Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

DEFF Research Database (Denmark)

Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt

2010-01-01

Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic...

Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells).

Science.gov (United States)

Howarth, Joanna L; Lee, Youn Bok; Uney, James B

2010-02-01

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described.

Viral Vectors for Use in the Development of Biodefense Vaccines

National Research Council Canada - National Science Library

Lee, John S; Hadjipanayis, Angela G; Parker, Michael D

2005-01-01

.... DNA vectors, live-attenuated viruses and bacteria, recombinant proteins combined with adjuvant, and viral- or bacterial-vectored vaccines have been developed as countermeasures against many potential...

Viral Hybrid Vectors for Somatic Integration - Are They the Better Solution?

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Anja Ehrhardt

2009-12-01

Full Text Available The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system.

Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

DEFF Research Database (Denmark)

Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt

2010-01-01

. Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity...

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

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Quirin, Kayla A; Kwon, Jason J; Alioufi, Arafat; Factora, Tricia; Temm, Constance J; Jacobsen, Max; Sandusky, George E; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Mendell, Joshua T; Korc, Murray; Kota, Janaiah

2018-03-16

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 10 12 viral genomes (vg). Intraductal delivery of 1 × 10 11 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 10 11 vg. In a Kras G12D -driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice

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Kayla A. Quirin

2018-03-01

Full Text Available Recombinant adeno-associated virus (rAAV-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9 expressing GFP in a self-complementary (sc AAV vector under an EF1α promoter (scAAV.GFP following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg. Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Progress in developing cationic vectors for non-viral systemic gene therapy against cancer.

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Morille, Marie; Passirani, Catherine; Vonarbourg, Arnaud; Clavreul, Anne; Benoit, Jean-Pierre

2008-01-01

Initially, gene therapy was viewed as an approach for treating hereditary diseases, but its potential role in the treatment of acquired diseases such as cancer is now widely recognized. The understanding of the molecular mechanisms involved in cancer and the development of nucleic acid delivery systems are two concepts that have led to this development. Systemic gene delivery systems are needed for therapeutic application to cells inaccessible by percutaneous injection and for multi-located tumor sites, i.e. metastases. Non-viral vectors based on the use of cationic lipids or polymers appear to have promising potential, given the problems of safety encountered with viral vectors. Using these non-viral vectors, the current challenge is to obtain a similarly effective transfection to viral ones. Based on the advantages and disadvantages of existing vectors and on the hurdles encountered with these carriers, the aim of this review is to describe the "perfect vector" for systemic gene therapy against cancer.

Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours.

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Buhles, Alexandra; Collins, Sara A; van Pijkeren, Jan P; Rajendran, Simon; Miles, Michelle; O'Sullivan, Gerald C; O'Hanlon, Deirdre M; Tangney, Mark

2009-03-09

The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate

Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

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Mayumi Oakland

2013-01-01

Full Text Available Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction.

An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector

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Vreugdenhil Erno

2010-07-01

Full Text Available Abstract Background This study compared the transduction efficiencies of an adeno-associated viral (AAV vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP, with a lentiviral (LV vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed, to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 × 108 or 1 × 109 genomic copies of AAV1-GFP and 1 × 105 transducing units of LV-dsRed. Results Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. Conclusions This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.

Lentiviral Vector Mediated Claudin1 Silencing Inhibits Epithelial to Mesenchymal Transition in Breast Cancer Cells

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Xianqi Zhao

2015-06-01

Full Text Available Breast cancer has a high incidence and mortality rate worldwide. Several viral vectors including lentiviral, adenoviral and adeno-associated viral vectors have been used in gene therapy for various forms of human cancer, and have shown promising effects in controlling tumor development. Claudin1 (CLDN1 is a member of the tetraspan transmembrane protein family that plays a major role in tight junctions and is associated with tumor metastasis. However, the role of CLDN1 in breast cancer is largely unexplored. In this study, we tested the therapeutic potential of silencing CLDN1 expression in two breast cancer (MDA-MB-231 and MCF7 cell lines using lentiviral vector mediated RNA interference. We found that a CLDN1 short hairpin (shRNA construct efficiently silenced CLDN1 expression in both breast cancer cell lines, and CLDN1 knockdown resulted in reduced cell proliferation, survival, migration and invasion. Furthermore, silencing CLDN1 inhibited epithelial to mesenchymal transition (EMT by upregulating the epithelial cell marker, E-cadherin, and downregulating mesenchymal markers, smooth muscle cell alpha-actin (SMA and Snai2. Our data demonstrated that lentiviral vector mediated CLDN1 RNA interference has great potential in breast cancer gene therapy by inhibiting EMT and controlling tumor cell growth.

Delivery of viral vectors to tumor cells: extracellular transport, systemic distribution, and strategies for improvement.

Science.gov (United States)

Wang, Yong; Yuan, Fan

2006-01-01

It is a challenge to deliver therapeutic genes to tumor cells using viral vectors because (i) the size of these vectors are close to or larger than the space between fibers in extracellular matrix and (ii) viral proteins are potentially toxic in normal tissues. In general, gene delivery is hindered by various physiological barriers to virus transport from the site of injection to the nucleus of tumor cells and is limited by normal tissue tolerance of toxicity determined by local concentrations of transgene products and viral proteins. To illustrate the obstacles encountered in the delivery and yet limit the scope of discussion, this review focuses only on extracellular transport in solid tumors and distribution of viral vectors in normal organs after they are injected intravenously or intratumorally. This review also discusses current strategies for improving intratumoral transport and specificity of viral vectors.

Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs.

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Masayuki Sano

Full Text Available Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp. Because of the capacity of Sendai virus (SeV nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications.

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

Science.gov (United States)

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

2017-03-01

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

Viral Vectors for Use in the Development of Biodefense Vaccines

National Research Council Canada - National Science Library

Lee, John S; Hadjipanayis, Angela G; Parker, Michael D

2005-01-01

... agents of bioterrorism or biowarfare. The use of viruses, for example adenovirus, vaccinia virus, and Venezuelan equine encephalitis virus, as vaccine-vectors has enabled researchers to develop effective means for countering the threat of bioterrorism and biowarfare. An overview of the different viral vectors and the threats they counter will be discussed.

CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

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Rasmus O. Bak

2017-07-01

Full Text Available The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV vectors to serve as donor template DNA during homologous recombination (HR. However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing. We use the methodology in primary human T cells and CD34+ hematopoietic stem and progenitor cells to site-specifically integrate an expression cassette that, as a single donor vector, would otherwise amount to a total of 6.5 kb. This approach now provides an efficient way to integrate large transgene cassettes into the genomes of primary human cells using HR-mediated genome editing with AAV vectors.

CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice

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Oscar Meca-Cortés

2017-09-01

Full Text Available The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs, using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene, under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells. Keywords: CRISPR/Cas9, cell therapy, mesenchymal stem cells, bystander suicide therapy, glioblastoma, non-invasive bioluminescence imaging, CRISPR/Cas9, CRISPR/Cas9 knockin

Viral vectors for production of recombinant proteins in plants.

Science.gov (United States)

Lico, Chiara; Chen, Qiang; Santi, Luca

2008-08-01

Global demand for recombinant proteins has steadily accelerated for the last 20 years. These recombinant proteins have a wide range of important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and components of novel nano-particles for various applications. The majority of recombinant proteins are produced by traditional biological "factories," that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. However, these traditional technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. During the last two decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe production of recombinant proteins. Although the genetic engineering of plant viral vectors for heterologous gene expression can be dated back to the early 1980s, recent understanding of plant virology and technical progress in molecular biology have allowed for significant improvements and fine tuning of these vectors. These breakthroughs enable the flourishing of a variety of new viral-based expression systems and their wide application by academic and industry groups. In this review, we describe the principal plant viral-based production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their applications. We will summarize the recent progress in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. (c) 2008 Wiley-Liss, Inc.

Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

Science.gov (United States)

Herzog, R W; Yang, E Y; Couto, L B; Hagstrom, J N; Elwell, D; Fields, P A; Burton, M; Bellinger, D A; Read, M S; Brinkhous, K M; Podsakoff, G M; Nichols, T C; Kurtzman, G J; High, K A

1999-01-01

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Brachial Artery Flow-mediated Dilation Following Exercise with Augmented Oscillatory and Retrograde Shear Rate

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Johnson Blair D

2012-08-01

Full Text Available Abstract Background Acute doses of elevated retrograde shear rate (SR appear to be detrimental to endothelial function in resting humans. However, retrograde shear increases during moderate intensity exercise which also enhances post-exercise endothelial function. Since SR patterns differ with the modality of exercise, it is important to determine if augmented retrograde SR during exercise influences post-exercise endothelial function. This study tested the hypothesis that (1 increased doses of retrograde SR in the brachial artery during lower body supine cycle ergometer exercise would attenuate post-exercise flow-mediated dilation (FMD in a dose-dependent manner, and (2 antioxidant vitamin C supplementation would prevent the attenuated post-exercise FMD response. Methods Twelve men participated in four randomized exercise sessions (90 W for 20 minutes on separate days. During three of the sessions, one arm was subjected to increased oscillatory and retrograde SR using three different forearm cuff pressures (20, 40, 60 mmHg (contralateral arm served as the control and subjects ingested placebo capsules prior to exercise. A fourth session with 60 mmHg cuff pressure was performed with 1 g of vitamin C ingested prior to the session. Results Post-exercise FMD following the placebo conditions were lower in the cuffed arm versus the control arm (arm main effect: P P > 0.05. Following vitamin C treatment, post-exercise FMD in the cuffed and control arm increased from baseline (P P > 0.05. Conclusions These results indicate that augmented oscillatory and retrograde SR in non-working limbs during lower body exercise attenuates post-exercise FMD without an evident dose–response in the range of cuff pressures evaluated. Vitamin C supplementation prevented the attenuation of FMD following exercise with augmented oscillatory and retrograde SR suggesting that oxidative stress contributes to the adverse effects of oscillatory and

Cell-mediated immune responses in rainbow trout after DNA immunization against the viral hemorrhagic septicemia virus

DEFF Research Database (Denmark)

Utke, Katrin; Kock, Holger; Schuetze, Heike

2008-01-01

injection site rather than to injection sites of heterologous vaccines, suggesting the antigen specificity of homing. By demonstrating CMC responses to distinct viral proteins and homing in rainbow trout, these results substantially contribute to the understanding of the teleost immune system.......To identify viral proteins that induce cell-mediated cytotoxicity (CMC) against viral hemorrhagic septicemia virus (VHSV)-infected cells, rainbow trout were immunized with DNA vectors encoding the glycoprotein G or the nucleocapsid protein N of VHSV. The G protein was a more potent trigger...... of cytotoxic cells than the N protein. Peripheral blood leukocytes (PBL) isolated from trout immunized against the G protein killed both VHSV-infected MHC class I matched (RTG-2) and VHSV-infected xenogeneic (EPC) target cells, suggesting the involvement of both cytotoxic T lymphocytes (CTL) and NK cells...

Novel non-viral vectors for gene delivery: synthesis of a second-generation library of mono-functionalized poly-(guanidinium)amines and their introduction into cationic lipids.

Science.gov (United States)

Byk, G; Soto, J; Mattler, C; Frederic, M; Scherman, D

1998-01-01

The development of new gene delivery technologies is a prerequisite towards gene therapy clinical trials. Because gene delivery mediated by viral vectors remains of limited scope due to immunological and propagation risks, the development of new non-viral gene delivery systems is of crucial importance. We have synthesized a secondary library of mono-functionalized poly-(guanidinium)amines generated from a library of mono-functionalized polyamines applying the concept of "libraries from libraries." The method allows a quick and easy access to mono-functionalized geometrically varied poly-(guanidinium)amines. The new building blocks were introduced into cationic lipids to obtain novel poly-(guanidinium)amine lipids, which are potential DNA vectors for gene delivery. Copyright 1998 John Wiley & Sons, Inc.

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

International Nuclear Information System (INIS)

Wang, Ying-Nai; Wang, Hongmei; Yamaguchi, Hirohito; Lee, Hong-Jen; Lee, Heng-Huan; Hung, Mien-Chie

2010-01-01

Research highlights: → ARF1 activation is involved in the EGFR transport to the ER and the nucleus. → Assembly of γ-COP coatomer mediates EGFR transport to the ER and the nucleus. → Golgi-to-ER retrograde trafficking regulates nuclear transport of EGFR. -- Abstract: Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH 2 -terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.

The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice.

Science.gov (United States)

Uchida, Kenzo; Nakajima, Hideaki; Hirai, Takayuki; Yayama, Takafumi; Chen, Kebing; Guerrero, Alexander Rodriguez; Johnson, William Eustace; Baba, Hisatoshi

2012-12-15

The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS

Chikungunya Virus Vaccines: Viral Vector-Based Approaches.

Science.gov (United States)

Ramsauer, Katrin; Tangy, Frédéric

2016-12-15

In 2013, a major chikungunya virus (CHIKV) epidemic reached the Americas. In the past 2 years, >1.7 million people have been infected. In light of the current epidemic, with millions of people in North and South America at risk, efforts to rapidly develop effective vaccines have increased. Here, we focus on CHIKV vaccines that use viral-vector technologies. This group of vaccine candidates shares an ability to potently induce humoral and cellular immune responses by use of highly attenuated and safe vaccine backbones. So far, well-described vectors such as modified vaccinia virus Ankara, complex adenovirus, vesicular stomatitis virus, alphavirus-based chimeras, and measles vaccine Schwarz strain (MV/Schw) have been described as potential vaccines. We summarize here the recent data on these experimental vaccines, with a focus on the preclinical and clinical activities on the MV/Schw-based candidate, which is the first CHIKV-vectored vaccine that has completed a clinical trial. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Insect symbiotic bacteria harbour viral pathogens for transovarial transmission.

Science.gov (United States)

Jia, Dongsheng; Mao, Qianzhuo; Chen, Yong; Liu, Yuyan; Chen, Qian; Wu, Wei; Zhang, Xiaofeng; Chen, Hongyan; Li, Yi; Wei, Taiyun

2017-03-06

Many insects, including mosquitoes, planthoppers, aphids and leafhoppers, are the hosts of bacterial symbionts and the vectors for transmitting viral pathogens 1-3 . In general, symbiotic bacteria can indirectly affect viral transmission by enhancing immunity and resistance to viruses in insects 3-5 . Whether symbiotic bacteria can directly interact with the virus and mediate its transmission has been unknown. Here, we show that an insect symbiotic bacterium directly harbours a viral pathogen and mediates its transovarial transmission to offspring. We observe rice dwarf virus (a plant reovirus) binding to the envelopes of the bacterium Sulcia, a common obligate symbiont of leafhoppers 6-8 , allowing the virus to exploit the ancient oocyte entry path of Sulcia in rice leafhopper vectors. Such virus-bacterium binding is mediated by the specific interaction of the viral capsid protein and the Sulcia outer membrane protein. Treatment with antibiotics or antibodies against Sulcia outer membrane protein interferes with this interaction and strongly prevents viral transmission to insect offspring. This newly discovered virus-bacterium interaction represents the first evidence that a viral pathogen can directly exploit a symbiotic bacterium for its transmission. We believe that such a model of virus-bacterium communication is a common phenomenon in nature.

Adeno-associated viral vectors as agents for gene delivery : application in disorders and trauma of the central nervous system

NARCIS (Netherlands)

Ruitenberg, Marc J; Eggers, Ruben; Boer, Gerard J; Verhaagen, J.

2002-01-01

The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors,

Development of Viral Vectors for Gene Therapy for Chronic Pain

Directory of Open Access Journals (Sweden)

Yu Huang

2011-01-01

Full Text Available Chronic pain is a major health concern that affects millions of people. There are no adequate long-term therapies for chronic pain sufferers, leading to significant cost for both society and the individual. The most commonly used therapy for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and may cause side effects. Further studies of the mechanisms of chronic pain have opened the way for development of new treatment strategies, one of which is gene therapy. The key to gene therapy is selecting safe and highly efficient gene delivery systems that can deliver therapeutic genes to overexpress or suppress relevant targets in specific cell types. Here we review several promising viral vectors that could be applied in gene transfer for the treatment of chronic pain and further discuss the possible mechanisms of genes of interest that could be delivered with viral vectors for the treatment of chronic pain.

P53-mediated rapid induction of apoptosis conveys resistance to viral infection in Drosophila melanogaster.

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Bo Liu

2013-02-01

Full Text Available Arthropod-borne pathogens account for millions of deaths each year. Understanding the genetic mechanisms controlling vector susceptibility to pathogens has profound implications for developing novel strategies for controlling insect-transmitted infectious diseases. The fact that many viruses carry genes that have anti-apoptotic activity has long led to the hypothesis that induction of apoptosis could be a fundamental innate immune response. However, the cellular mechanisms mediating the induction of apoptosis following viral infection remained enigmatic, which has prevented experimental verification of the functional significance of apoptosis in limiting viral infection in insects. In addition, studies with cultured insect cells have shown that there is sometimes a lack of apoptosis, or the pro-apoptotic response happens relatively late, thus casting doubt on the functional significance of apoptosis as an innate immunity. Using in vivo mosquito models and the native route of infection, we found that there is a rapid induction of reaper-like pro-apoptotic genes within a few hours following exposure to DNA or RNA viruses. Recapitulating a similar response in Drosophila, we found that this rapid induction of apoptosis requires the function of P53 and is mediated by a stress-responsive regulatory region upstream of reaper. More importantly, we showed that the rapid induction of apoptosis is responsible for preventing the expression of viral genes and blocking the infection. Genetic changes influencing this rapid induction of reaper-like pro-apoptotic genes led to significant differences in susceptibility to viral infection.

Viral Vector-Based Dissection of Marmoset GFAP Promoter in Mouse and Marmoset Brains.

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Yoichiro Shinohara

Full Text Available Adeno-associated virus (AAV vectors are small in diameter, diffuse easily in the brain, and represent a highly efficient means by which to transfer a transgene to the brain of a large animal. A major demerit of AAV vectors is their limited accommodation capacity for transgenes. Thus, a compact promoter is useful when delivering large transgenes via AAV vectors. In the present study, we aimed to identify the shortest astrocyte-specific GFAP promoter region that could be used for AAV-vector-mediated transgene expression in the marmoset brain. The 2.0-kb promoter region upstream of the GFAP gene was cloned from the marmoset genome, and short promoters (1.6 kb, 1.4 kb, 0.6 kb, 0.3 kb and 0.2 kb were obtained by progressively deleting the original 2.0-kb promoter from the 5' end. The short promoters were screened in the mouse cerebellum in terms of their strength and astrocyte specificity. We found that the 0.3-kb promoter maintained 40% of the strength of the original 2.0-kb promoter, and approximately 90% of its astrocyte specificity. These properties were superior to those of the 1.4-kb, 0.6-kb (20% promoter strength and 0.2-kb (70% astrocyte specificity promoters. Then, we verified whether the 0.3-kb GFAP promoter retained astrocyte specificity in the marmoset cerebral cortex. Injection of viral vectors carrying the 0.3-kb marmoset GFAP promoter specifically transduced astrocytes in both the cerebral cortex and cerebellar cortex of the marmoset. These results suggest that the compact 0.3-kb promoter region serves as an astrocyte-specific promoter in the marmoset brain, which permits us to express a large gene by AAV vectors that have a limited accommodation capacity.

Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

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Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

2014-07-01

The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL-1 during 40 days, and HSV-1, 2.7 Log10 PFU mL-1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL-1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

International Nuclear Information System (INIS)

Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

2010-01-01

Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

Methods of treating Parkinson's disease using viral vectors

Energy Technology Data Exchange (ETDEWEB)

Bankiewicz, Krystof; Cunningham, Janet

2016-11-15

Methods of delivering viral vectors, particularly recombinant adeno-associated virus (rAAV) virions, to the central nervous system (CNS) using convection enhanced delivery (CED) are provided. The rAAV virions include a nucleic acid sequence encoding a therapeutic polypeptide. The methods can be used for treating CNS disorders such as for treating Parkinson's Disease.

