque Brain

    PubMed Central

    Tanaka, Shingo; Koizumi, Masashi; Kikusui, Takefumi; Ichihara, Nobutsune; Kato, Shigeki; Kobayashi, Kazuto; Sakagami, Masamichi

    2015-01-01

    To precisely understand how higher cognitive functions are implemented in the prefrontal network of the brain, optogenetic and pharmacogenetic methods to manipulate the signal transmission of a specific neural pathway are required. The application of these methods, however, has been mostly restricted to animals other than the primate, which is the best animal model to investigate higher cognitive functions. In this study, we used a double viral vector infection method in the prefrontal network of the macaque brain. This enabled us to express specific constructs into specific neurons that constitute a target pathway without use of germline genetic manipulation. The double-infection technique utilizes two different virus vectors in two monosynaptically connected areas. One is a vector which can locally infect cell bodies of projection neurons (local vector) and the other can retrogradely infect from axon terminals of the same projection neurons (retrograde vector). The retrograde vector incorporates the sequence which encodes Cre recombinase and the local vector incorporates the “Cre-On” FLEX double-floxed sequence in which a reporter protein (mCherry) was encoded. mCherry thus came to be expressed only in doubly infected projection neurons with these vectors. We applied this method to two macaque monkeys and targeted two different pathways in the prefrontal network: The pathway from the lateral prefrontal cortex to the caudate nucleus and the pathway from the lateral prefrontal cortex to the frontal eye field. As a result, mCherry-positive cells were observed in the lateral prefrontal cortex in all of the four injected hemispheres, indicating that the double virus vector transfection is workable in the prefrontal network of the macaque brain. PMID:26193102

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

    PubMed Central

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

    2012-01-01

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

  16. Progress on adenovirus-vectored universal influenza vaccines

    PubMed Central

    Xiang, Kui; Ying, Guan; Yan, Zhou; Shanshan, Yan; Lei, Zhang; Hongjun, Li; Maosheng, Sun

    2015-01-01

    Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8+ T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides ‘self-adjuvanting’ activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches. PMID:25876176

  17. Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes.

    PubMed

    Unsinger, J; Kröger, A; Hauser, H; Wirth, D

    2001-11-01

    Regulated transgene expression is increasingly used in research but is also needed for certain therapies. Regulatory systems are usually composed of two expression units, one bearing the gene of interest under control of a regulatable promoter and the other, a constitutively expressed transactivator that modulates the activity of the regulatable promoter. Because the cotransfer of two independent elements is not efficient in primary cells, single transduction step vectors conferring regulatable gene expression cassettes would be helpful. We have developed retroviral vectors containing an autoregulatory bidirectional expression cassette that encodes all components necessary for regulated expression of a gene of interest. The influence of the orientation of the reporter gene with respect to the viral long terminal repeat (LTR) and the effect of transcriptionally inactive LTRs were investigated using mouse leukemia virus (MLV) and self-inactivating (SIN)-based retroviral vectors. Strict regulation was observed when the reporter was inserted in antisense orientation with respect to the LTR, whereas a sense arrangement of the reporter resulted in a loss of regulation capacity. Expression and regulation of the antisense-orientated reporter gene were homogenous in infected cell pools and investigated cell clones. Long-term observations of infected cells over a period of 30 passages revealed stable expression and regulation. These autoregulated, bidirectional retroviral vectors combine the advantages of single-step transduction with strict regulation of the gene of interest in the infected target cells. PMID:11708885

  18. Progress on adenovirus-vectored universal influenza vaccines.

    PubMed

    Xiang, Kui; Ying, Guan; Yan, Zhou; Shanshan, Yan; Lei, Zhang; Hongjun, Li; Maosheng, Sun

    2015-01-01

    Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8(+) T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides 'self-adjuvanting' activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches. PMID:25876176

  19. Hexon-modified recombinant E1-deleted adenovirus vectors as dual specificity vaccine carriers for influenza virus.

    PubMed

    Zhou, Dongming; Wu, Te-Lang; Emmer, Kristel L; Kurupati, Raj; Tuyishime, Steven; Li, Yan; Giles-Davis, Wynetta; Zhou, Xiangyang; Xiang, Zhiquan; Liu, Qin; Ratcliffe, Sarah J; Ertl, Hildegund C J