Neural stem cell-derived exosomes mediate viral entry

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Sims B

2014-10-01

Full Text Available Brian Sims,1,2,* Linlin Gu,3,* Alexandre Krendelchtchikov,3 Qiana L Matthews3,4 1Division of Neonatology, Department of Pediatrics, 2Department of Cell, Developmental, and Integrative Biology, 3Division of Infectious Diseases, Department of Medicine, 4Center for AIDS Research, University of Alabama at Birmingham, Birmingham, AL, USA *These authors contributed equally to this work Background: Viruses enter host cells through interactions of viral ligands with cellular receptors. Viruses can also enter cells in a receptor-independent fashion. Mechanisms regarding the receptor-independent viral entry into cells have not been fully elucidated. Exosomal trafficking between cells may offer a mechanism by which viruses can enter cells.Methods: To investigate the role of exosomes on cellular viral entry, we employed neural stem cell-derived exosomes and adenovirus type 5 (Ad5 for the proof-of-principle study. Results: Exosomes significantly enhanced Ad5 entry in Coxsackie virus and adenovirus receptor (CAR-deficient cells, in which Ad5 only had very limited entry. The exosomes were shown to contain T-cell immunoglobulin mucin protein 4 (TIM-4, which binds phosphatidylserine. Treatment with anti-TIM-4 antibody significantly blocked the exosome-mediated Ad5 entry.Conclusion: Neural stem cell-derived exosomes mediated significant cellular entry of Ad5 in a receptor-independent fashion. This mediation may be hampered by an antibody specifically targeting TIM-4 on exosomes. This set of results will benefit further elucidation of virus/exosome pathways, which would contribute to reducing natural viral infection by developing therapeutic agents or vaccines. Keywords: neural stem cell-derived exosomes, adenovirus type 5, TIM-4, viral entry, phospholipids

Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

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El Andaloussi Samir

2011-05-01

Full Text Available Abstract Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s. Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s, which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for

Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

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Busch Melanie

2009-12-01

Full Text Available Abstract Background Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as β-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS-coupled MIDGE encoding the β-endorphin precursor proopiomelanocortin (POMC on complete Freund's adjuvant-induced inflammatory pain in rats. Results POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of β-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing β-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of β-endorphin. Conclusion Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or β-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.

DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations

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Maria Schnödt

2016-01-01

Full Text Available Adeno-associated viral (AAV vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss and self-complementary (sc AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV. Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.

Enhancement of Mucosal Immunogenicity of Viral Vectored Vaccines by the NKT Cell Agonist Alpha-Galactosylceramide as Adjuvant

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Shailbala Singh

2014-10-01

Full Text Available Gene-based vaccination strategies, specifically viral vectors encoding vaccine immunogens are effective at priming strong immune responses. Mucosal routes offer practical advantages for vaccination by ease of needle-free administration, and immunogen delivery at readily accessible oral/nasal sites to efficiently induce immunity at distant gut and genital tissues. However, since mucosal tissues are inherently tolerant for induction of immune responses, incorporation of adjuvants for optimal mucosal vaccination strategies is important. We report here the effectiveness of alpha-galactosylceramide (α-GalCer, a synthetic glycolipid agonist of natural killer T (NKT cells, as an adjuvant for enhancing immunogenicity of vaccine antigens delivered using viral vectors by mucosal routes in murine and nonhuman primate models. Significant improvement in adaptive immune responses in systemic and mucosal tissues was observed by including α-GalCer adjuvant for intranasal immunization of mice with vesicular stomatitis virus vector encoding the model antigen ovalbumin and adenoviral vectors expressing HIV env and Gag antigens. Activation of NKT cells in systemic and mucosal tissues along with significant increases in adaptive immune responses were observed in rhesus macaques immunized by intranasal and sublingual routes with protein or adenovirus vectored antigens when combined with α-GalCer adjuvant. These results support the utility of α-GalCer adjuvant for enhancing immunogenicity of mucosal vaccines delivered using viral vectors.

Viral and vector zoonotic exploitation of a homo-sociome memetic complex.

Science.gov (United States)

Rupprecht, C E; Burgess, G W

2015-05-01

As most newly characterized emerging infectious diseases are considered to be zoonotic, a modern pre-eminence ascribed within this classification lies clearly within the viral taxonomic realm. In particular, RNA viruses deserve special concern given their documented impact on conservation biology, veterinary medicine and public health, with an unprecedented ability to promote an evolutionary host-pathogen arms race from the ultimate infection and immunity perspective. However, besides the requisite molecular/gross anatomical and physiological bases for infectious diseases to transmit from one host to another, both viral pathogens and their reservoirs/vectors exploit a complex anthropological, cultural, historical, psychological and social suite that specifically defines the phylodynamics within Homo sapiens, unlike any other species. Some of these variables include the ecological benefits of living in groups, decisions on hunting and foraging behaviours and dietary preferences, myths and religious doctrines, health economics, travel destinations, population planning, political decisions on agricultural product bans and many others, in a homo-sociome memetic complex. Taken to an extreme, such complexities elucidate the underpinnings of explanations as to why certain viral zoonoses reside in neglected people, places and things, whereas others are chosen selectively and prioritized for active mitigation. Canine-transmitted rabies serves as one prime example of how a neglected viral zoonosis may transition to greater attention on the basis of renewed advocacy, social media, local champions and vested international community engagement. In contrast, certain bat-associated and arboviral diseases suffer from basic ignorance and perpetuated misunderstanding of fundamental reservoir and vector ecology tenets, translated into failed control policies that only exacerbate the underlying environmental conditions of concern. Beyond applied biomedical knowledge, epidemiological

Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures

DEFF Research Database (Denmark)

Woldbye, David Paul Drucker; Ängehagen, Mikael; Gøtzsche, Casper René

2010-01-01

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure...... recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2...... and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment....

Self-interacting dark matter with a stable vector mediator

OpenAIRE

Duerr, Michael; Schmidt-Hoberg, Kai; Wild, Sebastian

2018-01-01

Light vector mediators can naturally induce velocity-dependent dark matter self-interactions while at the same time allowing for the correct dark matter relic abundance via thermal freeze-out. If these mediators subsequently decay into Standard Model states such as electrons or photons however, this is robustly excluded by constraints from the Cosmic Microwave Background. We study to what extent this conclusion can be circumvented if the vector mediator is stable and hence contributes to the ...

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

Science.gov (United States)

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

2017-12-07

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

An Update on Canine Adenovirus Type 2 and Its Vectors

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Bru, Thierry; Salinas, Sara; Kremer, Eric J.

2010-01-01

Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd) have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2) biology and gives an overview of the generation of early region 1 (E1)-deleted to helper-dependent (HD) CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors. PMID:21994722

Host and Viral Factors in HIV-Mediated Bystander Apoptosis

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Garg, Himanshu; Joshi, Anjali

2017-01-01

Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis. PMID:28829402

An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene.

Science.gov (United States)

Cui, Hongguang; Wang, Aiming

2017-03-01

RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus-induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense-orientated target gene sequence of 100-200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV-based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector-mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Neogenesis and proliferation of β-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

International Nuclear Information System (INIS)

Tokui, Yae; Kozawa, Junji; Yamagata, Kazuya; Zhang, Jun; Ohmoto, Hiroshi; Tochino, Yoshihiro; Okita, Kohei; Iwahashi, Hiromi; Namba, Mitsuyoshi; Shimomura, Iichiro; Miyagawa, Jun-ichiro

2006-01-01

Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of β-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a β-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of β-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of β-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted β-cell differentiation (mainly from duct cells) and proliferation of pre-existing β-cells, resulting in an increase of the β-cell mass that improved glucose tolerance in diabetic mice

Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep

Science.gov (United States)

Davey, Marcus G.; Riley, John S.; Andrews, Abigail; Tyminski, Alec; Limberis, Maria; Pogoriler, Jennifer E.; Partridge, Emily; Olive, Aliza; Hedrick, Holly L.; Flake, Alan W.; Peranteau, William H.

2017-01-01

A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens. PMID:28141818

Adeno-associated viral vector transduction of human mesenchymal stem cells

DEFF Research Database (Denmark)

Stender, Stefan; Murphy, Mary; O'Brien, Tim

2007-01-01

Mesenchymal stem cells (MSCs) have received considerable attention in the emerging field of regenerative medicine. One aspect of MSC research focuses on genetically modifying the cells with the aim of enhancing their regenerative potential. Adeno-associated virus (AAV) holds promise as a vector...... in human MSCs and to assess whether AAV transduction affects MSC multipotentiality. The results indicated that human MSCs could indeed be transiently transduced in vitro by the AAV2 vector with efficiencies of up to 65%. The percentage of GFP-positive cells peaked at 4 days post-transduction and declined...... rapidly towards 0% after day 8. The level of transgene expression in the GFP-positive population increased 4-fold over a 10,000 fold viral dose increase. This dose-response contrasted with the 200-fold increase observed in similarly transduced 293-cells, indicating a relatively restricted transgene...

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

Science.gov (United States)

Raisin, Sophie; Morille, Marie; Bony, Claire; Noël, Danièle; Devoisselle, Jean-Marie; Belamie, Emmanuel

2017-08-22

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Productive infection of human immunodeficiency virus type 1 in dendritic cells requires fusion-mediated viral entry

International Nuclear Information System (INIS)

Janas, Alicia M.; Dong, Chunsheng; Wang Jianhua; Wu Li

2008-01-01

Human immunodeficiency virus type 1 (HIV-1) enters dendritic cells (DCs) through endocytosis and viral receptor-mediated fusion. Although endocytosis-mediated HIV-1 entry can generate productive infection in certain cell types, including human monocyte-derived macrophages, productive HIV-1 infection in DCs appears to be dependent on fusion-mediated viral entry. It remains to be defined whether endocytosed HIV-1 in DCs can initiate productive infection. Using HIV-1 infection and cellular fractionation assays to measure productive viral infection and entry, here we show that HIV-1 enters monocyte-derived DCs predominately through endocytosis; however, endocytosed HIV-1 cannot initiate productive HIV-1 infection in DCs. In contrast, productive HIV-1 infection in DCs requires fusion-mediated viral entry. Together, these results provide functional evidence in understanding HIV-1 cis-infection of DCs, suggesting that different pathways of HIV-1 entry into DCs determine the outcome of viral infection

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

Science.gov (United States)

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

2007-07-30

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

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

Science.gov (United States)

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

2006-08-01

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

Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

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Chang Myint OO

2009-10-01

Full Text Available Abstract Background Hepatitis C virus (HCV is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1 and the origin of latent viral DNA replication (OriP sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV replicon cells. Results We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector. Conclusion These findings indicate that we successfully constructed a long-term transgene (shRNA expression vector (Ac-EP-shRNA452 using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA expression vector was evaluated with the view

Is passive transmission of non-viral vectors through artificial insemination of sperm-DNA mixtures sufficient for chicken transgenesis?

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CHAPARIAN, Shahram; ABDULAHNEJAD, Ahad; RASHIDI, Farzad; TOGHYANI, Majid; GHEISARI, Abbasali; EGHBALSAIED, Shahin

2016-01-01

DNA uptake in the post-acrosomal region of the spermatozoa takes place exclusively in immotile spermatozoa that are naturally unable to fertilize eggs. The present study aimed to assess whether passive transmission of non-viral vectors to the surrounding areas of chicken embryos could be an alternate mechanism in chicken sperm-mediated gene transfer. First, the presence of nucleases in rooster seminal plasma was evaluated. Semen ejaculates from five roosters were centrifuged and the supernatant was incubated with pBL2 for 1 h. A robust nuclease cocktail was detected in the rooster semen. To overcome these nucleases, plasmid-TransIT combinations were incubated with semen for 1 h. Incubation of exogenous DNA in the lipoplex structure could considerably bypass the semen nuclease effect. Then, intravaginal insemination of 1 × 109 sperm mixed with lipoplexes (40 µg pBL2:40 µl TransIT) was carried out in 15 virgin hens. Neither the epithelial tissue from the inseminated female reproductive tracts nor the produced embryos following artificial insemination showed the transgene. To remove any bias in the transgene transmission possibility, the plasmid-TransIT admixture was directly injected in close vicinity of the embryos in newly laid eggs. Nonetheless, none of the produced fetuses or chicks carried the transgene. In conclusion, the results of the present study revealed a nuclease admixture in rooster seminal plasma, and passive/active transmission of the non-viral vector into close vicinity of the chicken embryo was inefficient for producing transgenic chicks. PMID:26935324

Hybrid Lentivirus-transposon Vectors With a Random Integration Profile in Human Cells

DEFF Research Database (Denmark)

Staunstrup, Nicklas H; Moldt, Brian; Mátés, Lajos

2009-01-01

Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral...... Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase......-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans...

Construction of a recombinant viral vector containing part of the nucleocapsid protein gene of newcastle disease virus

Energy Technology Data Exchange (ETDEWEB)

Bader, D.E.

1995-09-01

This report describes the procedures used to clone a 673 base pair gene fragment of the major nucleocapsid protein gene of Newcastle disease virus into a viral vector molecule for the purpose of maintaining a stable, long-term, renewable source of this target sequence for gene probe studies. The gene fragment was prepared by reverse-transcription polymerase chain reaction of Newcastle disease virus RNA and was cloned into the viral DNA vector Ml3mp18 RF to produce a recombinant DNA molecule. The cloned fragment was shown to be present in the recombinant clones based on (i) clonal selection on indicator plates; (ii) restriction enzyme analysis; (iii) gene probe analysis and (iv) nested PCR amplification.

Adenovirus Vector-Derived VA-RNA-Mediated Innate Immune Responses

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Hiroyuki Mizuguchi

2011-07-01

Full Text Available The major limitation of the clinical use of replication-incompetent adenovirus (Ad vectors is the interference by innate immune responses, including induction of inflammatory cytokines and interferons (IFN, following in vivo application of Ad vectors. Ad vector-induced production of inflammatory cytokines and IFNs also results in severe organ damage and efficient induction of acquired immune responses against Ad proteins and transgene products. Ad vector-induced innate immune responses are triggered by the recognition of Ad components by pattern recognition receptors (PRRs. In order to reduce the side effects by Ad vector-induced innate immune responses and to develop safer Ad vectors, it is crucial to clarify which PRRs and which Ad components are involved in Ad vector-induced innate immune responses. Our group previously demonstrated that myeloid differentiating factor 88 (MyD88 and toll-like receptor 9 (TLR9 play crucial roles in the Ad vector-induced inflammatory cytokine production in mouse bone marrow-derived dendritic cells. Furthermore, our group recently found that virus associated-RNAs (VA-RNAs, which are about 160 nucleotide-long non-coding small RNAs encoded in the Ad genome, are involved in IFN production through the IFN-β promoter stimulator-1 (IPS-1-mediated signaling pathway following Ad vector transduction. The aim of this review is to highlight the Ad vector-induced innate immune responses following transduction, especially VA-RNA-mediated innate immune responses. Our findings on the mechanism of Ad vector-induced innate immune responses should make an important contribution to the development of safer Ad vectors, such as an Ad vector lacking expression of VA-RNAs.

Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus

International Nuclear Information System (INIS)

Zhang Jing; Yamada, Osamu; Sakamoto, Takashi; Yoshida, Hiroshi; Iwai, Takahiro; Matsushita, Yoshihisa; Shimamura, Hideo; Araki, Hiromasa; Shimotohno, Kunitada

2004-01-01

Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bγ), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects

A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons

NARCIS (Netherlands)

Fagoe, N D; Eggers, R; Verhaagen, J; Mason, M R J

Adeno-associated viral (AAV) vectors based on serotype 5 are an efficient means to target dorsal root ganglia (DRG) to study gene function in the primary sensory neurons of the peripheral nervous system. In this study, we have developed a compact AAV dual promoter vector composed of the

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells.

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Silva, J P Neves; Oliveira, A C N; Casal, M P P A; Gomes, A C; Coutinho, P J G; Coutinho, O P; Oliveira, M E C D Real

2011-10-01

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine™ LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the

Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

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Andreea Popa

2015-02-01

Full Text Available Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

pEPito: a significantly improved non-viral episomal expression vector for mammalian cells

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Ogris Manfred

2010-03-01

Full Text Available Abstract Background The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP and directed into a 2000 bp long matrix attachment region sequence (MARS derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression. Results Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both in vitro and in vivo. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies in vitro, as well as more persistent transgene expression profiles in vivo. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P element that is known to be less affected by epigenetic silencing events. Conclusions The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

A comparative analysis of constitutive promoters located in adeno-associated viral vectors.

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Lkhagvasuren Damdindorj

Full Text Available The properties of constitutive promoters within adeno-associated viral (AAV vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV, simian virus 40, and herpes simplex virus thymidine kinase, were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.

Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing

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Pavel I. Ortinski

2017-06-01

Full Text Available The CRISPR/Cas9 systems have revolutionized the field of genome editing by providing unprecedented control over gene sequences and gene expression in many species, including humans. Lentiviral vectors (LVs are one of the primary delivery platforms for the CRISPR/Cas9 system due to their ability to accommodate large DNA payloads and sustain robust expression in a wide range of dividing and non-dividing cells. However, long-term expression of LV-delivered Cas9/guide RNA may lead to undesirable off-target effects characterized by non-specific RNA-DNA interactions and off-target DNA cleavages. Integrase-deficient lentiviral vectors (IDLVs present an attractive means for delivery of CRISPR/Cas9 components because: (1 they are capable of transducing a broad range of cells and tissues, (2 have superior packaging capacity compared to other vectors (e.g., adeno-associated viral vectors, and (3 they are expressed transiently and demonstrate very weak integration capability. In this manuscript, we aimed to establish IDLVs as a means for safe and efficient delivery of CRISPR/Cas9. To this end, we developed an all-in-one vector cassette with increased production efficacy and demonstrated that CRISPR/Cas9 delivered by the improved IDLV vectors can mediate rapid and robust gene editing in human embryonic kidney (HEK293T cells and post-mitotic brain neurons in vivo, via transient expression and with higher gene-targeting specificity than the corresponding integrase-competent vectors.

Characterising dark matter searches at colliders and direct detection experiments: Vector mediators

International Nuclear Information System (INIS)

Buchmueller, Oliver; Dolan, Matthew J.; Malik, Sarah A.; McCabe, Christopher

2015-01-01

We introduce a Minimal Simplified Dark Matter (MSDM) framework to quantitatively characterise dark matter (DM) searches at the LHC. We study two MSDM models where the DM is a Dirac fermion which interacts with a vector and axial-vector mediator. The models are characterised by four parameters: m DM , M med, g DM and g q , the DM and mediator masses, and the mediator couplings to DM and quarks respectively. The MSDM models accurately capture the full event kinematics, and the dependence on all masses and couplings can be systematically studied. The interpretation of mono-jet searches in this framework can be used to establish an equal-footing comparison with direct detection experiments. For theories with a vector mediator, LHC mono-jet searches possess better sensitivity than direct detection searches for light DM masses (≲5 GeV). For axial-vector mediators, LHC and direct detection searches generally probe orthogonal directions in the parameter space. We explore the projected limits of these searches from the ultimate reach of the LHC and multi-ton xenon direct detection experiments, and find that the complementarity of the searches remains. In conclusion, we provide a comparison of limits in the MSDM and effective field theory (EFT) frameworks to highlight the deficiencies of the EFT framework, particularly when exploring the complementarity of mono-jet and direct detection searches

Safety of the novel influenza viral vector Brucella abortus vaccine in pregnant heifers

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Kaissar Tabynov

2016-01-01

Full Text Available ABSTRACT: The present study provides the first information about the safety of a new influenza viral vector vaccine expressing the Brucella ribosomal protein L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10 or subcutaneous (n=10 route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with B. abortus S19 (n=10 or B. abortus RB51 (n=10 and a negative (PBS+Montanide Gel01; n=10 control group. Clinical studies, thermometry, assessment of local reactogenicity and observation of abortion showed that the vector vaccine via the conjunctival or subcutaneous route was completely safe for pregnant heifers compared to the commercial vaccines B. abortus S19 or B. abortus RB51. The only single adverse event was the formation of infiltration at the site of subcutaneous injection; this reaction was not observed for the conjunctival route.

Promotion of Viral IRES-Mediated Translation Initiation under Mild Hypothermia.

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Maria Licursi

Full Text Available Internal ribosome entry site (IRES-mediated translation is an essential replication step for certain viruses. As IRES-mediated translation is regulated differently from cap-dependent translation under various cellular conditions, we sought to investigate whether temperature influences efficiency of viral IRES-mediated translation initiation by using bicistronic reporter constructs containing an IRES element of encephalomyocarditis virus (EMCV, foot-and-mouth disease virus (FMDV, hepatitis C virus (HCV, human rhinovirus (HRV or poliovirus (PV. Under mild hypothermic conditions (30 and 35°C, we observed increases in the efficiency of translation initiation by HCV and HRV IRES elements compared to translation initiation at 37°C. The promotion of HRV IRES activity was observed as early as 2 hours after exposure to mild hypothermia. We also confirmed the promotion of translation initiation by HRV IRES under mild hypothermia in multiple cell lines. The expression levels and locations of polypyrimidine tract-binding protein (PTB and upstream of N-Ras (unr, the IRES trans-acting factors (ITAFs of HCV and HRV IRES elements, were not modulated by the temperature shift from 37°C to 30°C. Taken together, this study demonstrates that efficiency of translation initiation by some viral IRES elements is temperature dependent.

Viral vector mediated continuous expression of interleukin-10 in DRG alleviates pain in type 1 diabetic animals.