    2013-03-01

    To determine if an ordered and repetitive display of an epitope promoted induction of superior antibody responses, we compared B-cell responses to an influenza A virus epitope that was either encoded as a transgene by an adenovirus (Ad) vector or expressed on the vector's surface. To this end, we constructed a panel of influenza A virus vaccines based on chimpanzee-derived replication-defective adenovirus (AdC) vectors of serotype SAd-V25 also called AdC68. AdC68 vectors were modified to express a linear B-cell epitope of the ectodomain of matrix 2 (M2e) within variable regions 1 (VR1) or 4 (VR4) of the adenovirus hexon. Additional vectors with wild-type or M2e-modified hexon encoded M2e fused to the influenza A virus nucleoprotein (NP) as a transgene product. Hexon-modified vectors were tested for immunogenicity and efficacy in mice in comparison to vectors with native hexon expressing the M2e-NP fusion protein. Upon priming, vectors expressing M2e within VR1 of hexon induced M2e-specific antibody responses of higher magnitude and avidity than those carrying M2e within VR4 or vectors expressing the M2e as part of a transgene product. CD8(+) T-cell responses to the transgenic NP were comparable between vectors. M2e-specific antibody responses could be boosted by a second dose of the VR1 hexon-modified vector but not by repeated immunization with the VR4 hexon-modified vector. PMID:23229092

  20. Source encoding for orbiter communications links

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The feasibility of using data compression to improve link efficiency as an alternative to increased transmitter power, reducing receiver noise figures, increasing antenna gain through more stringent orbiter attitude constraints, etc. is studied. A method of encoding digital data is developed which permits low band-width encoding as well as a unique system of adaptive run length encoding. The effectiveness of these techniques for the air-to-ground link and for the bandwidth-limited ground-to-ground data link used for the orbiter downlink data is evaluated. Results are presented.

  1. Programmable Pulse-Position-Modulation Encoder

    NASA Technical Reports Server (NTRS)

    Zhu, David; Farr, William

    2006-01-01

    A programmable pulse-position-modulation (PPM) encoder has been designed for use in testing an optical communication link. The encoder includes a programmable state machine and an electronic code book that can be updated to accommodate different PPM coding schemes. The encoder includes a field-programmable gate array (FPGA) that is programmed to step through the stored state machine and code book and that drives a custom high-speed serializer circuit board that is capable of generating subnanosecond pulses. The stored state machine and code book can be updated by means of a simple text interface through the serial port of a personal computer.

  2. VLSI Reed-Solomon Encoder With Interleaver

    NASA Technical Reports Server (NTRS)

    Hsu, In-Shek; Deutsch, L. J.; Truong, Trieu-Kie; Reed, I. S.

    1990-01-01

    Size, weight, and susceptibility to burst errors reduced. Encoding system built on single very-large-scale integrated (VLSI) circuit chip produces (255,223) Reed-Solomon (RS) code with programmable interleaving up to depth of 5. (225,223) RS encoder includes new remainder-and-interleaver unit providing programmable interleaving of code words. Remainder-and-interleaver unit contains shift registers and modulo-2 adders. Signals on "turn" and "no-turn" lines control depth of interleaving. Based on E. R. Berlekamp's bit-serial multiplication algorithm for (225,223) RS encoder over Galois Field (2 to the 8th power).

  3. Solid rocket thrust vector control

    NASA Technical Reports Server (NTRS)

    1974-01-01

    Thrust vector control systems that superimpose a side force on the motor thrust, steering being achieved by the side force causing a moment about the vehicle center of gravity are described. A brief review of thrust vector control systems is presented, and two systems, flexible joint and liquid injection, are treated in detail. Treatment of the flexible-joint thrust vector control system is limited to the design of the flexible joint and its insulation against hot motor gases. Treatment of the liquid injection thrust vector control system is limited to discussion of the injectant, valves, piping, storage tanks, and pressurization system; no evaluation is presented of the nozzle except for (1) the effect of the injectant and erosion at the injection port and (2) the effect of injection on pressure distribution within the nozzle.

  4. Experiments With Magnetic Vector Potential

    ERIC Educational Resources Information Center

    Skinner, J. W.

    1975-01-01

    Describes the experimental apparatus and method for the study of magnetic vector potential (MVP). Includes a discussion of inherent errors in the calculations involved, precision of the results, and further applications of MVP. (GS)