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Thakur, Vikram; Gonzalez, Mayra; Pennington, Kristen; Chattopadhyay, Munmun

2016-04-01

Painful diabetic neuropathy is a common and difficult to treat complication of diabetes. A growing body of evidence implicates the role of inflammatory mediators in the damage to the peripheral axons and in the pathogenesis of neuropathic pain. Increased expression of pro-inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the peripheral nervous system suggests the possibility of change in pain perception in diabetes. In this study we investigated that continuous delivery of IL10 in the nerve fibers achieved by HSV vector mediated transduction of dorsal root ganglion (DRG) in animals with Type 1 diabetes, blocks the nociceptive and stress responses in the DRG neurons by reducing IL1β expression along with inhibition of phosphorylation of p38 MAPK (mitogen-activated protein kinase) and protein kinase C (PKC). The continuous expression of IL10 also alters Toll like receptor (TLR)-4 expression in the DRG with increased expression of heat shock protein (HSP)-70 in conjunction with the reduction of pain. Taken together, this study suggests that macrophage activation in the peripheral nervous system may be involved in the pathogenesis of pain in Type 1 diabetes and therapeutic benefits of HSV mediated local expression of IL10 in the DRG with the reduction of a number of proinflammatory cytokines, subsequently inhibits the development of painful neuropathy along with a decrease in stress associated markers in the DRG. This basic and preclinical study provides an important evidence for a novel treatment strategy that could lead to a clinical trial for what is currently a treatment resistant complication of diabetes. Copyright © 2016 Elsevier Inc. All rights reserved.

Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection.

Science.gov (United States)

Kuroda, Hitoshi; Kutner, Robert H; Bazan, Nicolas G; Reiser, Jakob

2009-05-01

During the past 12 years, lentiviral vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression. Despite significant progress, the production of high-titer high-quality lentiviral vectors is cumbersome and costly. The most commonly used method to produce lentiviral vectors involves transient transfection using calcium phosphate (CaP)-mediated precipitation of plasmid DNAs. However, inconsistencies in pH can cause significant batch-to-batch variations in lentiviral vector titers, making this method unreliable. This study describes optimized protocols for lentiviral vector production based on polyethylenimine (PEI)-mediated transfection, resulting in more consistent lentiviral vector stocks. To achieve this goal, simple production methods for high-titer lentiviral vector production involving transfection of HEK 293T cells immediately after plating were developed. Importantly, high titers were obtained with cell culture media lacking serum or other protein additives altogether. As a consequence, large-scale lentiviral vector stocks can now be generated with fewer batch-to-batch variations and at reduced costs and with less labor compared to the standard protocols.

Role of complement and antibodies in controlling infection with pathogenic simian immunodeficiency virus (SIV in macaques vaccinated with replication-deficient viral vectors

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Strasak Alexander

2009-06-01

Full Text Available Abstract Background We investigated the interplay between complement and antibodies upon priming with single-cycle replicating viral vectors (SCIV encoding SIV antigens combined with Adeno5-SIV or SCIV pseudotyped with murine leukemia virus envelope boosting strategies. The vaccine was applied via spray-immunization to the tonsils of rhesus macaques and compared with systemic regimens. Results Independent of the application regimen or route, viral loads were significantly reduced after challenge with SIVmac239 (p Conclusion The heterologous prime-boost strategy with replication-deficient viral vectors administered exclusively via the tonsils did not induce any neutralizing antibodies before challenge. However, after challenge, comparable SIV-specific humoral immune responses were observed in all vaccinated animals. Immunization with single cycle immunodeficiency viruses mounts humoral immune responses comparable to live-attenuated immunodeficiency virus vaccines.

Chikungunya viral fitness measures within the vector and subsequent transmission potential.

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Rebecca C Christofferson

Full Text Available Given the recent emergence of chikungunya in the Americas, the accuracy of forecasting and prediction of chikungunya transmission potential in the U.S. requires urgent assessment. The La Reunion-associated sub-lineage of chikungunya (with a valine substitution in the envelope protein was shown to increase viral fitness in the secondary vector, Ae. albopictus. Subsequently, a majority of experimental and modeling efforts focused on this combination of a sub-lineage of the East-Central-South African genotype (ECSA-V-Ae. albopictus, despite the Asian genotype being the etiologic agent of recent chikungunya outbreaks world-wide. We explore a collection of data to investigate relative transmission efficiencies of the three major genotypes/sub-lineages of chikungunya and found difference in the extrinsic incubation periods to be largely overstated. However, there is strong evidence supporting the role of Ae. albopictus in the expansion of chikungunya that our R0 calculations cannot attribute to fitness increases in one vector over another. This suggests other ecological factors associated with the Ae. albopictus-ECSA-V cycle may drive transmission intensity differences. With the apparent bias in literature, however, we are less prepared to evaluate transmission where Ae. aegypti plays a significant role. Holistic investigations of CHIKV transmission cycle(s will allow for more complete assessment of transmission risk in areas affected by either or both competent vectors.

Adeno-Associated Viral Vector-Induced Overexpression of Neuropeptide Y Y2 Receptors in the Hippocampus Suppresses Seizures

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Woldbye, David P. D.; Angehagen, Mikael; Gotzsche, Casper R.; Elbrond-Bek, Heidi; Sorensen, Andreas T.; Christiansen, Soren H.; Olesen, Mikkel V.; Nikitidou, Litsa; Hansen, Thomas v. O.; Kanter-Schlifke, Irene; Kokaia, Merab

2010-01-01

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is…

Viral Inhibition of PRR-Mediated Innate Immune Response: Learning from KSHV Evasion Strategies.

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Lee, Hye-Ra; Choi, Un Yung; Hwang, Sung-Woo; Kim, Stephanie; Jung, Jae U

2016-11-30

The innate immune system has evolved to detect and destroy invading pathogens before they can establish systemic infection. To successfully eradicate pathogens, including viruses, host innate immunity is activated through diverse pattern recognition receptors (PRRs) which detect conserved viral signatures and trigger the production of type I interferon (IFN) and pro-inflammatory cytokines to mediate viral clearance. Viral persistence requires that viruses co-opt cellular pathways and activities for their benefit. In particular, due to the potent antiviral activities of IFN and cytokines, viruses have developed various strategies to meticulously modulate intracellular innate immune sensing mechanisms to facilitate efficient viral replication and persistence. In this review, we highlight recent advances in the study of viral immune evasion strategies with a specific focus on how Kaposi's sarcoma-associated herpesvirus (KSHV) effectively targets host PRR signaling pathways.

Presencia de rotavirus durante un proceso de compostaje. Abonos como vectores de contaminación viral

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María Mercedes Martínez

2009-12-01

Full Text Available Rotavirus presence in a waste composting process. Organic fertilizers as vehicles for viral contamination. Objective. To show thepresence of rotavirus in different stages of a composting process: matrices used as raw material, mixture to be composted and the finalproduct. Materials and methods. Immunochromatography, ELISA and RT-PCR were used for viral detection. Results. Rotavirus wasfound in the first composting step, no virus was found in the second step, and some inhibitory substances were found in the third step thatposed difficulties in interpreting the PCR results and therefore providing a concluding result on rotavirus presence in the final product.Conclusions. Organic fertilizers can be vectors of human pathogenic viruses; therefore quality control tests must be implemented to avoidfurther viral dissemination. There are inhibitory substances present in organic fertilizers capable of interfering with the detection tests.

Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein

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Gary R. Whittaker

2012-06-01

Full Text Available Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes—A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.

Targeted viral-mediated plant genome editing using crispr/cas9

KAUST Repository

Mahfouz, Magdy M.; Ali, Zahir

2015-01-01

The present disclosure provides a viral-mediated genome-editing platform that facilitates multiplexing, obviates stable transformation, and is applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and systemically deliver a guide RNA molecules into plants overexpressing Cas9 endonuclease. High genomic modification frequencies were observed in inoculated as well as systemic leaves including the plant growing points. This system facilitates multiplexing and can lead to germinal transmission of the genomic modifications in the progeny, thereby obviating the requirements of repeated transformations and tissue culture. The editing platform of the disclosure is useful in plant genome engineering and applicable across plant species amenable to viral infections for agricultural biotechnology applications.

Targeted viral-mediated plant genome editing using crispr/cas9

KAUST Repository

Mahfouz, Magdy M.

2015-12-17

The present disclosure provides a viral-mediated genome-editing platform that facilitates multiplexing, obviates stable transformation, and is applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and systemically deliver a guide RNA molecules into plants overexpressing Cas9 endonuclease. High genomic modification frequencies were observed in inoculated as well as systemic leaves including the plant growing points. This system facilitates multiplexing and can lead to germinal transmission of the genomic modifications in the progeny, thereby obviating the requirements of repeated transformations and tissue culture. The editing platform of the disclosure is useful in plant genome engineering and applicable across plant species amenable to viral infections for agricultural biotechnology applications.

Retrograde peri-implantitis

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Mohamed Jumshad

2010-01-01

Full Text Available Retrograde peri-implantitis constitutes an important cause for implant failure. Retrograde peri-implantitis may sometimes prove difficult to identify and hence institution of early treatment may not be possible. This paper presents a report of four cases of (the implant placed developing to retrograde peri-implantitis. Three of these implants were successfully restored to their fully functional state while one was lost due to extensive damage. The paper highlights the importance of recognizing the etiopathogenic mechanisms, preoperative assessment, and a strong postoperative maintenance protocol to avoid retrograde peri-implant inflammation.

Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

International Nuclear Information System (INIS)

L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon

2007-01-01

NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application

The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more.

Science.gov (United States)

Johannes, Ludger

2002-07-01

Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

(AAV)-mediated expression of small interfering RNA

African Journals Online (AJOL)

Effective inhibition of specific gene by adenoassociated virus (AAV)-mediated expression of small interfering RNA. ... To perform functional tests on siRNA, which was expressed by the viral vector, recombinant AAVs, coding for siRNA against exogenous gene, EGFP, and endogenous gene, p53, were established and ...

Current good manufacturing practice production of an oncolytic recombinant vesicular stomatitis viral vector for cancer treatment.

Science.gov (United States)

Ausubel, L J; Meseck, M; Derecho, I; Lopez, P; Knoblauch, C; McMahon, R; Anderson, J; Dunphy, N; Quezada, V; Khan, R; Huang, P; Dang, W; Luo, M; Hsu, D; Woo, S L C; Couture, L

2011-04-01

Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 10(9) plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 10(10) PFU/ml (total yield, 1 × 10(13) PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC.

Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups.

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Stefania Piersanti

Full Text Available Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD canine adenovirus type 2 vectors (CAV-2 are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.

Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

Science.gov (United States)

Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

2014-08-01

We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell

Modified montmorillonite as vector for gene delivery.

Science.gov (United States)

Lin, Feng-Huei; Chen, Chia-Hao; Cheng, Winston T K; Kuo, Tzang-Fu

2006-06-01

Currently, gene delivery systems can be divided into two parts: viral or non-viral vectors. In general, viral vectors have a higher efficiency on gene delivery. However, they may sometimes provoke mutagenesis and carcinogenesis once re-activating in human body. Lots of non-viral vectors have been developed that tried to solve the problems happened on viral vectors. Unfortunately, most of non-viral vectors showed relatively lower transfection rate. The aim of this study is to develop a non-viral vector for gene delivery system. Montmorillonite (MMT) is one of clay minerals that consist of hydrated aluminum with Si-O tetrahedrons on the bottom of the layer and Al-O(OH)2 octahedrons on the top. The inter-layer space is about 12 A. The room is not enough to accommodate DNA for gene delivery. In the study, the cationic hexadecyltrimethylammonium (HDTMA) will be intercalated into the interlayer of MMT as a layer expander to expand the layer space for DNA accommodation. The optimal condition for the preparation of DNA-HDTMA-MMT is as follows: 1 mg of 1.5CEC HDTMA-MMT was prepared under pH value of 10.7 and with soaking time for 2 h. The DNA molecules can be protected from nuclease degradation, which can be proven by the electrophoresis analysis. DNA was successfully transfected into the nucleus of human dermal fibroblast and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. The HDTMA-MMT has a great potential as a vector for gene delivery in the future.

Viral Determinants and Vector Competence of Zika Virus Transmission

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Hong-Wai Tham

2018-05-01

Full Text Available Zika virus (ZIKV has emerged as a new global health threat. Since its first discovery in Zika forest in Uganda, this virus has been isolated from several mosquito species, including Aedes aegypti and Aedes albopictus. The geographical distribution of these mosquito species across tropical and subtropical regions has led to several outbreaks, including the recent pandemic in Brazil, followed by the Pacific islands and other areas of North and South America. This has gained attention of the scientific community to elucidate the epidemiology and transmission of ZIKV. Despite its strong attention on clinical aspects for healthcare professionals, the relationships between ZIKV and its principal vectors, A. aegypti and A. albopictus, have not gained substantial interest in the scientific research community. As such, this review aims to summarize the current knowledge on ZIKV tropism and some important mechanisms which may be employed by the virus for effective strategies on viral survival in mosquitoes. In addition, this review identifies the areas of research that should be placed attention to, for which to be exploited for novel mosquito control strategies.

Viral Determinants and Vector Competence of Zika Virus Transmission

Science.gov (United States)

Tham, Hong-Wai; Balasubramaniam, Vinod; Ooi, Man K.; Chew, Miaw-Fang

2018-01-01

Zika virus (ZIKV) has emerged as a new global health threat. Since its first discovery in Zika forest in Uganda, this virus has been isolated from several mosquito species, including Aedes aegypti and Aedes albopictus. The geographical distribution of these mosquito species across tropical and subtropical regions has led to several outbreaks, including the recent pandemic in Brazil, followed by the Pacific islands and other areas of North and South America. This has gained attention of the scientific community to elucidate the epidemiology and transmission of ZIKV. Despite its strong attention on clinical aspects for healthcare professionals, the relationships between ZIKV and its principal vectors, A. aegypti and A. albopictus, have not gained substantial interest in the scientific research community. As such, this review aims to summarize the current knowledge on ZIKV tropism and some important mechanisms which may be employed by the virus for effective strategies on viral survival in mosquitoes. In addition, this review identifies the areas of research that should be placed attention to, for which to be exploited for novel mosquito control strategies. PMID:29875751

Retrograde Signals: Integrators of Interorganellar Communication and Orchestrators of Plant Development.

Science.gov (United States)

de Souza, Amancio; Wang, Jin-Zheng; Dehesh, Katayoon

2017-04-28

Interorganellar cooperation maintained via exquisitely controlled retrograde-signaling pathways is an evolutionary necessity for maintenance of cellular homeostasis. This signaling feature has therefore attracted much research attention aimed at improving understanding of the nature of these communication signals, how the signals are sensed, and ultimately the mechanism by which they integrate targeted processes that collectively culminate in organellar cooperativity. The answers to these questions will provide insight into how retrograde-signal-mediated regulatory mechanisms are recruited and which biological processes are targeted, and will advance our understanding of how organisms balance metabolic investments in growth against adaptation to environmental stress. This review summarizes the present understanding of the nature and the functional complexity of retrograde signals as integrators of interorganellar communication and orchestrators of plant development, and offers a perspective on the future of this critical and dynamic area of research.

The double-stranded RNA-activated protein kinase mediates viral-induced encephalitis

International Nuclear Information System (INIS)

Scheuner, Donalyn; Gromeier, Matthias; Davies, Monique V.; Dorner, Andrew J.; Song Benbo; Patel, Rupali V.; Wimmer, Eckard J.; McLendon, Roger E.; Kaufman, Randal J.

2003-01-01

The double-stranded (ds) RNA-activated protein kinase (PKR) plays an important role in control of viral infections and cell growth. We have studied the role of PKR in viral infection in mice that are defective in the PKR signaling pathway. Transgenic mice were derived that constitutively express a trans-dominant-negative kinase-defective mutant PKR under control of the β-actin promoter. The trans-dominant-negative PKR mutant expressing transgenic mice do not have a detectable phenotype, similar to observations with PKR knock-out mice. The requirement for PKR in viral pathogenesis was studied by intracerebral infection of mice with a mouse-adapted poliovirus. Histopathological analysis revealed diffuse encephalomyelitis with severe inflammatory lesions throughout the central nervous system (CNS) in infected wild-type mice. In contrast, histopathological evaluation of virus-injected trans-dominant-negative PKR transgenic mice as well as PKR knock-out mice yielded no signs of tissue damage associated with inflammatory host responses. However, the virus did replicate in both models of PKR-deficient mice at a level equal to that observed in wild-type infected mice. Although the results indicate a clear difference in susceptibility to poliovirus-induced encephalitis, this difference manifests clinically as a slight delay in fatal neuropathy in trans-dominant-negative PKR transgenic and PKR knock-out animals. Our observations support the finding that viral-induced PKR activation may play a significant role in pathogenesis by mediating the host response to viral CNS infection. They support PKR to be an effective target to control tissue damage due to deleterious host responses to viral infection

Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity

NARCIS (Netherlands)

Roberts, Diane M.; Nanda, Anjali; Havenga, Menzo J. E.; Abbink, Peter; Lynch, Diana M.; Ewald, Bonnie A.; Liu, Jinyan; Thorner, Anna R.; Swanson, Patricia E.; Gorgone, Darci A.; Lifton, Michelle A.; Lemckert, Angelique A. C.; Holterman, Lennart; Chen, Bing; Dilraj, Athmanundh; Carville, Angela; Mansfield, Keith G.; Goudsmit, Jaap; Barouch, Dan H.

2006-01-01

A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of

Cooler temperatures destabilize RNA interference and increase susceptibility of disease vector mosquitoes to viral infection.

Directory of Open Access Journals (Sweden)

Zach N Adelman

Full Text Available The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus, exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.We utilized transgenic "sensor" strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor" strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2 or Argonaute-2 (AGO-2. We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses.This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.

Advances in Viral Vector-Based TRAIL Gene Therapy for Cancer

International Nuclear Information System (INIS)

Norian, Lyse A.; James, Britnie R.; Griffith, Thomas S.

2011-01-01

Numerous biologic approaches are being investigated as anti-cancer therapies in an attempt to induce tumor regression while circumventing the toxic side effects associated with standard chemo- or radiotherapies. Among these, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown particular promise in pre-clinical and early clinical trials, due to its preferential ability to induce apoptotic cell death in cancer cells and its minimal toxicity. One limitation of TRAIL use is the fact that many tumor types display an inherent resistance to TRAIL-induced apoptosis. To circumvent this problem, researchers have explored a number of strategies to optimize TRAIL delivery and to improve its efficacy via co-administration with other anti-cancer agents. In this review, we will focus on TRAIL-based gene therapy approaches for the treatment of malignancies. We will discuss the main viral vectors that are being used for TRAIL gene therapy and the strategies that are currently being attempted to improve the efficacy of TRAIL as an anti-cancer therapeutic

The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector

Directory of Open Access Journals (Sweden)

Galler R.

1997-01-01

Full Text Available The yellow fever (YF virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed

Transport According to GARP: Receiving Retrograde Cargo at the Trans-Golgi Network

Science.gov (United States)

Bonifacino, Juan S.; Hierro, Aitor

2010-01-01

Tethering factors are large protein complexes that capture transport vesicles and enable their fusion with acceptor organelles at different stages of the endomembrane system. Recent studies have shed new light on the structure and function of a heterotetrameric tethering factor named Golgi-associated retrograde protein (GARP), which promotes fusion of endosome-derived, retrograde transport carriers to the trans-Golgi network (TGN). X-ray crystallography of the Vps53 and Vps54 subunits of GARP has revealed that this complex is structurally related to other tethering factors such as the exocyst, COG and Dsl1, indicating that they all might work by a similar mechanism. Loss of GARP function compromises the growth, fertility and/or viability of the defective organisms, underscoring the essential nature of GARP-mediated retrograde transport. PMID:21183348

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

Science.gov (United States)

Lv, Lulu; Zhao, Huiqing

2014-01-01

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes. PMID:24392152

Recombinant viruses as vaccines against viral diseases

Directory of Open Access Journals (Sweden)

A.P.D. Souza

2005-04-01

Full Text Available Vaccine approaches to infectious diseases are widely applied and appreciated. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens and induce specific immunological responses against these antigens in vivo. Generally, gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application, and thus far none of them have proven to be ideal vaccine carriers. In this review we describe the potential, as well as some of the foreseeable obstacles associated with viral vaccine vectors and their use in preventive medicine.

Physical non-viral gene delivery methods for tissue engineering.

Science.gov (United States)

Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

2013-03-01

The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

Physical non-viral gene delivery methods for tissue engineering

Science.gov (United States)

Mellott, Adam J.; Forrest, M. Laird; Detamore, Michael S.