  5. Generation of high-titer pseudotyped retroviral vectors with very broad host range.

    PubMed

    Yee, J K; Friedmann, T; Burns, J C

    1994-01-01

    Encapsidation of the VSV G protein into the virions of MoMLV-derived retroviral vectors in the absence of other VSV-encoded proteins is shown to be an efficient process, although the exact mechanism for this process is currently unclear. Unlike the conventional retroviral vectors bearing the amphotropic envelope protein, the pseudotyped virus has the ability to withstand the shearing forces encountered during ultracentrifugation. This property of the pseudotyped virus enables the generation of high-titer retroviral vector stocks and has potential application for in vivo gene therapy studies. We have found as many as four copies of a pseudotyped vector to integrate into the genome of a single cell when a high multiplicity of infection was used to infect the cells. Multiple integration events were not observed with amphotropic retroviral vectors, probably because of their low virus titers. In addition, when retroviral vectors are pseudotyped with the VSV G protein, they acquire the host range of VSV and are able to infect nonmammalian cells derived from fish, Xenopus, mosquito, and Lepidoptera. Since techniques for efficient gene transfer in some of these nonmammalian systems are not currently available, retrovirus-mediated gene transfer described here should be useful for transgenic and other genetic studies in lower vertebrate species. The inability to establish a stable cell line expressing the VSV G protein, however, limits large-scale production of the pseudotyped retroviral vectors. Generation of stable packaging cell lines for the pseudotyped retroviral vectors is a major challenge for the future. PMID:7823872

  6. Gene transduction efficiency in cells of different species by HIV and EIAV vectors.

    PubMed

    Ikeda, Y; Collins, M K L; Radcliffe, P A; Mitrophanous, K A; Takeuchi, Y

    2002-07-01

    The ability of human immunodeficiency virus (HIV)- and equine infectious anaemia virus (EIAV)-based vectors to transduce cell lines from a range of species was compared. Both vectors carried the vesicular stomatitis virus G (VSV-G) envelope protein and encoded an enhanced green fluorescent protein (eGFP) gene driven by a human cytomegalovirus (CMV) early promoter. Immunostaining for viral core proteins and VSV-G was used to demonstrate that the HIV and EIAV vector preparations contained similar numbers of virus particles. Various cell lines were transduced with these vectors and the transduction efficiency was estimated by measuring eGFP expression. Efficient transduction by both vectors was observed in human, hamster, pig, horse, cat and dog cell lines, although EIAV vector was about 10-fold less efficient in human, hamster and pig cells normalised to the total number of viral particles. This could be partly explained by the lower RNA genome levels per particle for EIAV as measured by real-time RT-PCR. Rodent cells appeared to be transduced inefficiently with both vectors, but when the CMV promoter was substituted with the EF1alpha promoter in the HIV vectors, the expression level increased leading to an increase in the measurable level of transduction. PMID:12085241

  7. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    SciTech Connect

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M.

    2014-04-15

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

  8. Effective Masses of Vector Polarons

    NASA Astrophysics Data System (ADS)

    Foell, Charles; Clougherty, Dennis

    2006-03-01

    We consider the vector polarons of a one-dimensional model of an electron in a doubly (or nearly) degenerate band that couples to two elastic distortions, as described previously by Clougherty and Foell [1]. A variational approach is used to analytically and numerically calculate effective masses of the three types of vector polarons. [1] D. P. Clougherty and C. A. Foell, Phys. Rev. B 70, 052301 (2004).

  9. Coulomb problem for vector bosons

    SciTech Connect

    Kuchiev, M.Yu.; Flambaum, V.V.

    2006-05-01

    The Coulomb problem for vector bosons W{sup {+-}} incorporates a well-known difficulty; the charge of the boson localized in a close vicinity of the attractive Coulomb center proves to be infinite. The paradox is shown to be resolved by the QED vacuum polarization, which brings in a strong effective repulsion that eradicates the infinite charge of the boson on the Coulomb center. This property allows one to define the Coulomb problem for vector bosons properly.

  10. Molecular dynamics on vector computers

    NASA Astrophysics Data System (ADS)

    Sullivan, F.; Mountain, R. D.; Oconnell, J.

    1985-10-01

    An algorithm called the method of lights (MOL) has been developed for the computerized simulation of molecular dynamics. The MOL, implemented on the CYBER 205 computer, is based on sorting and reformulating the manner in which neighbor lists are compiled, and it uses data structures compatible with specialized vector statements that perform parallel computations. The MOL is found to reduce running time over standard methods in scalar form, and vectorization is shown to produce an order-of-magnitude reduction in execution time.

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

    PubMed Central

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

    2015-01-01

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

  12. Axisymmetric Coanda-assisted vectoring

    NASA Astrophysics Data System (ADS)

    Allen, Dustin; Smith, Barton L.