2016-01-01

The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications. PMID:23099792

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

Directory of Open Access Journals (Sweden)

Claudia Merkl

Full Text Available Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4 into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

International Nuclear Information System (INIS)

Zhao Weihong; Zhong Li; Wu Jianqing; Chen Linyuan; Qing Keyun; Weigel-Kelley, Kirsten A.; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H.; Srivastava, Arun

2006-01-01

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by ∼25-fold in WT MEFs, but only by ∼4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency ∼23-fold in WT MEFs, but only ∼4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, ∼59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only ∼28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene

Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

International Nuclear Information System (INIS)

Shitara, Shingo; Kakeda, Minoru; Nagata, Keiko; Hiratsuka, Masaharu; Sano, Akiko; Osawa, Kanako; Okazaki, Akiyo; Katoh, Motonobu; Kazuki, Yasuhiro; Oshimura, Mitsuo; Tomizuka, Kazuma

2008-01-01

Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-β-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120 days after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70 days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells

HIV-derived vectors for gene therapy targeting dendritic cells.

Science.gov (United States)

Rossetti, Maura; Cavarelli, Mariangela; Gregori, Silvia; Scarlatti, Gabriella

2013-01-01

Human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) have the potential to mediate stable therapeutic gene transfer. However, similarly to other viral vectors, their benefit is compromised by the induction of an immune response toward transgene-expressing cells that closely mimics antiviral immunity. LV share with the parental HIV the ability to activate dendritic cells (DC), while lack the peculiar ability of subverting DC functions, which is responsible for HIV immune escape. Understanding the interaction between LV and DC, with plasmacytoid and myeloid DC playing fundamental and distinct roles, has paved the way to novel approaches aimed at regulating transgene-specific immune responses. Thanks to the ability to target either DC subsets LV might be a powerful tool to induce immunity (i.e., gene therapy of cancer), cell death (i.e., in HIV/AIDS infection), or tolerance (i.e., gene therapy strategies for monogenic diseases). In this chapter, similarities and differences between the LV-mediated and HIV-mediated induction of immune responses, with specific focus on their interactions with DC, are discussed.

Cellular sources and targets of IFN-γ-mediated protection against viral demyelination and neurological deficits

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Murray, Paul D.; McGavern, Dorian B.; Pease, Larry R.; Rodriguez, Moses

2017-01-01

IFN-γ is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-γ, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler’s virus model of multiple sclerosis. During viral infections, IFN-γ is produced by natural killer (NK) cells, CD4+ and CD8+ T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-γ-mediated protection. To determine the lymphocyte subsets that produce IFN-γ to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-γ-production. We demonstrate that IFN-γ production by both CD4+ and CD8+ T cell subsets is critical for resistance to Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4+ T cells make a greater contribution to IFN-γ-mediated protection. To determine the cellular targets of IFN-γ-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-γ receptor. We demonstrate that IFN-γ receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination. PMID:11857334

Direct Neural Conversion from Human Fibroblasts Using Self-Regulating and Nonintegrating Viral Vectors

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Shong Lau

2014-12-01

Full Text Available Summary: Recent findings show that human fibroblasts can be directly programmed into functional neurons without passing via a proliferative stem cell intermediate. These findings open up the possibility of generating subtype-specific neurons of human origin for therapeutic use from fetal cell, from patients themselves, or from matched donors. In this study, we present an improved system for direct neural conversion of human fibroblasts. The neural reprogramming genes are regulated by the neuron-specific microRNA, miR-124, such that each cell turns off expression of the reprogramming genes once the cell has reached a stable neuronal fate. The regulated system can be combined with integrase-deficient vectors, providing a nonintegrative and self-regulated conversion system that rids problems associated with the integration of viral transgenes into the host genome. These modifications make the system suitable for clinical use and therefore represent a major step forward in the development of induced neurons for cell therapy. : Lau et al. now use miRNA targeting to build a self-regulating neural conversion system. Combined with nonintegrating vectors, this system can efficiently drive conversion of human fibroblasts into functional induced neurons (iNs suitable for clinical applications.

A multi-layered mechanistic modelling approach to understand how effector genes extend beyond phytoplasma to modulate plant hosts, insect vectors and the environment.

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Tomkins, Melissa; Kliot, Adi; Marée, Athanasius Fm; Hogenhout, Saskia A

2018-03-13

Members of the Candidatus genus Phytoplasma are small bacterial pathogens that hijack their plant hosts via the secretion of virulence proteins (effectors) leading to a fascinating array of plant phenotypes, such as witch's brooms (stem proliferations) and phyllody (retrograde development of flowers into vegetative tissues). Phytoplasma depend on insect vectors for transmission, and interestingly, these insect vectors were found to be (in)directly attracted to plants with these phenotypes. Therefore, phytoplasma effectors appear to reprogram plant development and defence to lure insect vectors, similarly to social engineering malware, which employs tricks to lure people to infected computers and webpages. A multi-layered mechanistic modelling approach will enable a better understanding of how phytoplasma effector-mediated modulations of plant host development and insect vector behaviour contribute to phytoplasma spread, and ultimately to predict the long reach of phytoplasma effector genes. Copyright © 2018. Published by Elsevier Ltd.

You can hide but you have to run: direct detection with vector mediators

Energy Technology Data Exchange (ETDEWEB)

D’Eramo, Francesco [Department of Physics, University of California Santa Cruz,1156 High St., Santa Cruz, CA 95064 (United States); Santa Cruz Institute for Particle Physics,1156 High St., Santa Cruz, CA 95064 (United States); Kavanagh, Bradley J. [Laboratoire de Physique Théorique et Hautes Energies, CNRS, UMR 7589,4 Place Jussieu, F-75252, Paris (France); Institut de Physique Théorique, Université Paris Saclay, CNRS, CEA,Orme des Merisiers batiment 774, F-91191 Gif-sur-Yvette Cedex (France); Panci, Paolo [Institut d’Astrophysique de Paris, UMR 7095 CNRS, Université Pierre et Marie Curie,98 bis Boulevard Arago, Paris 75014 (France)

2016-08-18

We study direct detection in simplified models of Dark Matter (DM) in which interactions with Standard Model (SM) fermions are mediated by a heavy vector boson. We consider fully general, gauge-invariant couplings between the SM, the mediator and both scalar and fermion DM. We account for the evolution of the couplings between the energy scale of the mediator mass and the nuclear energy scale. This running arises from virtual effects of SM particles and its inclusion is not optional. We compare bounds on the mediator mass from direct detection experiments with and without accounting for the running. In some cases the inclusion of these effects changes the bounds by several orders of magnitude, as a consequence of operator mixing which generates new interactions at low energy. We also highlight the importance of these effects when translating LHC limits on the mediator mass into bounds on the direct detection cross section. For an axial-vector mediator, the running can alter the derived bounds on the spin-dependent DM-nucleon cross section by a factor of two or more. Finally, we provide tools to facilitate the inclusion of these effects in future studies: general approximate expressions for the low energy couplings and a public code runDM to evolve the couplings between arbitrary energy scales.

Chikungunya Virus–Vector Interactions

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Lark L. Coffey

2014-11-01

Full Text Available 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.

Adenosine Triphosphate (ATP Is a Candidate Signaling Molecule in the Mitochondria-to-Nucleus Retrograde Response Pathway

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Zhengchang Liu

2013-03-01

Full Text Available Intracellular communication from the mitochondria to the nucleus is achieved via the retrograde response. In budding yeast, the retrograde response, also known as the RTG pathway, is regulated positively by Rtg1, Rtg2, Rtg3 and Grr1 and negatively by Mks1, Lst8 and two 14-3-3 proteins, Bmh1/2. Activation of retrograde signaling leads to activation of Rtg1/3, two basic helix-loop-helix leucine zipper transcription factors. Rtg1/3 activation requires Rtg2, a cytoplasmic protein with an N-terminal adenosine triphosphate (ATP binding domain belonging to the actin/Hsp70/sugar kinase superfamily. The critical regulatory step of the retrograde response is the interaction between Rtg2 and Mks1. Rtg2 binds to and inactivates Mks1, allowing for activation of Rtg1/3 and the RTG pathway. When the pathway is inactive, Mks1 has dissociated from Rtg2 and bound to Bmh1/2, preventing activation of Rtg1/3. What signals association or disassociation of Mks1 and Rtg2 is unknown. Here, we show that ATP at physiological concentrations dissociates Mks1 from Rtg2 in a highly cooperative fashion. We report that ATP-mediated dissociation of Mks1 from Rtg2 is conserved in two other fungal species, K. lactis and K. waltii. Activation of Rtg1/3 upregulates expression of genes encoding enzymes catalyzing the first three reactions of the Krebs cycle, which is coupled to ATP synthesis through oxidative phosphorylation. Therefore, we propose that the retrograde response is an ATP homeostasis pathway coupling ATP production with ATP-mediated repression of the retrograde response by releasing Mks1 from Rtg2.

Generation of a human induced pluripotent stem cell line (MUSIi001-A from caesarean section scar fibroblasts using Sendai viral vectors

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Methichit Wattanapanitch

2018-03-01

Full Text Available We generated a human induced pluripotent stem cell (iPSC line from caesarean section scar fibroblasts of a 33-year-old healthy woman using transgene-free Sendai viral vectors under feeder-free condition. The established iPSC line, designated as MUSIi001-A, exhibited a normal karyotype, expressed pluripotent markers, differentiated into cells of three embryonic germ layers. Further analyses showed that the Sendai viral genome was absent at passage 25. The MUSIi001-A line can serve as a control for studying developmental biology and phenotypic comparison with disease-specific iPSCs.

Gene Therapy with Helper-Dependent Adenoviral Vectors: Current Advances and Future Perspectives

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Philip Ng

2010-09-01

Full Text Available Recombinant Adenoviral vectors represent one of the best gene transfer platforms due to their ability to efficiently transduce a wide range of quiescent and proliferating cell types from various tissues and species. The activation of an adaptive immune response against the transduced cells is one of the major drawbacks of first generation Adenovirus vectors and has been overcome by the latest generation of recombinant Adenovirus, the Helper-Dependent Adenoviral (HDAd vectors. HDAds have innovative features including the complete absence of viral coding sequences and the ability to mediate high level transgene expression with negligible chronic toxicity. This review summarizes the many aspects of HDAd biology and structure with a major focus on in vivo gene therapy application and with an emphasis on the unsolved issues that these vectors still presents toward clinical application.

Popliteal versus tibial retrograde access for subintimal arterial flossing with antegrade-retrograde intervention (SAFARI) technique.

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Hua, W R; Yi, M Q; Min, T L; Feng, S N; Xuan, L Z; Xing, J

2013-08-01

This study aimed to ascertain differences in benefit and effectiveness of popliteal versus tibial retrograde access in subintimal arterial flossing with the antegrade-retrograde intervention (SAFARI) technique. This was a retrospective study of SAFARI-assisted stenting for long chronic total occlusion (CTO) of TASC C and D superficial femoral lesions. 38 cases had superficial femoral artery lesions (23 TASC C and 15 TASC D). All 38 cases underwent SAFARI-assisted stenting. The ipsilateral popliteal artery was retrogradely punctured in 17 patients. A distal posterior tibial (PT) or dorsalis pedis (DP) artery was retrogradely punctured in 21 patients, and 16 of them were punctured after open surgical exposure. SAFARI technical success was achieved in all cases. There was no significant difference in 1-year primary patency (75% vs. 78.9%, p = .86), secondary patency (81.2% vs. 84.2%, p = .91) and access complications (p = 1.00) between popliteal and tibial retrograde access. There was statistical difference in operation time between popliteal (140.1 ± 28.4 min) and tibial retrograde access with PT/DP punctures after surgical vessel exposure (120.4 ± 23.0 min, p = .04). The SAFARI technique is a safe and feasible option for patients with infrainguinal CTO (TASC II C and D). The PT or DP as the retrograde access after surgical vessel exposure is a good choice when using the SAFARI technique. Copyright © 2013 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Retrograde pulmonary arteriography

International Nuclear Information System (INIS)

Calcaterra, G.; Lam, J.; Losekoot, T.G.

1984-01-01

The authors performed retrograde pulmonary arteriography by means of a pulmonary venous wedge injection in 10 patients with no demonstrable intrapericardial pulmonary arteries by 'conventional' angiographic techniques. In all cases but one, the procedure demonstrated the feasibility of a further operation. No complications were observed. Retrograde pulmonary arteriography is an important additional method for determining the existence of surgically accessible pulmonary arteries when other techniques have failed. (Auth.)

Retrograde curves of solidus and solubility

International Nuclear Information System (INIS)

Vasil'ev, M.V.

1979-01-01

The investigation was concerned with the constitutional diagrams of the eutectic type with ''retrograde solidus'' and ''retrograde solubility curve'' which must be considered as diagrams with degenerate monotectic transformation. The solidus and the solubility curves form a retrograde curve with a common retrograde point representing the solubility maximum. The two branches of the Aetrograde curve can be described with the aid of two similar equations. Presented are corresponding equations for the Cd-Zn system and shown is the possibility of predicting the run of the solubility curve

Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

OpenAIRE

Peluffo, Hugo; Acarin, Laia; Arís, Anna; González, Pau; Villaverde, Antoni; Castellano, Bernardo; González, Berta

2006-01-01

Abstract Background Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn S...

Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.

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Zihua Wang

Full Text Available Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV, a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg RNA which is also required as bicistronic mRNA for the capsid (core protein and the reverse transcriptase (Pol; their open reading frames (ORFs overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES. We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR and humanized Renilla green fluorescent protein (hrGFP produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to

Discovery of Cationic Polymers for Non-viral Gene Delivery using Combinatorial Approaches

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Barua, Sutapa; Ramos, James; Potta, Thrimoorthy; Taylor, David; Huang, Huang-Chiao; Montanez, Gabriela; Rege, Kaushal

2015-01-01

Gene therapy is an attractive treatment option for diseases of genetic origin, including several cancers and cardiovascular diseases. While viruses are effective vectors for delivering exogenous genes to cells, concerns related to insertional mutagenesis, immunogenicity, lack of tropism, decay and high production costs necessitate the discovery of non-viral methods. Significant efforts have been focused on cationic polymers as non-viral alternatives for gene delivery. Recent studies have employed combinatorial syntheses and parallel screening methods for enhancing the efficacy of gene delivery, biocompatibility of the delivery vehicle, and overcoming cellular level barriers as they relate to polymer-mediated transgene uptake, transport, transcription, and expression. This review summarizes and discusses recent advances in combinatorial syntheses and parallel screening of cationic polymer libraries for the discovery of efficient and safe gene delivery systems. PMID:21843141

Non-viral Nucleic Acid Delivery Strategies to the Central Nervous System

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James-Kevin Tan

2016-11-01

Full Text Available With an increased prevalence and understanding of central nervous system injuries and neurological disorders, nucleic acid therapies are gaining promise as a way to regenerate lost neurons or halt disease progression. While more viral vectors have been used clinically as tools for gene delivery, non-viral vectors are gaining interest due to lower safety concerns and the ability to deliver all types of nucleic acids. Nevertheless, there are still a number of barriers to nucleic acid delivery. In this focused review, we explore the in vivo challenges hindering non-viral nucleic acid delivery to the central nervous system and the strategies and vehicles used to overcome them. Advantages and disadvantages of different routes of administration including: systemic injection, cerebrospinal fluid injection, intraparenchymal injection, and peripheral administration are discussed. Non-viral vehicles and treatment strategies that have overcome delivery barriers and demonstrated in vivo gene transfer to the central nervous system are presented. These approaches can be used as guidelines in developing synthetic gene delivery vectors for central nervous system applications and will ultimately bring non-viral vectors closer to clinical application.

Constraining flavor changing interactions from LHC Run-2 dilepton bounds with vector mediators

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Farinaldo S. Queiroz

2016-12-01

Full Text Available Within the context of vector mediators, is a new signal observed in flavor changing interactions, particularly in the neutral mesons systems K0−K¯0, D0−D¯0 and B0−B0¯, consistent with dilepton resonance searches at the LHC? In the attempt to address this very simple question, we discuss the complementarity between flavor changing neutral current (FCNC and dilepton resonance searches at the LHC run 2 at 13 TeV with 3.2 fb−1 of integrated luminosity, in the context of vector mediators at tree level. Vector mediators, are often studied in the flavor changing framework, specially in the light of the recent LHCb anomaly observed at the rare B decay. However, the existence of stringent dilepton bound severely constrains flavor changing interactions, due to restrictive limits on the Z′ mass. We discuss this interplay explicitly in the well motivated framework of a 3-3-1 scheme, where fermions and scalars are arranged in the fundamental representation of the weak SU(3 gauge group. Due to the paucity of relevant parameters, we conclude that dilepton data leave little room for a possible new physics signal stemming from these systems, unless a very peculiar texture parametrization is used in the diagonalization of the CKM matrix. In other words, if a signal is observed in such flavor changing interactions, it unlikely comes from a 3-3-1 model.

High-Throughput Agrobacterium-mediated Transformation of Medicago Truncatula in Comparison to Two Expression Vectors

International Nuclear Information System (INIS)

Sultana, T.; Deeba, F.; Naqvi, S. M. S.

2016-01-01

Legumes have been turbulent to efficient Agrobacterium-mediated transformation for a long time. The selection of Medicago truncatula as a model legume plant for molecular analysis resulted in the development of efficient Agrobacterium-mediated transformation protocols. In current study, M. truncatula transformed plants expressing OsRGLP1 were obtained through GATEWAY technology using pGOsRGLP1 (pH7WG2.0=OsRGLP1). The transformation efficiency of this vector was compared with expression vector from pCAMBIA series over-expressing same gene (pCOsRGLP1). A lower number of explants generated hygromycin resistant plantlet for instance, 18.3 with pGOsRGLP1 vector as compared to 35.5 percent with pCOsRGLP1 vector. Transformation efficiency of PCR positive plants generated was 9.4 percent for pGOsRGLP1 while 21.6 percent for pCOsRGLP1. Furthermore 24.4 percent of explants generated antibiotic resistant plantlet on 20 mgl/sup -1/ of hygromycin which was higher than on 15 mgl/sup -1/ of hygromycin such as 12.2 percent. T/sub 1/ progeny analysis indicated that the transgene was inherited in Mendelian manner. The functionally active status of transgene was monitored by high level of Superoxide dismutase (SOD) activity in transformed progeny. (author)

Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

Science.gov (United States)

Molinier-Frenkel, Valérie; Gahery-Segard, Hanne; Mehtali, Majid; Le Boulaire, Christophe; Ribault, Sébastien; Boulanger, Pierre; Tursz, Thomas; Guillet, Jean-Gérard; Farace, Françoise

2000-01-01

We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+ CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses. PMID:10906225

Characterization of rice black-streaked dwarf virus- and rice stripe virus-derived siRNAs in singly and doubly infected insect vector Laodelphax striatellus.

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Junmin Li

Full Text Available Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi which generates viral-derived small interfering RNAs (siRNAs. However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus was infected by Rice black-streaked dwarf virus (RBSDV (Reoviridae; Fijivirus, more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV, a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5'- and 3'-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.

A retrograde object near Jupiter's orbit

Science.gov (United States)

Connors, M.; Wiegert, P.

2018-02-01

Asteroid 2007 VW266 is among the rare objects with a heliocentric retrograde orbit, and its semimajor axis is within a Hill sphere radius of that of Jupiter. This raised the interesting possibility that it could be in co-orbital retrograde resonance with Jupiter, a second "counter-orbital" object in addition to recently discovered 2015 BZ509. We find instead that the object is in 13/14 retrograde mean motion resonance (also referred to as 13/-14). The object is shown to have entered its present orbit about 1700 years ago, and it will leave it in about 8000 years, both through close approach to Jupiter. Entry and exit states both avoid 1:1 retrograde resonance, but the retrograde nature is preserved. The temporary stable state is due to an elliptic orbit with high inclination keeping nodal passages far from the associated planet. We discuss the motion of this unusual object based on modeling and theory, and its observational prospects.

A molecular toolbox for rapid generation of viral vectors to up- or down-regulate in vivo neuronal gene expression

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Melanie D. White

2011-07-01

Full Text Available We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1 and Kir3.2 and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miR. We show that AAV assembled to express HCN1 miR produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miR with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

Dexamethasone rapidly increases GABA release in the dorsal motor nucleus of the vagus via retrograde messenger-mediated enhancement of TRPV1 activity.