    2009-01-01

    An experimental demonstration of a jet vectoring technique used in our novel spray method called Coanda-assisted Spray Manipulation (CSM) is presented. CSM makes use of the Coanda effect on axisymmetric geometries through the interaction of two jets: a primary jet and a control jet. The primary jet has larger volume flow rate but generally a smaller momentum flux than the control jet. The primary jet flows through the center of a rounded collar. The control jet is parallel to the primary and is adjacent to the convex collar. The Reynolds number range for the primary jet at the exit plane was between 20,000 and 80,000. The flow was in the incompressible Mach number range (Mach < 0.3). The control jet attaches to the convex wall and vectors according to known Coanda effect principles, entraining and vectoring the primary jet, resulting in controllable r - θ directional spraying. Several annular control slots and collar radii were tested over a range of momentum flux ratios to determine the effects of these variables on the vectored jet angle and spreading. Two and Three-component Particle Image Velocimetry systems were used to determine the vectoring angle and the profile of the combined jet in each experiment. The experiments show that the control slot and expansion radius, along with the momentum ratios of the two jets predominantly affected the vectoring angle and profile of the combined jets.

  13. Vectoring of parallel synthetic jets

    NASA Astrophysics Data System (ADS)

    Berk, Tim; Ganapathisubramani, Bharathram; Gomit, Guillaume

    2015-11-01

    A pair of parallel synthetic jets can be vectored by applying a phase difference between the two driving signals. The resulting jet can be merged or bifurcated and either vectored towards the actuator leading in phase or the actuator lagging in phase. In the present study, the influence of phase difference and Strouhal number on the vectoring behaviour is examined experimentally. Phase-locked vorticity fields, measured using Particle Image Velocimetry (PIV), are used to track vortex pairs. The physical mechanisms that explain the diversity in vectoring behaviour are observed based on the vortex trajectories. For a fixed phase difference, the vectoring behaviour is shown to be primarily influenced by pinch-off time of vortex rings generated by the synthetic jets. Beyond a certain formation number, the pinch-off timescale becomes invariant. In this region, the vectoring behaviour is determined by the distance between subsequent vortex rings. We acknowledge the financial support from the European Research Council (ERC grant agreement no. 277472).

  14. Phase function encoding of diffractive structures.

    PubMed

    Schilling, A; Herzig, H P

    2000-10-10

    We analyzed the direct sampling (DS) method for diffractive lens encoding, using exact electromagnetic diffraction theory. In addition to previously published research [Pure Appl. Opt. 7, 565 (1998)] we present what we believe to be new results for TM polarization. We found that the validity of the scalar-based DS method is even more extended for TM than for TE polarization. Additionally, we fabricated and characterized DS-encoded blazed gratings and found good agreement between the experimental and theoretical diffraction efficiencies. We analyzed quantitatively the influence of the encoding schemes DS and analytic quantization (AQ) on the quality of the focal spot. We also investigated the focal spot sizes (FWHM) and the Strehl ratios of the DS- and the AQ-encoded cylindrical lenses. PMID:18354523

  15. Beyond Oncolytics: E1B55K-Deleted Adenovirus as a Vaccine Delivery Vector.

    PubMed

    Thomas, Michael A; Nyanhete, Tinashe; Tuero, Iskra; Venzon, David; Robert-Guroff, Marjorie

    2016-01-01

    Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 (dl1520) and H101 are best known for their oncolytic potential. As a vaccine vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors. PMID:27391605

  16. Beyond Oncolytics: E1B55K-Deleted Adenovirus as a Vaccine Delivery Vector

    PubMed Central

    Thomas, Michael A.; Nyanhete, Tinashe; Tuero, Iskra; Venzon, David; Robert-Guroff, Marjorie

    2016-01-01

    Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 (dl1520) and H101 are best known for their oncolytic potential. As a vaccine vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors. PMID:27391605

  17. An essential receptor for adeno-associated virus infection.

    PubMed

    Pillay, S; Meyer, N L; Puschnik, A S; Davulcu, O; Diep, J; Ishikawa, Y; Jae, L T; Wosen, J E; Nagamine, C M; Chapman, M S; Carette, J E

    2016-02-01

    Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr(-/-) (also known as Au040320(-/-) and Kiaa0319l(-/-)) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection. PMID:26814968

  18. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  19. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  20. Encoding and decoding of femtosecond pulses.

    PubMed

    Weiner, A M; Heritage, J P; Salehi, J A

    1988-04-01

    We demonstrate the spreading of femtosecond optical pulses into picosecond-duration pseudonoise bursts. Spreading is accomplished by encoding pseudorandom binary phase codes onto the optical frequency spectrum. Subsequent decoding of the spectral phases restores the original pulse. We propose that frequency-domain encoding and decoding of coherent ultrashort pulses could form the basis for a rapidly reconfigurable, code-division multiple-access optical telecommunications network. PMID:19745879