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Andrei V Derbenev

Full Text Available Glucocorticoids influence vagal parasympathetic output to the viscera via mechanisms that include modulation of neural circuitry in the dorsal vagal complex, a principal autonomic regulatory center. Glucocorticoids can modulate synaptic neurotransmitter release elsewhere in the brain by inducing release of retrograde signalling molecules. We tested the hypothesis that the glucocorticoid agonist dexamethasone (DEX modulates GABA release in the rat dorsal motor nucleus of the vagus (DMV. Whole-cell patch-clamp recordings revealed that DEX (1-10 µM rapidly (i.e. within three minutes increased the frequency of tetrodotoxin-resistant, miniature IPSCs (mIPSCs in 67% of DMV neurons recorded in acutely prepared slices. Glutamate-mediated mEPSCs were also enhanced by DEX (10 µM, and blockade of ionotropic glutamate receptors reduced the DEX effect on mIPSC frequency. Antagonists of type I or II corticosteroid receptors blocked the effect of DEX on mIPSCs. The effect was mimicked by application of the membrane-impermeant BSA-conjugated DEX, and intracellular blockade of G protein function with GDP βS in the recorded cell prevented the effect of DEX. The enhancement of GABA release was blocked by the TRPV1 antagonists, 5'-iodoresiniferatoxin or capsazepine, but was not altered by the cannabinoid type 1 receptor antagonist AM251. The DEX effect was prevented by blocking fatty acid amide hydrolysis or by inhibiting anandamide transport, implicating involvement of the endocannabinoid system in the response. These findings indicate that DEX induces an enhancement of GABA release in the DMV, which is mediated by activation of TRPV1 receptors on afferent terminals. The effect is likely induced by anandamide or other 'endovanilloid', suggesting activation of a local retrograde signal originating from DMV neurons to enhance synaptic inhibition locally in response to glucocorticoids.

Effective photodynamic therapy in drug-resistant prostate cancer cells utilizing a non-viral antitumor vector (a secondary publication).

Science.gov (United States)

Yamauchi, Masaya; Honda, Norihiro; Hazama, Hisanao; Tachikawa, Shoji; Nakamura, Hiroyuki; Kaneda, Yasufumi; Awazu, Kunio

2016-03-31

There is an urgent need to develop an efficient strategy for the treatment of drug-resistant prostate cancer. Photodynamic therapy (PDT), in which low incident levels of laser energy are used to activate a photosensitizer taken up by tumor cells, is expected as a novel therapy for the treatment of prostate cancer because of the minimal invasive nature of PDT. The present study was designed to assess the efficacy of a novel vector approach combined with a conventional porphyrin-based photosensitizer. Our group focused on a non-viral vector (hemagglutinating virus of Japan envelope; HVJ-E) combined with protoporphyrin IX (PpIX) lipid, termed the porphyrus envelope (PE). It has been previously confirmed that HVJ-E has drug-delivering properties and can induce cancer-specific cell death. The PE (HVJ-E contained in PpIX lipid) was developed as a novel photosensitizer. In this study, the antitumor and PDT efficacy of the PE against hormone-antagonistic human prostate cancer cells (PC-3) were evaluated. Our results demonstrated that, under specific circumstances, PDT using the PE was very effective against PC-3 cells. A novel therapy for drug-resistant prostate cancer based on this vector approach is eagerly anticipated.

Recombinant vaccines against T. gondii: comparison between homologous and heterologous vaccination protocols using two viral vectors expressing SAG1.

Science.gov (United States)

Mendes, Érica Araújo; Fonseca, Flavio G; Casério, Bárbara M; Colina, Janaína P; Gazzinelli, Ricardo Tostes; Caetano, Braulia C

2013-01-01

The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1) of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector) is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination). Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1), to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

Recombinant vaccines against T. gondii: comparison between homologous and heterologous vaccination protocols using two viral vectors expressing SAG1.

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Érica Araújo Mendes

Full Text Available The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1 of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination. Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1, to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

Receptor-mediated gene delivery using chemically modified chitosan

International Nuclear Information System (INIS)

Kim, T H; Jiang, H L; Nah, J W; Cho, M H; Akaike, T; Cho, C S

2007-01-01

Chitosan has been investigated as a non-viral vector because it has several advantages such as biocompatibility, biodegradability and low toxicity with high cationic potential. However, the low specificity and low transfection efficiency of chitosan need to be solved prior to clinical application. In this paper, we focused on the galactose or mannose ligand modification of chitosan for enhancement of cell specificity and transfection efficiency via receptor-mediated endocytosis in vitro and in vivo

Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

Science.gov (United States)

Turunen, Tytteli Anni Kaarina; Laakkonen, Johanna Päivikki; Alasaarela, Laura; Airenne, Kari Juhani; Ylä-Herttuala, Seppo

2014-01-01

A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

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

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Giridhar Murlidharan

2016-01-01

Full Text Available 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.

Progress on adenovirus-vectored universal influenza vaccines.

Science.gov (United States)

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.

NYVAC vector modified by C7L viral gene insertion improves T cell immune responses and effectiveness against leishmaniasis.

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Sánchez-Sampedro, L; Mejías-Pérez, E; S Sorzano, Carlos Óscar; Nájera, J L; Esteban, M

2016-07-15

The NYVAC poxvirus vector is used as vaccine candidate for HIV and other diseases, although there is only limited experimental information on its immunogenicity and effectiveness for use against human pathogens. Here we defined the selective advantage of NYVAC vectors in a mouse model by comparing the immune responses and protection induced by vectors that express the LACK (Leishmania-activated C-kinase antigen), alone or with insertion of the viral host range gene C7L that allows the virus to replicate in human cells. Using DNA prime/virus boost protocols, we show that replication-competent NYVAC-LACK that expresses C7L (NYVAC-LACK-C7L) induced higher-magnitude polyfunctional CD8(+) and CD4(+) primary adaptive and effector memory T cell responses (IFNγ, TNFα, IL-2, CD107a) to LACK antigen than non-replicating NYVAC-LACK. Compared to NYVAC-LACK, the NYVAC-LACK-C7L-induced CD8(+) T cell population also showed higher proliferation when stimulated with LACK antigen. After a challenge by subcutaneous Leishmania major metacyclic promastigotes, NYVAC-LACK-C7L-vaccinated mouse groups showed greater protection than the NYVAC-LACK-vaccinated group. Our results indicate that the type and potency of immune responses induced by LACK-expressing NYVAC vectors is improved by insertion of the C7L gene, and that a replication-competent vector as a vaccine renders greater protection against a human pathogen than a non-replicating vector. Copyright © 2016 Elsevier B.V. All rights reserved.

Zn(II)-dipicolylamine-based metallo-lipids as novel non-viral gene vectors.

Science.gov (United States)

Su, Rong-Chuan; Liu, Qiang; Yi, Wen-Jing; Zhao, Zhi-Gang

2017-08-01

In this study, a series of Zn(II)-dipicolylamine (Zn-DPA) based cationic lipids bearing different hydrophobic tails (long chains, α-tocopherol, cholesterol or diosgenin) were synthesized. Structure-activity relationship (SAR) of these lipids was studied in detail by investigating the effects of several structural aspects including the type of hydrophobic tails, the chain length and saturation degree. In addition, several assays were used to study their interactions with plasmid DNA, and results reveal that these lipids could condense DNA into nanosized particles with appropriate size and zeta-potentials. MTT-based cell viability assays showed that lipoplexes 5 had low cytotoxicity. The in vitro gene transfection studies showed the hydrophobic tails clearly affected the TE, and hexadecanol-containing lipid 5b gives the best TE, which was 2.2 times higher than bPEI 25k in the presence of 10% serum. The results not only demonstrate that these lipids might be promising non-viral gene vectors, but also afford us clues for further optimization of lipidic gene delivery materials.

Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis.

Science.gov (United States)

Mason, J B; Gurda, B L; Van Wettere, A; Engiles, J B; Wilson, J M; Richardson, D W

2017-01-01

Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness. The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot. Randomised in vivo experiment. We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse. Vector serotypes rAAV2/1, 2/8 and 2/9 all successfully transduced equine foot tissues and displayed similar levels and patterns of transduction. The regional distribution of transduction within the foot decreased with decreasing vector dose. The highest transduction values were seen in the sole and coronary regions and the lowest transduction values were detected in the dorsal hoof-wall region. The use of a surfactant-enriched vector diluent increased regional distribution of the vector and improved the transduction in the hoof-wall region. The hoof-wall region of the foot, which exhibited the lowest levels of transduction using saline as the vector diluent, displayed a dramatic increase in transduction when surfactant was included in the vector diluent (9- to 81-fold increase). In transduced tissues, no significant difference was observed between promoters (chicken β-actin vs. cytomegalovirus) for gene expression. All horses tested for vector-neutralising antibodies were positive for serotype-specific neutralising antibodies to rAAV2/5. The current experiments demonstrate that transgenes can be successfully delivered to the equine distal extremity using rAAV vectors and that serotypes 2/8, 2/9 and 2/1 can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant-containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the

RUNX2 Mediates Plasmacytoid Dendritic Cell Egress from the Bone Marrow and Controls Viral Immunity

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Michaël Chopin

2016-04-01

Full Text Available Plasmacytoid dendritic cells (pDCs represent a unique immune cell type that responds to viral nucleic acids through the rapid production of type I interferons. Within the hematopoietic system, the transcription factor RUNX2 is exclusively expressed in pDCs and is required for their peripheral homeostasis. Here, we show that RUNX2 plays an essential role in promoting pDC localization and function. RUNX2 is required for the appropriate expression of the integrin-mediated adhesion machinery, as well as for the down-modulation of the chemokine receptor CXCR4, which allows pDC egress into the circulation. RUNX2 also facilitates the robust response to viral infection through the control of IRF7, the major regulator of type I interferon production. Mice lacking one copy of Runx2 have reduced numbers of peripheral pDCs and IFN-α expression, which might contribute to the reported difficulties of individuals with cleidocranial dysplasia, who are haploinsufficient for RUNX2, to clear viral infections.

Prospects for Foamy Viral Vector Anti-HIV Gene Therapy

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Arun K. Nalla

2016-03-01

Full Text Available Stem cell gene therapy approaches for Human Immunodeficiency Virus (HIV infection have been explored in clinical trials and several anti-HIV genes delivered by retroviral vectors were shown to block HIV replication. However, gammaretroviral and lentiviral based retroviral vectors have limitations for delivery of anti-HIV genes into hematopoietic stem cells (HSC. Foamy virus vectors have several advantages including efficient delivery of transgenes into HSC in large animal models, and a potentially safer integration profile. This review focuses on novel anti-HIV transgenes and the potential of foamy virus vectors for HSC gene therapy of HIV.

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

Science.gov (United States)

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

2008-12-01

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

Hatchery Vaccination Against Poultry Viral Diseases: Potential Mechanisms and Limitations.

Science.gov (United States)

Abdul-Cader, Mohamed Sarjoon; Palomino-Tapia, Victor; Amarasinghe, Aruna; Ahmed-Hassan, Hanaa; De Silva Senapathi, Upasama; Abdul-Careem, Mohamed Faizal

Commercial broiler and layer chickens are heavily vaccinated against economically important viral diseases with a view of preventing morbidity, mortality, and production impacts encountered during short production cycles. Hatchery vaccination is performed through in ovo embryo vaccination prehatch or spray and subcutaneous vaccinations performed at the day of hatch before the day-old chickens are being placed in barns with potentially contaminated environments. Commercially, multiple vaccines (e.g., live, live attenuated, and viral vectored vaccines) are available to administer through these routes within a short period (embryo day 18 prehatch to day 1 posthatch). Although the ability to mount immune response, especially the adaptive immune response, is not optimal around the hatch, it is possible that the efficacy of these vaccines depends partly on innate host responses elicited in response to replicating vaccine viruses. This review focuses on the current knowledge of hatchery vaccination in poultry and potential mechanisms of hatchery vaccine-mediated protective responses and limitations.

PRODUCTION OF NEAR-EARTH ASTEROIDS ON RETROGRADE ORBITS

International Nuclear Information System (INIS)

Greenstreet, S.; Gladman, B.; Ngo, H.; Granvik, M.; Larson, S.

2012-01-01

While computing an improved near-Earth object (NEO) steady-state orbital distribution model, we discovered in the numerical integrations the unexpected production of retrograde orbits for asteroids that had originally exited from the accepted main-belt source regions. Our model indicates that ∼0.1% (a factor of two uncertainty) of the steady-state NEO population (perihelion q < 1.3 AU) is on retrograde orbits. These rare outcomes typically happen when asteroid orbits flip to a retrograde configuration while in the 3:1 mean-motion resonance with Jupiter and then live for ∼0.001 to 100 Myr. The model predicts, given the estimated near-Earth asteroid (NEA) population, that a few retrograde 0.1-1 km NEAs should exist. Currently, there are two known MPC NEOs with asteroidal designations on retrograde orbits which we therefore claim could be escaped asteroids instead of devolatilized comets. This retrograde NEA population may also answer a long-standing question in the meteoritical literature regarding the origin of high-strength, high-velocity meteoroids on retrograde orbits.

Immune responses to rAAV6: The influence of canine parvovirus vaccination and neonatal administration of viral vector

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Andrea L H Arnett

2011-11-01

Full Text Available Recombinant adeno-associated viral (rAAV vectors promote long-term gene transfer in many animal species. Significant effort has focused on the evaluation of rAAV delivery and the immune response in both murine and canine models of neuromuscular disease. However, canines provided for research purposes are routinely vaccinated against canine parvovirus (CPV. rAAV and CPV possess significant homology and are both parvoviruses. Thus, any immune response generated to CPV vaccination has the potential to cross-react with rAAV vectors. In this study, we investigated the immune response to rAAV6 delivery in a cohort of CPV-vaccinated canines and evaluated multiple vaccination regimens in a mouse model of CPV-vaccination. We show that CPV-vaccination stimulates production of neutralizing antibodies with minimal cross-reactivity to rAAV6. In addition, no significant differences were observed in the magnitude of the rAAV6-directed immune response between CPV-vaccinated animals and controls. Moreover, CPV-vaccination did not inhibit rAAV6-mediated transduction. We also evaluated the immune response to early rAAV6-vaccination in neonatal mice. The influence of maternal hormones and cytokines leads to a relatively permissive state in the neonate. We hypothesized that immaturity of the immune system would permit induction of tolerance to rAAV6 when delivered during the neonatal period. Mice were vaccinated with rAAV6 at 1 or 5 days of age, and subsequently challenged with rAAV6 exposure during adulthood via two sequential IM injections, one month apart. All vaccinated animals generated a significant neutralizing antibody response to rAAV6-vaccination that was enhanced following IM injection in adulthood. Taken together, these data demonstrate that the immune response raised against rAAV6 is distinct from that which is elicited by the standard parvoviral vaccines and is sufficient to prevent stable tolerization in neonatal mice.

Large-scale adenovirus and poxvirus-vectored vaccine manufacturing to enable clinical trials.

Science.gov (United States)

Kallel, Héla; Kamen, Amine A

2015-05-01

Efforts to make vaccines against infectious diseases and immunotherapies for cancer have evolved to utilize a variety of heterologous expression systems such as viral vectors. These vectors are often attenuated or engineered to safely deliver genes encoding antigens of different pathogens. Adenovirus and poxvirus vectors are among the viral vectors that are most frequently used to develop prophylactic vaccines against infectious diseases as well as therapeutic cancer vaccines. This mini-review describes the trends and processes in large-scale production of adenovirus and poxvirus vectors to meet the needs of clinical applications. We briefly describe the general principles for the production and purification of adenovirus and poxvirus viral vectors. Currently, adenovirus and poxvirus vector manufacturing methods rely on well-established cell culture technologies. Several improvements have been evaluated to increase the yield and to reduce the overall manufacturing cost, such as cultivation at high cell densities and continuous downstream processing. Additionally, advancements in vector characterization will greatly facilitate the development of novel vectored vaccine candidates. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

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Akkina Ramesh

2005-01-01

Full Text Available Abstract Background RNA interference (RNAi mediated by small interfering RNAs (siRNAs has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4 and monocyte tropic (R5 viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. Results To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. Conclusions Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

Adenoviral vector-mediated gene transfer and neurotransplantation : possibilities and limitations in grafting of the fetal rat suprachiasmatic nucleus

NARCIS (Netherlands)

van Esseveldt, K E; Liu, R.; Hermens, W.T.J.M.C.; Verhaagen, J; Boer, G J

Several studies have reported on the use of primary neural cells transduced by adenoviral vectors as donor cells in neurotransplantation. In the present investigation, we examined whether adenoviral vector-mediated gene transfer could be used to introduce and express a foreign gene in solid neural

MicroRNA-Mediated Gene Silencing in Plant Defense and Viral Counter-Defense

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Sheng-Rui Liu

2017-09-01

Full Text Available MicroRNAs (miRNAs are non-coding RNAs of approximately 20–24 nucleotides in length that serve as central regulators of eukaryotic gene expression by targeting mRNAs for cleavage or translational repression. In plants, miRNAs are associated with numerous regulatory pathways in growth and development processes, and defensive responses in plant–pathogen interactions. Recently, significant progress has been made in understanding miRNA-mediated gene silencing and how viruses counter this defense mechanism. Here, we summarize the current knowledge and recent advances in understanding the roles of miRNAs involved in the plant defense against viruses and viral counter-defense. We also document the application of miRNAs in plant antiviral defense. This review discusses the current understanding of the mechanisms of miRNA-mediated gene silencing and provides insights on the never-ending arms race between plants and viruses.

A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

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Baodong Wu

2009-03-01

Full Text Available Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

Virus Database and Online Inquiry System Based on Natural Vectors.

Science.gov (United States)

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

2017-01-01

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

Studies of retrograde memory: A long-term view

OpenAIRE

Warrington, Elizabeth K.

1996-01-01

Studies of retrograde amnesia are reviewed. First, the issues of temporal gradients of retrograde amnesia are discussed. Second, the question of the anatomical substrates of this syndrome are considered. Finally, some evidence for fractionation of different classes of memoranda within the retrograde time period are presented.

Solid lipid nanoparticles mediate non-viral delivery of plasmid DNA to dendritic cells

Science.gov (United States)

Penumarthi, Alekhya; Parashar, Deepti; Abraham, Amanda N.; Dekiwadia, Chaitali; Macreadie, Ian; Shukla, Ravi; Smooker, Peter M.

2017-06-01

There is an increasing demand for novel DNA vaccine delivery systems, mainly for the non-viral type as they are considered relatively safe. Therefore, solid lipid nanoparticles (SLNs) were investigated for their suitability as a non-viral DNA vaccine delivery system. SLNs were synthesised by a modified solvent-emulsification method in order to study their potential to conjugate with plasmid DNA and deliver them in vitro to dendritic cells using eGFP as the reporter plasmid. The DNA-SLN complexes were characterised by electron microscopy, gel retardation assays and dynamic light scattering. The cytotoxicity assay data supported their biocompatibility and was used to estimate safe threshold concentration resulting in high transfection rate. The transfection efficiency of these complexes in a dendritic cell line was shown to increase significantly compared to plasmid alone, and was comparable to that mediated by lipofectamine. Transmission electron microscopy studies delineated the pathway of cellular uptake. Endosomal escape was observed supporting the mechanism of transfection.

Restoration of central nervous system alpha-N-acetylglucosaminidase activity and therapeutic benefits in mucopolysaccharidosis IIIB mice by a single intracisternal recombinant adeno-associated viral type 2 vector delivery.

Science.gov (United States)

Fu, Haiyan; DiRosario, Julianne; Kang, Lu; Muenzer, Joseph; McCarty, Douglas M

2010-07-01

Finding efficient central nervous system (CNS) delivery approaches has been the major challenge facing therapeutic development for treating diseases with global neurological manifestation, such as mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease, caused by autosomal recessive defect of alpha-N-acetylglucosaminidase (NaGlu). Previously, we developed an approach, intracisternal (i.c.) injection, to deliver recombinant adeno-associated viral (rAAV) vector to the CNS of mice, leading to a widespread periventricular distribution of transduction. In the present study, we delivered rAAV2 vector expressing human NaGlu into the CNS of MPS IIIB mice by an i.c. injection approach, to test its therapeutic efficacy and feasibility for treating the neurological manifestation of the disease. We demonstrated significant functional neurological benefits of a single i.c. vector infusion in adult MPS IIIB mice. The treatment slowed the disease progression by mediating widespread recombinant NaGlu expression in the CNS, resulting in the reduction of brain lysosomal storage pathology, significantly improved cognitive function and prolonged survival. However, persisting motor function deficits suggested that pathology in areas outside the CNS contributes to the MPS IIIB behavioral phenotype. The therapeutic benefit of i.c. rAAV2 delivery was dose-dependent and could be attribute solely to the CNS transduction because the procedure did not lead to detectable transduction in somatic tissues. A single IC rAAV2 gene delivery is functionally beneficial for treating the CNS disease of MPS IIIB in mice. It is immediately clinically translatable, with the potential of improving the quality of life for patients with MPS IIIB.

CRISPR/Cas9-mediated viral interference in plants

KAUST Repository

Ali, Zahir; Abulfaraj, Aala A.; Idris, Ali; Ali, Shawkat; Tashkandi, Manal; Mahfouz, Magdy M.

2015-01-01

These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution

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Yang Lin

2013-02-01

Full Text Available Abstract Adeno-associated virus (AAV is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolution of viral vectors. We further attempted to evolve the AAV using DNA shuffling and in vivo biopanning in a mouse model. An AAVM41 mutant was characterized, which was found to have improved transduction efficiency and specificity in myocardium, an attribute unknown for any natural AAV serotypes. This review focuses on the development of AAV vector for cardiac gene transfer, the history of directed evolution of viral vectors, and our creation of a cardiotropic AAV, which might have implications for the future design and application of viral vectors.

Retrograde vs. Antegrade Puncture for Infra-Inguinal Angioplasty

International Nuclear Information System (INIS)

Nice, C.; Timmons, G.; Bartholemew, P.; Uberoi, R.

2003-01-01

This study was done to compare antegrade punctures with a retrograde puncture technique for infrainguinal angioplasty. A group of 100 consecutive patients (71 men, 29 women) were randomized for antegrade puncture or retrograde puncture of the common femoral artery. Following retrograde puncture the guidewire was 'turned' and placed into the superficial femoral artery. The time for gaining access, screening time, radiation dose, patient height, weight and complications were recorded. All patients were reviewed the day after the procedure and within 3 months. Data from 46 patients (34 males and 12 females) in the retrograde group and 44 (28 males and 16 females) in the antegrade group were available for analysis. Mean procedure time,screening time, radiation dose, height and weight were 8.3 minutes(range 3-22), 2.1 minutes (0.3-6.5), 7950 mGy cm -2 (820-71250), 169 cm (149-204) and 79 kg (32-108) for retrograde puncture and 8 min (2-60), 0.7 min (0.0-3.2), 1069 mGycm -2 (0-15400), 169 cm (152-186) and 75 kg (39-125) for antegrade punctures, respectively. An average of 1.2 (1-2) punctures was required for retrograde and 1.75 (1-8) for antegrade. Seven small hematomas occurred with antegrade and three for retrograde puncture.Retrograde puncture is technically easier with a tendency to fewer complications but results in a higher radiation dose. This technique should be used in difficult patients at high risk of haematoma formation

The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

Directory of Open Access Journals (Sweden)

Blaise Ndjamen

2014-03-01

Full Text Available The Herpes Simplex Virus 1 (HSV-1 glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG. gE-gI can also participate in antibody bipolar bridging (ABB, a process by which the antigen-binding fragments (Fabs of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

Improved vaccine protection against retrovirus infection after co-administration of adenoviral vectors encoding viral antigens and type I interferon subtypes

Directory of Open Access Journals (Sweden)

Groitl Peter

2011-09-01

Full Text Available Abstract Background Type I interferons (IFNs exhibit direct antiviral effects, but also distinct immunomodulatory properties. In this study, we analyzed type I IFN subtypes for their effect on prophylactic adenovirus-based anti-retroviral vaccination of mice against Friend retrovirus (FV or HIV. Results Mice were vaccinated with adenoviral vectors encoding FV Env and Gag proteins alone or in combination with vectors encoding IFNα1, IFNα2, IFNα4, IFNα5, IFNα6, IFNα9 or IFNβ. Only the co-administration of adenoviral vectors encoding IFNα2, IFNα4, IFNα6 and IFNα9 resulted in strongly improved immune protection of vaccinated mice from subsequent FV challenge infection with high control over FV-induced splenomegaly and reduced viral loads. The level of protection correlated with augmented virus-specific CD4+ T cell responses and enhanced antibody titers. Similar results were obtained when mice were vaccinated against HIV with adenoviral vectors encoding HIV Env and Gag-Pol in combination with various type I IFN encoding vectors. Here mainly CD4+ T cell responses were enhanced by IFNα subtypes. Conclusions Our results indicate that certain IFNα subtypes have the potential to improve the protective effect of adenovirus-based vaccines against retroviruses. This correlated with augmented virus-specific CD4+ T cell and antibody responses. Thus, co-expression of select type I IFNs may be a valuable tool for the development of anti-retroviral vaccines.

Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin

Science.gov (United States)

Leu, Chia-Hsing; Yang, Mei-Lin; Chung, Nai-Hui; Huang, Yen-Jang; Su, Yu-Chu; Chen, Yi-Cheng; Lin, Chia-Cheng; Shieh, Gia-Shing; Chang, Meng-Ya; Wang, Shainn-Wei; Chang, Yao; Chao, Julie; Chao, Lee

2015-01-01

Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses. PMID:26149981

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein

International Nuclear Information System (INIS)

Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro; Ogihara, Takuo

2016-01-01

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein

Energy Technology Data Exchange (ETDEWEB)

Tomono, Takumi [Laboratory of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan); Kajita, Masahiro [Laboratory of Molecular Pharmaceutics and Technology, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan); Yano, Kentaro [Laboratory of Biopharmaceutics, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan); Ogihara, Takuo, E-mail: togihara@takasaki-u.ac.jp [Laboratory of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033 (Japan)

2016-08-05

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.

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

Science.gov (United States)

2013-01-01

Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

Magnetic concentration of a retroviral vector using magnetite cationic liposomes.

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Ito, Akira; Takahashi, Tetsuya; Kameyama, Yujiro; Kawabe, Yoshinori; Kamihira, Masamichi

2009-03-01

For tissue engineering purposes, retroviral vectors represent an efficient method of delivering exogenous genes such as growth factors to injured tissues because gene-transduced cells can produce stable and constant levels of the gene product. However, retroviral vector technology suffers from low yields. In the present study, we used magnetite nanoparticles and magnetic force to concentrate the retroviral vectors to enhance the transduction efficiency and to enable their magnetic manipulation. Magnetite nanoparticles modified with cationic liposomes were added to a solution containing a retroviral vector pseudotyped with vesicular stomatitis virus glycoprotein. The magnetic particles that captured the viral vectors were collected using a magnetic force and seeded into mouse neuroblastoma Neuro2a cells. The viral titer was up to 55 times greater (up to 3 x 10(8) infectious units/mL). Additionally, the magnetically labeled retroviral vectors can be directed to the desired regions for infection by applying magnetic fields, and micro-patterns of gene-transduced cell regions could be created on a cellular monolayer using micro-patterned magnetic concentrators. These results suggest that this technique provides a promising approach to capturing and concentrating viral vectors, thus achieving high transduction efficiency and the ability to deliver genes to a specific injured site by applying a magnetic field.

The specific transmission of Grapevine fanleaf virus by its nematode vector Xiphinema index is solely determined by the viral coat protein

International Nuclear Information System (INIS)

Andret-Link, Peggy; Schmitt-Keichinger, Corinne; Demangeat, Gerard; Komar, Veronique; Fuchs, Marc

2004-01-01

The viral determinants involved in the specific transmission of Grapevine fanleaf virus (GFLV) by its nematode vector Xiphinema index are located within the 513 C-terminal residues of the RNA2-encoded polyprotein, that is, the 9 C-terminal amino acids of the movement protein (2B MP ) and contiguous 504 amino acids of the coat protein (2C CP ) [Virology 291 (2001) 161]. To further delineate the viral determinants responsible for the specific spread, the four amino acids that are different within the 9 C-terminal 2B MP residues between GFLV and Arabis mosaic virus (ArMV), another nepovirus which is transmitted by Xiphinema diversicaudatum but not by X. index, were subjected to mutational analysis. Of the recombinant viruses derived from transcripts of GFLV RNA1 and RNA2 mutants that systemically infected herbaceous host plants, all with the 2C CP of GFLV were transmitted by X. index unlike none with the 2C CP of ArMV, regardless of the mutations within the 2B MP C-terminus. These results demonstrate that the coat protein is the sole viral determinant for the specific spread of GFLV by X. index

A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation

DEFF Research Database (Denmark)

Frandsen, Rasmus John Normand

2011-01-01

Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi has become a common technique for the study of a wide variety of different fungal species over the past 12years. The discovery that the host range of A. tumefaciens could be extended to include fungi provided an efficient transform......Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi has become a common technique for the study of a wide variety of different fungal species over the past 12years. The discovery that the host range of A. tumefaciens could be extended to include fungi provided an efficient......-regulation of gene expression. This review summarizes the technical advances within the field from 1998 to the summer of 2011, focusing on the development of binary vectors that are compatible with fungal transformation (over 180 general vectors) and methods for constructing binary vectors for targeted integration...

Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

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Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

2014-04-01

Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution

Modeling electric bicycle's lane-changing and retrograde behaviors

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Tang, Tie-Qiao; Luo, Xiao-Feng; Zhang, Jian; Chen, Liang

2018-01-01

Recently, electric bicycle (EB) has been one important traffic tool due to its own merits. However, EB's motion behaviors (especially at a signalized/non-signalized intersection) are more complex than those of vehicle since it always has lane-changing and retrograde behaviors. In this paper, we propose a model to explore EB's lane-changing and retrograde behaviors on a road with a signalized intersection. The numerical results indicate that the proposed model can qualitatively describe each EB's lane-changing and retrograde behaviors near a signalized intersection, and that lane-changing and retrograde behaviors have prominent impacts on the signalized intersection (i.e., prominent jams and congestions occur). The above results show that EB should be controlled as a vehicle, i.e., lane-changing and retrograde behaviors at a signalized intersection should strictly be prohibited to improve the operational efficiency and traffic safety at the signalized intersection.

Pharmacologic Effects in vivo in Brain by Vector-Mediated Peptide Drug Delivery

Science.gov (United States)

Bickel, Ulrich; Yoshikawa, Takayoshi; Landaw, Elliot M.; Faull, Kym F.; Pardridge, William M.

1993-04-01

Pharmacologic effects in brain caused by systemic administration of neuropeptides are prevented by poor transport of the peptide through the brain vascular endothelium, which comprises the blood-brain barrier in vivo. In the present study, successful application of a chimeric peptide approach to enhance drug delivery through the blood-brain barrier for the purpose of achieving a central nervous system pharmacologic effect is described. The chimeric peptide was formed by linkage of a potent vasoactive intestinal peptide (VIP) analogue, which had been monobiotinylated, to a drug transport vector. The vector consisted of a covalent conjugate of avidin and the OX26 monoclonal antibody to the transferrin receptor. Owing to the high concentration of transferrin receptors on brain capillary endothelia, OX26 targets brain and undergoes receptor-mediated transcytosis through the blood-brain barrier. Systemic infusion of low doses (12 μg/kg) of the VIP chimeric peptide in rats resulted in an in vivo central nervous system pharmacologic effect: a 65% increase in cerebral blood flow. Biotinylated VIP analogue without the brain transport vector was ineffective.

Doppler-guided retrograde catheterization system

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Frazin, Leon J.; Vonesh, Michael J.; Chandran, Krishnan B.; Khasho, Fouad; Lanza, George M.; Talano, James V.; McPherson, David D.

1991-05-01

The purpose of this study was to investigate a Doppler guided catheterization system as an adjunctive or alternative methodology to overcome the disadvantages of left heart catheterization and angiography. These disadvantages include the biological effects of radiation and the toxic and volume effects of iodine contrast. Doppler retrograde guidance uses a 20 MHz circular pulsed Doppler crystal incorporated into the tip of a triple lumen multipurpose catheter and is advanced retrogradely using the directional flow information provided by the Doppler waveform. The velocity detection limits are either 1 m/second or 4 m/second depending upon the instrumentation. In a physiologic flow model of the human aortic arch, multiple data points revealed a positive wave form when flow was traveling toward the catheter tip indicating proper alignment for retrograde advancement. There was a negative wave form when flow was traveling away from the catheter tip if the catheter was in a branch or bent upon itself indicating improper catheter tip position for retrograde advancement. In a series of six dogs, the catheter was able to be accurately advanced from the femoral artery to the left ventricular chamber under Doppler signal guidance without the use of x-ray. The potential applications of a Doppler guided retrograde catheterization system include decreasing time requirements and allowing safer catheter guidance in patients with atherosclerotic vascular disease and suspected aortic dissection. The Doppler system may allow left ventricular pressure monitoring in the intensive care unit without the need for x-ray and it may allow left sided contrast echocardiography. With pulse velocity detection limits of 4 m/second, this system may allow catheter direction and passage into the aortic root and left ventricle in patients with aortic stenosis. A modification of the Doppler catheter may include transponder technology which would allow precise catheter tip localization once the

Quantum dot coating of baculoviral vectors enables visualization of transduced cells and tissues

International Nuclear Information System (INIS)

Zhao, Ying; Lo, Seong Loong; Zheng, Yuangang; Lam, Dang Hoang; Wu, Chunxiao; Han, Ming Yong; Wang, Shu

2013-01-01

Highlights: •The use of quantum dot (QD)-labeled viral vectors for in vivo imaging is not well investigated. •A new method to label enveloped baculovirus with glutathione-capped CdTe QDs is developed. •The labeling enables the identification of transduced, cultured cells based on fluorescence. •The labeling also allows evaluation of viral transduction in a real-time manner in living mice. •The method has the potential to assess viral vector-based gene therapy protocols in future. -- Abstract: Imaging of transduced cells and tissues is valuable in developing gene transfer vectors and evaluating gene therapy efficacy. We report here a simple method to use bright and photostable quantum dots to label baculovirus, an emerging gene therapy vector. The labeling was achieved through the non-covalent interaction of glutathione-capped CdTe quantum dots with the virus envelope, without the use of chemical conjugation. The quantum dot labeling was nondestructive to viral transduction function and enabled the identification of baculoviral vector-transduced, living cells based on red fluorescence. When the labeled baculoviral vectors were injected intravenously or intraventricularly for in vivo delivery of a transgene into mice, quantum dot fluorescence signals allow us monitor whether or not the injected tissues were transduced. More importantly, using a dual-color whole-body imaging technology, we demonstrated that in vivo viral transduction could be evaluated in a real-time manner in living mice. Thus, our method of labeling a read-to-use gene delivery vector with quantum dots could be useful towards the improvement of vector design and will have the potential to assess baculovirus-based gene therapy protocols in future

Zika and its vector mosquitoes in Mexico

Directory of Open Access Journals (Sweden)

Luis del Carpio-Orantes

2018-06-01

Full Text Available In the present study, we carried out a review on the potential vectors of the Zika virus in the Americas, specifically in Mexico. Being vectors of the Culicidae family, they have great predominance in those territories, which could facilitate viral dissemination.

Diverse Array of New Viral Sequences Identified in Worldwide Populations of the Asian Citrus Psyllid (Diaphorina citri) Using Viral Metagenomics.

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Nouri, Shahideh; Salem, Nidá; Nigg, Jared C; Falk, Bryce W

2015-12-16

The Asian citrus psyllid, Diaphorina citri, is the natural vector of the causal agent of Huanglongbing (HLB), or citrus greening disease. Together; HLB and D. citri represent a major threat to world citrus production. As there is no cure for HLB, insect vector management is considered one strategy to help control the disease, and D. citri viruses might be useful. In this study, we used a metagenomic approach to analyze viral sequences associated with the global population of D. citri. By sequencing small RNAs and the transcriptome coupled with bioinformatics analysis, we showed that the virus-like sequences of D. citri are diverse. We identified novel viral sequences belonging to the picornavirus superfamily, the Reoviridae, Parvoviridae, and Bunyaviridae families, and an unclassified positive-sense single-stranded RNA virus. Moreover, a Wolbachia prophage-related sequence was identified. This is the first comprehensive survey to assess the viral community from worldwide populations of an agricultural insect pest. Our results provide valuable information on new putative viruses, some of which may have the potential to be used as biocontrol agents. Insects have the most species of all animals, and are hosts to, and vectors of, a great variety of known and unknown viruses. Some of these most likely have the potential to be important fundamental and/or practical resources. In this study, we used high-throughput next-generation sequencing (NGS) technology and bioinformatics analysis to identify putative viruses associated with Diaphorina citri, the Asian citrus psyllid. D. citri is the vector of the bacterium causing Huanglongbing (HLB), currently the most serious threat to citrus worldwide. Here, we report several novel viral sequences associated with D. citri. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Setting Up Shop: The Formation and Function of the Viral Factories of Cauliflower mosaic virus

Directory of Open Access Journals (Sweden)

James E. Schoelz

2017-10-01

Full Text Available Similar to cells, viruses often compartmentalize specific functions such as genome replication or particle assembly. Viral compartments may contain host organelle membranes or they may be mainly composed of viral proteins. These compartments are often termed: inclusion bodies (IBs, viroplasms or viral factories. The same virus may form more than one type of IB, each with different functions, as illustrated by the plant pararetrovirus, Cauliflower mosaic virus (CaMV. CaMV forms two distinct types of IBs in infected plant cells, those composed mainly of the viral proteins P2 (which are responsible for transmission of CaMV by insect vectors and P6 (required for viral intra-and inter-cellular infection, respectively. P6 IBs are the major focus of this review. Much of our understanding of the formation and function of P6 IBs comes from the analyses of their major protein component, P6. Over time, the interactions and functions of P6 have been gradually elucidated. Coupled with new technologies, such as fluorescence microscopy with fluorophore-tagged viral proteins, these data complement earlier work and provide a clearer picture of P6 IB formation. As the activities and interactions of the viral proteins have gradually been determined, the functions of P6 IBs have become clearer. This review integrates the current state of knowledge on the formation and function of P6 IBs to produce a coherent model for the activities mediated by these sophisticated virus-manufacturing machines.

PRMT5 Is Upregulated in HTLV-1-Mediated T-Cell Transformation and Selective Inhibition Alters Viral Gene Expression and Infected Cell Survival

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Amanda R. Panfil

2015-12-01

Full Text Available Human T-cell leukemia virus type-1 (HTLV-1 is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL. This disease manifests after a long clinical latency period of up to 2–3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5 on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment.

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

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Maarit Neuvonen

2011-11-01

Full Text Available Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3 domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV, Sindbis (SINV, and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.

Retrograde amnesia in patients with Alzheimer's disease

NARCIS (Netherlands)

Meeter, M.; Eijsackers, E; Mulder, J

2006-01-01

Patients with mild to moderate Alzheimer's disease and normal controls were tested on two retrograde memory tests, one based on public events, and the other querying autobiographical memory. On both tests, patients showed strong decrements as compared to normal controls, pointing to retrograde

Improved osteogenic vector for non-viral gene therapy

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ARA Hacobian

2016-03-01

Full Text Available Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2 gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp. Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches.

Insecticides promote viral outbreaks by altering herbivore competition.

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Pan, Huipeng; Preisser, Evan L; Chu, Dong; Wang, Shaoli; Wu, Qingjun; Carriére, Yves; Zhou, Xuguo; Zhang, Youjun

2015-09-01

While the management of biological invasions is often characterized by a series of single-specieg decisions, invasive species exist within larger food webs. These biotic interactions can alter the impact of control/eradication programs and may cause suppression efforts to inadvertently facilitate invasion spread and impact. We document the rapid replacement of the invasive Bemisia Middle East-Asia Minor I (MEAM1) cryptic biotype by the cryptic Mediterranean (MED) biotype throughout China and demonstrate that MED is more tolerant of insecticides and a better vector of tomato yellow leaf curl virus (TYLCV) than MEAMJ. While MEAM1 usually excludes MED under natural conditions, insecticide application reverses the MEAM1-MED competitive hierarchy and allows MED to exclude MEAMI. The insecticide-mediated success of MED has led to TYLCV outbreaks throughout China. Our work strongly supports the hypothesis that insecticide use in China reverses the MEAMl-MED competitive hierarchy and allows MED to displace MEAM1 in managed landscapes. By promoting the dominance of a Bemisia species that is a competent viral vector, insecticides thus increase the spread and impact of TYLCV in heterogeneous agroecosystems.

Development and applications of VSV vectors based on cell tropism

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Hideki eTani

2012-01-01

Full Text Available Viral vectors have been available in various fields such as medical and biological research or gene therapy applications. Targeting vectors pseudotyped with distinct viral envelope proteins that influence cell tropism and transfection efficiency is a useful tool not only for examining entry mechanisms or cell tropisms but also for vaccine vector development. Vesicular stomatitis virus (VSV is an excellent candidate for development as a pseudotype vector. A recombinant VSV lacking its own envelope (G gene has been used to produce a pseudotype or recombinant VSV possessing the envelope proteins of heterologous viruses. These viruses possess a reporter gene instead of a VSV G gene in their genome, and therefore it is easy to evaluate their infectivity in the study of viral entry, including identification of viral receptors. Furthermore, advantage can be taken of a property of the pseudotype VSV, which is competence for single-round infection, in handling many different viruses that are either difficult to amplify in cultured cells or animals or that require specialized containment facilities. Here we describe procedures for producing pseudotype or recombinant VSVs and a few of the more prominent examples from among envelope viruses, such as hepatitis C virus, Japanese encephalitis virus, baculovirus, and hemorrhagic fever viruses.

Dynamics of the retrograde 1/1 mean motion resonance

Science.gov (United States)

Huang, Yukun; Li, Miao; Li, Junfeng; Gong, Shengping

2018-04-01

Mean motion resonances are very common in the solar system. Asteroids in mean motion resonances with giant planets have been studied for centuries. But it was not until recently that asteroids in retrograde mean motion resonances with Jupiter and Saturn were discovered. The newly discovered asteroid, 2015 BZ509 is confirmed to be the first asteroid in retrograde 1:1 mean motion resonance (or retrograde co-orbital resonance) with Jupiter, which gives rise to our interests in its unique resonant dynamics. In this study, we thoroughly investigate the phase-space structure of the retrograde 1:1 resonance within the framework of the circular restricted three-body problem. We begin by constructing a simple integrable approximation for the planar retrograde resonance with the Hamiltonian approach and show that the variables definition of the retrograde resonance is very different to the prograde one. When it comes to the disturbing function, we abandon the classical series expansion approach, whereas numerically carry out the averaging process on the disturbing function in closed form. The phase portrait of the retrograde 1:1 resonance is depicted with the level curves of the averaged Hamiltonian. We find that the topological structure of phase space for the retrograde 1:1 resonance is very different to other resonances, due to the consistent existence of the collision separatrix. And the surprising bifurcation of equilibrium point around 180° (i.e., the apocentric libration center) has never been found in any other mean motion resonances before. We thoroughly analyze the novel apocentric librations and find that close encounter with the planet does not always lead to the disruption of a stable apocentric libration. Afterwards, we examine the Kozai dynamics inside the mean motion resonance with the similar Hamiltonian approach and explain why the exact resonant point does not exist in the 3D retrograde 1:1 resonance model.

Retrograde amnesia for semantic information in Alzheimer's disease

OpenAIRE

Meeter, M.; Kollen, A.; Scheltens, P.

2005-01-01

Patients with mild to moderate Alzheimer's disease and normal controls were tested on a retrograde amnesia test with semantic content (Neologism and Vocabulary Test, or NVT), consisting of neologisms to be defined. Patients showed a decrement as compared to normal controls, pointing to retrograde amnesia within semantic memory. No evidence for a gradient within this amnesia was found, although one was present on an autobiographic test of retrograde amnesia that had a wider time scale. Several...

Mismatch analysis of humeral nailing. Antegrade versus retrograde insertion

International Nuclear Information System (INIS)

Mahaisavariya, B.; Jiamwatthanachai, P.; Aroonjarattham, P.; Aroonjarattham, K.; Wongcumchang, M.; Sitthiseripratip, K.

2011-01-01

Closed humeral nailing is now considered an alternative treatment for humeral-shaft fracture. The nail can be inserted with either the antegrade or retrograde method. We investigated and compared the problem of geometric mismatch of the humeral nail to the humerus between the two methods of insertion. The study was performed using virtual simulation based on computed tomography (CT) data of 76 Thai cadaveric humeri and the commonly used Russell-Taylor humeral nail 8 mm in diameter and 220 mm long. Mismatch of the nail to the intact humerus was analyzed and compared between the antegrade and retrograde nailing approaches. The results showed: the diameter of the medullary canal averaged 7.9-13.8 mm; the minimal reaming diameter to accommodate virtual nail insertion averaged 8.8-14.8 mm for the antegrade and 8.8-29.3 mm for the retrograde approach; the minimal reaming thickness of the inner cortex averaged 0.1-1.5 mm for the antegrade and 0.1-9.9 mm for the retrograde approach; the percentages of cortical bone removed prior to nail insertion were 3.8-107.1% and 3.8-1,287.6% for the antegrade and retrograde approaches, respectively; the eccentricity of the nail-medullary canal center were 0.4-3.4 and 0.4-10.6 mm for the antegrade and retrograde approaches, respectively. Less mismatching occurred with antegrade nailing than with the retrograde approach. Retrograde nailing requires excessive reaming at the distal part of the humerus to accommodate nail insertion. This may create bone weakness and the risk of supracondylar fracture. (author)

Retrograde prostatic urethroplasty with balloon catheter

International Nuclear Information System (INIS)

Castaneda, F.; Reddy, P.; Hulbert, J.; Letourneau, J.G.; Hunter, D.W.; Castaneda-Zuniga, W.R.; Amplatz, K.

1987-01-01

The authors performed retrograde prostatic urethroplasty in 18 patients using a 25-mm urethroplasty balloon catheter. The procedure was performed on an outpatient basis under local anesthesia. Voiding cystourethrography, retrograde urethrography, rectal US, and MRE imaging were performed before and immediately after the procedure and at 2 weeks and 3, 6, 12, and 18 months. Long-term results at 18 months and possible clinical implications are discussed

Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infection.

Science.gov (United States)

Tabynov, Kaissar; Sansyzbay, Abylai; Kydyrbayev, Zhailaubay; Yespembetov, Bolat; Ryskeldinova, Sholpan; Zinina, Nadezhda; Assanzhanova, Nurika; Sultankulova, Kulaisan; Sandybayev, Nurlan; Khairullin, Berik; Kuznetsova, Irina; Ferko, Boris; Egorov, Andrej

2014-04-10

We generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals. Four recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by

Preclinical assessment of viral vectored and protein vaccines targeting the Duffy-binding protein region II of Plasmodium vivax

Directory of Open Access Journals (Sweden)

Simone C de Cassan

2015-07-01

Full Text Available Malaria vaccine development has largely focused on Plasmodium falciparum; however a reawakening to the importance of P. vivax has spurred efforts to develop vaccines against this difficult to treat and at times severe form of relapsing malaria, which constitutes a significant proportion of human malaria cases worldwide. The almost complete dependence of P. vivax red blood cell invasion on the interaction of the P. vivax Duffy-binding protein region II (PvDBP_RII with the human Duffy antigen receptor for chemokines (DARC, makes this antigen an attractive vaccine candidate against blood-stage P. vivax. Here, we generated both preclinical and clinically-compatible adenoviral and poxviral vectored vaccine candidates expressing the Salvador I allele of PvDBP_RII – including human adenovirus serotype 5 (HAdV5, chimpanzee adenovirus serotype 63 (ChAd63 and modified vaccinia virus Ankara (MVA vectors. We report on the antibody and T cell immunogenicity of these vaccines in mice or rabbits, either used alone in a viral vectored prime-boost regime, or in ‘mixed-modality’ adenovirus prime – protein-in-adjuvant boost regimes (using a recombinant protein PvDBP_RII protein antigen formulated in Montanide®ISA720 or Abisco®100 adjuvants. Antibodies induced by these regimes were found to bind to native parasite antigen from P. vivax infected Thai patients and were capable of inhibiting the binding of PvDBP_RII to its receptor DARC using an in vitro binding inhibition assay. In recent years, recombinant ChAd63 and MVA vectors have been quickly translated into human clinical trials for numerous antigens from P. falciparum as well as a growing number of other pathogens. The vectors reported here are immunogenic in small animals, elicit antibodies against PvDBP_RII and have recently entered clinical trials which will provide the first assessment of the safety and immunogenicity of the PvDBP_RII antigen in humans.

Retrograde transurethral balloon dilation of the prostate

International Nuclear Information System (INIS)

Castaneda, F.; Reddy, P.; Wasserman, N.F.; Lund, G.; Hulbert, J.; Hunter, D.; Castaneda-Zuniga, W.R.; Amplatz, K.

1986-01-01

A series of patients with documented benign prostatic hypertrophy evaluated by urodynamic studies, voiding cystourethrography, retrograde urethrography, and MR imaging underwent dilation performed using a retrograde transurethral approach with 25-mm balloon dilators inflated at a pressure of 3-4 atm for 10 minutes. Immediately after the procedure, retrograde and voiding cystourethrography as well as MR imaging were performed. A Foley catheter was left in place for 24 hours. Complete relief of symptoms has occurred in all of the patients during the follow-up period. No significant complications other than transient hematuria resulted from the procedure. Results of the comparison studies and of MR imaging are discussed

Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

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Peluffo, Hugo; Acarin, Laia; Arís, Anna; González, Pau; Villaverde, Antoni; Castellano, Bernardo; González, Berta

2006-01-01

Background Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA) administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated. Results Overexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced. Conclusion When the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene. PMID:16638118

Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

Directory of Open Access Journals (Sweden)

Castellano Bernardo

2006-04-01

Full Text Available Abstract Background Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated. Results Overexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced. Conclusion When the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene.

Synaptic plasticity in the medial vestibular nuclei: role of glutamate receptors and retrograde messengers in rat brainstem slices.

Science.gov (United States)

Grassi, S; Pettorossi, V E

2001-08-01

The analysis of cellular-molecular events mediating synaptic plasticity within vestibular nuclei is an attempt to explain the mechanisms underlying vestibular plasticity phenomena. The present review is meant to illustrate the main results, obtained in vitro, on the mechanisms underlying long-term changes in synaptic strength within the medial vestibular nuclei. The synaptic plasticity phenomena taking place at the level of vestibular nuclei could be useful for adapting and consolidating the efficacy of vestibular neuron responsiveness to environmental requirements, as during visuo-vestibular recalibration and vestibular compensation. Following a general introduction on the most salient features of vestibular compensation and visuo-vestibular adaptation, which are two plastic events involving neuronal circuitry within the medial vestibular nuclei, the second and third sections describe the results from rat brainstem slice studies, demonstrating the possibility to induce long-term potentiation and depression in the medial vestibular nuclei, following high frequency stimulation of the primary vestibular afferents. In particular the mechanisms sustaining the induction and expression of vestibular long-term potentiation and depression, such as the role of various glutamate receptors and retrograde messengers have been described. The relevant role of the interaction between the platelet-activating factor, acting as a retrograde messenger, and the presynaptic metabotropic glutamate receptors, in determining the full expression of vestibular long-term potentiation is also underlined. In addition, the mechanisms involved in vestibular long-term potentiation have been compared with those leading to long-term potentiation in the hippocampus to emphasize the most significant differences emerging from vestibular studies. The fourth part, describes recent results demonstrating the essential role of nitric oxide, another retrograde messenger, in the induction of vestibular

Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells

DEFF Research Database (Denmark)

Stiehler, Maik; Duch, Mogens R.; Mygind, Tina

2006-01-01

-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno...

Dynamic Portrait of the Retrograde 1:1 Mean Motion Resonance

Science.gov (United States)

Huang, Yukun; Li, Miao; Li, Junfeng; Gong, Shengping

2018-06-01

Asteroids in mean motion resonances with giant planets are common in the solar system, but it was not until recently that several asteroids in retrograde mean motion resonances with Jupiter and Saturn were discovered. A retrograde co-orbital asteroid of Jupiter, 2015 BZ509 is confirmed to be in a long-term stable retrograde 1:1 mean motion resonance with Jupiter, which gives rise to our interests in its unique resonant dynamics. In this paper, we investigate the phase-space structure of the retrograde 1:1 resonance in detail within the framework of the circular restricted three-body problem. We construct a simple integrable approximation for the planar retrograde resonance using canonical contact transformation and numerically employ the averaging procedure in closed form. The phase portrait of the retrograde 1:1 resonance is depicted with the level curves of the averaged Hamiltonian. We thoroughly analyze all possible librations in the co-orbital region and uncover a new apocentric libration for the retrograde 1:1 resonance inside the planet’s orbit. We also observe the significant jumps in orbital elements for outer and inner apocentric librations, which are caused by close encounters with the perturber.

Antegrade or Retrograde Accessory Pathway Conduction: Who Dies First?

Directory of Open Access Journals (Sweden)

Claudio Hadid, MD

2012-05-01

Full Text Available A 36 year-old man with Wolff Parkinson White syndrome due to a left-sided accessory pathway (AP was referred for catheter ablation. Whether abolition of antegrade and retrograde AP conduction during ablation therapy occurs simultaneously, is unclear. At the ablation procedure, radiofrequency delivery resulted in loss of preexcitation followed by a short run of orthodromic tachycardia with eccentric atrial activation, demonstrating persistence of retrograde conduction over the AP after abolition of its antegrade conduction. During continued radiofrequency delivery at the same position, the fifth non-preexcitated beat failed to conduct retrogradely and the tachycardia ended. In this case, antegrade AP conduction was abolished earlier than retrograde conduction.

Construction and expression of secreting type human TRAIL gene vector mediated by hypoxia/radiation double sensitive promoter

International Nuclear Information System (INIS)

Yang Yanming; Jia Xiaojing; Qu Yaqin; Li Yanbo

2009-01-01

Objective: To construct secreting type human TRAIL (shTRAIL) gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter, and observe the effect of hypoxia and radiation on shTRAIL. Methods: HRE upper and lower strands were gotten by chemical synthesis, double strands HRE was gotten by PCR; pMD19T-Egr1 was digested by Sac I and Hind III, then Egr1 was obtained, pshuttle-shTRAIL was digested by Kpn I and BamH I, then shTRAIL was obtained; HRE/Egr1 double sensitive promoter mediated shTRAIL expression vector pcDNA3.1-HRE/Egr1-shTRAIL was constructed by gene recombination technique, it was identified correctly by enzyme digestion, PCR and sequencing. A549 cells were divided into normal, hypoxia (0.1%), irradiation (6 Gy) and hypoxia + irradiation groups. Results: After enzyme digestion by BamH I and Sma I, the fragments which lengths were 1284 bp and 4 998 bp, 2 292 bp and 3 990 bp were obtained; the vector was amplified by PCR with Egr1 and shTRAIL primer, the products which lengthens were 469 bp and 820 bp were obtained; pcDNA3.1-HRE/Egr1-shTRAIL was sequenced, the result was same to designed, this demonstrated that the construction was right. The vectors were transfected into A549 cells of adenocarcinoma of lung, the expression levels of shTRAIL mRNA and protein were increased after treated with hypoxia and radiation, it had statistically significant differences compared with normal group (P<0.05), and when they were combinated, the effect was more obvious. Conclusion: Secreting type human TRAIL gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter is constructed successfully, and hypoxia and radiation could increase the expression of TRAIL, and they have synergetic effect. (authors)

A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors.

Science.gov (United States)

Nair, Ayyappan; Xie, Jinger; Joshi, Sarasijam; Harden, Paul; Davies, Joan; Hermiston, Terry

2008-11-01

A robust assay to titer lentiviral vectors is imperative to qualifying their use in drug discovery, target validation and clinical applications. In this study, a novel branched DNA based hybridization assay was developed to titer lentiviral vectors by quantifying viral RNA genome copy numbers from viral lysates without having to purify viral RNA, and this approach was compared with other non-functional (p24 protein ELISA and viral RT-qPCR) and a functional method (reporter gene expression) used commonly. The RT-qPCR method requires purification of viral RNA and the accuracy of titration therefore depends on the efficiency of purification; this requirement is ameliorated in the hybridization assay as RNA is measured directly in viral lysates. The present study indicates that the hybridization based titration assay performed on viral lysates was more accurate and has additional advantages of being rapid, robust and not dependent on transduction efficiency in different cell types.

Retrograde Renal Cooling to Minimize Ischemia

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Janet L. Colli

2013-01-01

Full Text Available Objective: During partial nephrectomy, renal hypothermia has been shown to decrease ischemia induced renal damage which occurs from renal hilar clamping. In this study we investigate the infusion rate required to safely cool the entire renal unit in a porcine model using retrograde irrigation of iced saline via dual-lumen ureteral catheter. Materials and Methods: Renal cortical, renal medullary, bowel and rectal temperatures during retrograde cooling in a laparoscopic porcine model were monitored in six renal units. Iced normal saline was infused at 300 cc/hour, 600 cc/hour, 1000 cc/hour and gravity (800 cc/hour for 600 seconds with and without hilar clamping. Results: Retrograde cooling with hilar clamping provided rapid medullary renal cooling and significant hypothermia of the medulla and cortex at infusion rates ≥ 600 cc/hour. With hilar clamping, cortical temperatures decreased at -0.9° C/min. reaching a threshold temperature of 26.9° C, and medullary temperatures decreased at -0.90 C/min. reaching a temperature of 26.1° C over 600 seconds on average for combined data at infusion rates ≥ 600 cc/hour. The lowest renal temperatures were achieved with gravity infusion. Without renal hilum clamping, retrograde cooling was minimal at all infusion rates. Conclusions: Significant renal cooling by gravity infusion of iced cold saline via a duel lumen catheter with a clamped renal hilum was achieved in a porcine model. Continuous retrograde irrigation with iced saline via a two way ureteral catheter may be an effective method to induce renal hypothermia in patients undergoing robotic assisted and/or laparoscopic partial nephrectomy.

Vector-like fields, messenger mixing and the Higgs mass in gauge mediation

Energy Technology Data Exchange (ETDEWEB)

Fischler, Willy; Tangarife, Walter [Department of Physics and Texas Cosmology Center,The University of Texas at Austin,TX 78712 (United States)

2014-05-30

In order to generate, in the context of gauge mediation, a Higgs mass around 126 GeV that avoids the little hierarchy problem, we explore a set of models where the messengers are directly coupled to new vector-like fields at the TeV scale in addition to the usual low energy degrees of freedom. We find that in this context, stop masses lighter than 2 TeV and large A-terms are generated, thereby improving issues of fine tuning.

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

Science.gov (United States)

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

2009-11-12

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

The inherent catastrophic traps in retrograde CTO PCI.

Science.gov (United States)

Wu, Eugene B; Tsuchikane, Etsuo

2018-05-01

When we learn to drive, our driving instructor tells us how to check the side mirror and turn your head to check the blind spot before changing lanes. He tells us how to stop at stop signs, how to drive in slippery conditions, the safe stopping distances, and these all make our driving safe. Similarly, when we learn PCI, our mentors teach us to seat the guiding catheter co-axially, to wire the vessel safely, to deliver balloon and stents over the wire, to watch the pressure of the guiding, in order that we perform PCI safely and evade complications. In retrograde CTO PCI, there is no such published teaching. Also many individual mentors have not had the wide experience to see all the possible complications of retrograde CTO PCI and, therefore, may not be able to warn their apprentice. As the number of retrograde procedures increase worldwide, there is a corresponding increase in catastrophic complications, many of which, we as experts, can see are easily avoidable. To breach this gap in knowledge, this article describes 12 commonly met inherent traps in retrograde CTO PCI. They are inherent because by arranging our equipment in the manner to perform retrograde CTO PCI, these complications are either induced directly or happen easily. We hope this work will enhance safety of retrograde CTO PCI and avoid many catastrophic complications for our readers and operators. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Criteria for retrograde rotation of accreting black holes

Science.gov (United States)

Mikhailov, A. G.; Piotrovich, M. Yu; Gnedin, Yu N.; Natsvlishvili, T. M.; Buliga, S. D.

2018-06-01

Rotating supermassive black holes produce jets and their origin is connected to the magnetic field that is generated by accreting matter flow. There is a point of view that electromagnetic fields around rotating black holes are brought to the hole by accretion. In this situation the prograde accreting discs produce weaker large-scale black hole threading magnetic fields, implying weaker jets than in retrograde regimes. The basic goal of this paper is to find the best candidates for retrograde accreting systems in observed active galactic nuclei. We show that active galactic nuclei with low Eddington ratio are really the best candidates for retrograde systems. This conclusion is obtained for kinetically dominated Fanaroff-Riley class II radio galaxies, flat-spectrum radio-loud narrow-line Seyfert I galaxies and a number of nearby galaxies. Our conclusion is that the best candidates for retrograde systems are the noticeable population of active galactic nuclei in the Universe. This result corresponds to the conclusion that in the merging process the interaction of merging black holes with a retrograde circumbinary disc is considerably more effective for shrinking the binary system.

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

Science.gov (United States)

Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian; Wei, Taiyun

2016-01-15

Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an

Retrograde amnesia after electroconvulsive therapy: a temporary effect?

NARCIS (Netherlands)

Meeter, M.; Murre, J.M.J.; Janssen, S.M.J.; Birkenhager, T.; van den Broek, W.W.

2011-01-01

Objective: Although electroconvulsive therapy (ECT) is generally considered effective against depression, it remains controversial because of its association with retrograde memory loss. Here, we assessed memory after ECT in circumstances most likely to yield strong retrograde amnesia. Method: A

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

Science.gov (United States)

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

2018-01-01

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

The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export.

Science.gov (United States)

Brunotte, Linda; Flies, Joe; Bolte, Hardin; Reuther, Peter; Vreede, Frank; Schwemmle, Martin

2014-07-18

In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

Science.gov (United States)

Geis, Franziska K; Galla, Melanie; Hoffmann, Dirk; Kuehle, Johannes; Zychlinski, Daniela; Maetzig, Tobias; Schott, Juliane W; Schwarzer, Adrian; Goffinet, Christine; Goff, Stephen P; Schambach, Axel

2017-05-31

Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

Retrograde amnesia for semantic information in Alzheimer's disease

NARCIS (Netherlands)

Meeter, M.; Kollen, A.; Scheltens, P.

2005-01-01

Patients with mild to moderate Alzheimer's disease and normal controls were tested on a retrograde amnesia test with semantic content (Neologism and Vocabulary Test, or NVT), consisting of neologisms to be defined. Patients showed a decrement as compared to normal controls, pointing to retrograde

Disrupting circadian rhythms in rats induces retrograde amnesia

NARCIS (Netherlands)

Fekete, Mátyás; Ree, J.M. van; Niesink, Raymond J.M.; Wied, D. de

1985-01-01

Disrupting circadian organization in rats by phase-shifting the illumination cycle or by exposure to a reversed day/night cycle or to continuous light, resulted in retrograde amnesia for passive avoidance behavior. This retrograde amnesia induced by phase-shifting lasted at least 2 days, and

Antiretroviral treatment adherence as a mediating factor between psychosocial variables and HIV viral load.

Science.gov (United States)

Attonito, Jennifer; Dévieux, Jessy G; Lerner, Brenda D G; Hospital, Michelle M; Rosenberg, Rhonda

2014-01-01

Psychosocial factors may directly impact HIV health measures such as viral load (VL) whether or not patients are taking antiretroviral treatment (ART) consistently. Structural equation modeling plus Baron and Kenny's (1986) four-step approach were used to test a mediated model predicting VL among 246 HIV-infected adults who were on ART. Exogenous variables were social support, barriers to adherence, and stress. Moderators were alcohol use, marijuana use, and neurocognitive impairment. A small positive association between marijuana use and ART adherence approached significance. Only barriers to adherence predicted a decrease in adherence rates and an increase in VL. No other factors were significantly associated with either VL or adherence, and no interaction effects between exogenous variables and moderators were identified. The association between barriers to adherence and VL was partially mediated by ART adherence. Findings provide modest support for a direct link between psychosocial variables and a virologic response to ART. Copyright © 2014 Association of Nurses in AIDS Care. Published by Elsevier Inc. All rights reserved.

Development of Recombinant Vaccine Using Herpesvirus of Turkey (Hvt as Vector for Several Viral Diseases in Poultry Industry

Directory of Open Access Journals (Sweden)

Risza Hartawan

2011-03-01

Full Text Available Herpesvirus of turkey (HVT has been utilised as live vaccine against Marek’s disease in poultry industry world-wide for many years. However, the potency of HVT is not limited on the Marek’s disease only. Along with rapid development of recombinant technique, the potency of HVT can be broaden for other diseases. As naturally apathogenic virus, HVT is a suitable candidate as vector vaccine to express important antigens of viral pathogens. Several researches have been dedicated to design HVT recombinant vaccine by inserting gene of important virus, such as Marek’s disease virus (MDV, immuno bursal disease virus (IBDV, Newcastle disease virus (NDV and Avian Influenza virus (AIV. Therefore, the future recombinant of HVT has been expected to be better in performance along with the improvement of recombinant technique.

Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells

DEFF Research Database (Denmark)

Stiehler, Maik; Duch, Mogens; Mygind, Tina

2006-01-01

INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors...... viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells. MATERIALS AND METHODS: MSCs were purified from bone marrow aspirates from the proximal tibiae of four 3-month......-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno...

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

LENUS (Irish Health Repository)

McGinley, Lisa

2012-01-31

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

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

LENUS (Irish Health Repository)

McGinley, Lisa

2011-03-07

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

Functional Outcomes of the Knee after Retrograde and Antegrade ...

African Journals Online (AJOL)

of femur shaft fractures although retrograde technique is gaining acceptance. Although ... Antegrade group, while the rate of knee stiffness was higher in the retrograde .... reaching direct and indirect social economic effect within the society.

AAV Vectorization of DSB-mediated Gene Editing Technologies.

Science.gov (United States)

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.

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

Directory of Open Access Journals (Sweden)

Stefania Piersanti

Full Text Available Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD human (HAd and canine (CAV-2 adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS. With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.

Arthropod Innate Immune Systems and Vector-Borne Diseases.

Science.gov (United States)

Baxter, Richard H G; Contet, Alicia; Krueger, Kathryn

2017-02-21

Arthropods, especially ticks and mosquitoes, are the vectors for a number of parasitic and viral human diseases, including malaria, sleeping sickness, Dengue, and Zika, yet arthropods show tremendous individual variation in their capacity to transmit disease. A key factor in this capacity is the group of genetically encoded immune factors that counteract infection by the pathogen. Arthropod-specific pattern recognition receptors and protease cascades detect and respond to infection. Proteins such as antimicrobial peptides, thioester-containing proteins, and transglutaminases effect responses such as lysis, phagocytosis, melanization, and agglutination. Effector responses are initiated by damage signals such as reactive oxygen species signaling from epithelial cells and recognized by cell surface receptors on hemocytes. Antiviral immunity is primarily mediated by siRNA pathways but coupled with interferon-like signaling, antimicrobial peptides, and thioester-containing proteins. Molecular mechanisms of immunity are closely linked to related traits of longevity and fertility, and arthropods have the capacity for innate immunological memory. Advances in understanding vector immunity can be leveraged to develop novel control strategies for reducing the rate of transmission of both ancient and emerging threats to global health.

Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector

Directory of Open Access Journals (Sweden)

Samuel Campbell

2018-04-01

Full Text Available The previously developed adeno-associated virus/phage (AAVP vector, a hybrid between M13 bacteriophage (phage viruses that infect bacteria only and human Adeno-Associated Virus (AAV, is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC. We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

Implications of gauge-mediated supersymmetry breaking with vector-like quarks and a ~125 GeV Higgs boson

Energy Technology Data Exchange (ETDEWEB)

Martin, Stephen P. [Fermi National Accelerator Laboratory (FNAL), Batavia, IL (United States); Wells, James D. [CERN, Geneva (Switzerland)

2012-08-01

We investigate the implications of models that achieve a Standard Model-like Higgs boson of mass near 125 GeV by introducing additional TeV-scale supermultiplets in the vector-like 10+\\bar{10} representation of SU(5), within the context of gauge-mediated supersymmetry breaking. We study the resulting mass spectrum of superpartners, comparing and contrasting to the usual gauge-mediated and CMSSM scenarios, and discuss implications for LHC supersymmetry searches. This approach implies that exotic vector-like fermions t'_{1,2}, b',and \\tau' should be within the reach of the LHC. We discuss the masses, the couplings to electroweak bosons, and the decay branching ratios of the exotic fermions, with and without various unification assumptions for the mass and mixing parameters. We comment on LHC prospects for discovery of the exotic fermion states, both for decays that are prompt and non-prompt on detector-crossing time scales.

Vaxvec: The first web-based recombinant vaccine vector database and its data analysis

Science.gov (United States)

Deng, Shunzhou; Martin, Carly; Patil, Rasika; Zhu, Felix; Zhao, Bin; Xiang, Zuoshuang; He, Yongqun

2015-01-01

A recombinant vector vaccine uses an attenuated virus, bacterium, or parasite as the carrier to express a heterologous antigen(s). Many recombinant vaccine vectors and related vaccines have been developed and extensively investigated. To compare and better understand recombinant vectors and vaccines, we have generated Vaxvec (http://www.violinet.org/vaxvec), the first web-based database that stores various recombinant vaccine vectors and those experimentally verified vaccines that use these vectors. Vaxvec has now included 59 vaccine vectors that have been used in 196 recombinant vector vaccines against 66 pathogens and cancers. These vectors are classified to 41 viral vectors, 15 bacterial vectors, 1 parasitic vector, and 1 fungal vector. The most commonly used viral vaccine vectors are double-stranded DNA viruses, including herpesviruses, adenoviruses, and poxviruses. For example, Vaxvec includes 63 poxvirus-based recombinant vaccines for over 20 pathogens and cancers. Vaxvec collects 30 recombinant vector influenza vaccines that use 17 recombinant vectors and were experimentally tested in 7 animal models. In addition, over 60 protective antigens used in recombinant vector vaccines are annotated and analyzed. User-friendly web-interfaces are available for querying various data in Vaxvec. To support data exchange, the information of vaccine vectors, vaccines, and related information is stored in the Vaccine Ontology (VO). Vaxvec is a timely and vital source of vaccine vector database and facilitates efficient vaccine vector research and development. PMID:26403370

Characterization of infectivity of knob-modified adenoviral vectors in glioma

NARCIS (Netherlands)

C.P.L. Paul (C. P L); M. Everts (M.); J.N. Glasgow (J.); P. Dent (P.); P.B. Fisher (P.); I.V. Ulasov (I.); M.S. Lesniak (M.); M.A. Stoff-Khalili (M.); J.C. Roth (J.); M. Preuss (Michael); C.M.F. Dirven (Clemens); M.L.M. Lamfers (Martine); T. Siegal (Tali); Z.B. Zhu (Z.); R.E. Curiel (Rafael E.)

2008-01-01

textabstractMalignant glioma continues to be a major target for gene therapy and virotherapy due to its aggressive growth and the current lack of effective treatment. However, these approaches have been hampered by inefficient infection of glioma cells by viral vectors, particularly vectors derived

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

Science.gov (United States)

Bai, Zhiyong; Grant, Barth D.

2015-01-01

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling. PMID:25775511

Independent predictors of retrograde failure in CTO-PCI after successful collateral channel crossing.

Science.gov (United States)

Suzuki, Yoriyasu; Muto, Makoto; Yamane, Masahisa; Muramatsu, Toshiya; Okamura, Atsunori; Igarashi, Yasumi; Fujita, Tsutomu; Nakamura, Shigeru; Oida, Akitsugu; Tsuchikane, Etsuo

2017-07-01

To evaluate factors for predicting retrograde CTO-PCI failure after successful collateral channel crossing. Successful guidewire/catheter collateral channel crossing is important for the retrograde approach in percutaneous coronary intervention (PCI) for chronic total occlusion (CTO). A total of 5984 CTO-PCI procedures performed in 45 centers in Japan from 2009 to 2012 were studied. The retrograde approach was used in 1656 CTO-PCIs (27.7%). We investigated these retrograde procedures to evaluate factors for predicting retrograde CTO-PCI failure even after successful collateral channel crossing. Successful guidewire/catheter collateral crossing was achieved in 77.1% (n = 1,276) of 1656 retrograde CTO-PCI procedures. Retrograde procedural success after successful collateral crossing was achieved in 89.4% (n = 1,141). Univariate analysis showed that the predictors for retrograde CTO-PCI failure were in-stent occlusion (OR = 1.9829, 95%CI = 1.1783 - 3.3370 P = 0.0088), calcified lesions (OR = 1.9233, 95%CI = 1.2463 - 2.9679, P = 0.0027), and lesion tortuosity (OR = 1.5244, 95%CI = 1.0618 - 2.1883, P = 0.0216). On multivariate analysis, lesion calcification was an independent predictor of retrograde CTO-PCI failure after successful collateral channel crossing (OR = 1.3472, 95%CI = 1.0614 - 1.7169, P = 0.0141). The success rate of retrograde CTO-PCI following successful guidewire/catheter collateral channel crossing was high in this registry. Lesion calcification was an independent predictor of retrograde CTO-PCI failure after successful collateral channel crossing. Devices and techniques to overcome complex CTO lesion morphology, such as lesion calcification, are required to further improve the retrograde CTO-PCI success rate. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities.

Science.gov (United States)

Li, Ling; Hu, Shuo; Chen, Xiaoyuan

2018-07-01

In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed. Published by Elsevier Ltd.

Retrograde amnesia for semantic information in Alzheimer’s disease

NARCIS (Netherlands)

Meeter, M.; Knollen, A.; Scheltens, P.

2005-01-01

Patients with mild to moderate Alzheimer's disease and normal controls were tested on a retrograde amnesia test with semantic content (Neologism and Vocabulary Test, or NVT), consisting of neologisms to be defined. Patients showed a decrement as compared to normal controls, pointing to retrograde

Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy.

Science.gov (United States)

Osada, Takuya; Morse, Michael A; Hobeika, Amy; Lyerly, H Kim

2012-06-01

Although cellular immunotherapy based on autolgous dendritic cells (DCs) targeting antigens expressed by metastatic cancer has demonstrated clinical efficacy, the logistical challenges in generating an individualized cell product create an imperative to develop alternatives to DC-based cancer vaccines. Particularly attractive alternatives include in situ delivery of antigen and activation signals to resident antigen-presenting cells (APCs), which can be achieved by novel fusion molecules targeting the mannose receptor and by recombinant viral vectors expressing the antigen of interest and capable of infecting DCs. A particular challenge in the use of viral vectors is the well-appreciated clinical obstacles to their efficacy, specifically vector-specific neutralizing immune responses. Because heterologous prime and boost strategies have been demonstrated to be particularly potent, we developed two novel recombinant vectors based on alphaviral replicon particles and a next-generation adenovirus encoding an antigen commonly overexpressed in many human cancers, carcinoembryonic antigen (CEA). The rationale for developing these vectors, their unique characteristics, the preclinical studies and early clinical experience with each, and opportunities to enhance their effectiveness will be reviewed. The potential of each of these potent recombinant vectors to efficiently generate clinically active anti-tumor immune response alone, or in combination, will be discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

Mercury Retrograde Effect in Capital Markets: Truth or Illusion?

Directory of Open Access Journals (Sweden)

Murgea Aurora

2016-06-01

Full Text Available From the most ancient times, the astrological beliefs have played an important role in human history, thinking, world-views, language and other elements of social culture. The practice of relating the movement of celestial bodies to events in financial markets is relatively newer but despite the inconsistency between financial astrology and standard economic or financial theory, it seems to be largely spread among capital market traders. This paper evaluates one of the astrological effects on the capital market, more precisely the Mercury retrograde effect on US capital market. Despite the fact that it is just an optical illusion the astrological tradition says that Mercury retrograde periods are characterized by confusion and miscommunications. The trades could be less effective, the individuals more prone to make mistakes so there is a long-held belief that it is better to avoid set plans during Mercury retrograde, signing contracts, starting new ventures or open new stock market positions. The main findings of this study are lower return’s volatilities in the Mercury retrograde periods, inconsistent with the astrologic theories assumptions but consistent with the idea that trader’s beliefs in Mercury retrograde effect could change the market volatility exactly in the opposite sense than the predicted one.

A sight on the current nanoparticle-based gene delivery vectors

Science.gov (United States)

Dizaj, Solmaz Maleki; Jafari, Samira; Khosroushahi, Ahmad Yari

2014-05-01

Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/ in vivo transformation efficiency.

Increased sinusoidal volume and solute extraction during retrograde liver perfusion

International Nuclear Information System (INIS)

Bass, N.M.; Manning, J.A.; Weisiger, R.A.

1989-01-01

Retrograde isolated liver perfusion has been used to probe acinar functional heterogeneity, but the hemodynamic effects of backward flow have not been characterized. In this study, extraction of a long-chain fatty acid derivative, 12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazol-amino stearate (12-NBDS), was greater during retrograde than during anterograde perfusion of isolated rat liver. To determine whether hemodynamic differences between anterograde and retrograde perfused livers could account for this finding, the hepatic extracellular space was measured for both directions of flow by means of [ 14 C]sucrose washout during perfusion as well as by direct measurement of [ 14 C]sucrose entrapped during perfusion. A three- to fourfold enlargement of the total hepatic extracellular space was found during retrograde perfusion by both approaches. Examination of perfusion-fixed livers by light microscopy and morphometry revealed that marked distension of the sinusoids occurred during retrograde perfusion and that this accounts for the observed increase in the [ 14 C]sucrose space. These findings support the hypothesis that maximum resistance to perfusate flow in the isolated perfused rat liver is located at the presinusoidal level. In addition, increased transit time of perfusate through the liver and greater sinusoidal surface area resulting from sinusoidal distension may account for the higher extraction of 12-NBDS and possibly other compounds by retrograde perfused liver

Kinematics of turnaround and retrograde axonal transport

International Nuclear Information System (INIS)

Snyder, R.E.

1986-01-01

Rapid axonal transport of a pulse of 35 S-methionine-labelled material was studied in vitro in the sensory neurons of amphibian sciatic nerve using a position-sensitive detector. For 10 nerves studied at 23.0 +/- 0.2 degrees C it was found that a pulse moved in the anterograde direction characterized by front edge, peak, and trailing edge transport rates of (mm/d) 180.8 +/- 2.2 (+/- SEM), 176.6 +/- 2.3, and 153.7 +/- 3.0, respectively. Following its arrival at a distal ligature, a smaller pulse was observed to move in the retrograde direction characterized by front edge and peak transport rates of 158.0 +/- 7.3 and 110.3 +/- 3.5, respectively, indicating that retrograde transport proceeds at a rate of 0.88 +/- 0.04 that of anterograde. The retrograde pulse was observed to disperse at a rate greater than the anterograde. Reversal of radiolabel at the distal ligature began 1.49 +/- 0.15 h following arrival of the first radiolabel. Considerable variation was seen between preparations in the way radiolabel accumulated in the end (ligature) regions of the nerve. Although a retrograde pulse was seen in all preparations, in 7 of 10 preparations there was no evidence of this pulse accumulating within less than 2-3 mm of a proximal ligature; however, accumulation was observed within less than 5 mm in all preparations

CRISPR/Cas9-mediated viral interference in plants

KAUST Repository

Ali, Zahir

2015-11-11

Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

Diseño y construcción de vectores de transferencia para la obtención de virus vaccinia Ankara modificado (MVA recombinantes Design and construction of transfer vectors in order to obtain recombinant modified vaccinia virus Ankara (MVA

Directory of Open Access Journals (Sweden)

M. F. Ferrer

2007-09-01

Full Text Available El virus vaccinia Ankara modificado (MVA constituye un buen candidato para el desarrollo de vectores virales de expresión no replicativos porque no replica en la mayoría de las células de mamíferos. Para la producción de MVA recombinantes es fundamental disponer de vectores de transferencia que, por recombinación homóloga con el genoma viral, permitan introducir los genes de interés en regiones no esenciales para la replicación in vitro. En este trabajo se diseñaron y obtuvieron los vectores de transferencia denominados VT-MHA y VT-MTK que portan las regiones correspondientes a las posiciones 1-303 y 608-948 del gen MVA165R y 1-244 y 325-534 del gen MVA086R, respectivamente, las que flanquean un sitio de clonado múltiple para la inserción de los genes foráneos. En dichos vectores se clonaron los casetes para la expresión de los genes lac Z o uid A, y la actividad de las enzimas marcadoras b-galactosidasa y b-glucuronidasa se confirmó in situ. Además, utilizando el vector denominado VT-MTK-GUS, se obtuvieron y aislaron MVA recombinantes puros que portan y expresan el gen uid A. Los resultados obtenidos constituyen las herramientas básicas para establecer la metodología de obtención de MVA recombinantes, con el propósito de desarrollar localmente vectores virales no replicativos candidatos a vacunas.Modified Vaccinia virus Ankara (MVA constitutes a good candidate for the development of non-replicative expression viral vectors because it does not replicate in most of mammalian cells. It is essential, for the production of recombinant MVA, the availability of transfer vectors which allow the introduction of desired genes into non-essential regions for in vitro viral replication, by homologous recombination with the viral genome. In the present work, the transfer vectors named VT-MHA and VT-MTK were designed and obtained. They carried genomic regions corresponding to 1- 303 and 608-948 positions of the MVA165R gene and 1-244 and

A retrograde co-orbital asteroid of Jupiter.

Science.gov (United States)

Wiegert, Paul; Connors, Martin; Veillet, Christian

2017-03-29

Recent theoretical work in celestial mechanics has revealed that an asteroid may orbit stably in the same region as a planet, despite revolving around the Sun in the sense opposite to that of the planet itself. Asteroid 2015 BZ 509 was discovered in 2015, but with too much uncertainty in its measured orbit to establish whether it was such a retrograde co-orbital body. Here we report observations and analysis that demonstrates that asteroid 2015 BZ 509 is indeed a retrograde co-orbital asteroid of the planet Jupiter. We find that 2015 BZ 509 has long-term stability, having been in its current, resonant state for around a million years. This is long enough to preclude precise calculation of the time or mechanism of its injection to its present state, but it may be a Halley-family comet that entered the resonance through an interaction with Saturn. Retrograde co-orbital asteroids of Jupiter and other planets may be more common than previously expected.

Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes.

Science.gov (United States)

Iizuka, Shunsuke; Sakurai, Fuminori; Tachibana, Masashi; Ohashi, Kazuo; Mizuguchi, Hiroyuki

2017-09-15

Gene therapy during neonatal and infant stages is a promising approach for hemophilia B, a congenital disorder caused by deficiency of blood coagulation factor IX (FIX). An adenovirus (Ad) vector has high potential for use in neonatal or infant gene therapy for hemophilia B due to its superior transduction properties; however, leaky expression of Ad genes often reduces the transduction efficiencies by Ad protein-mediated tissue damage. Here, we used a novel Ad vector, Ad-E4-122aT, which exhibits a reduction in the leaky expression of Ad genes in liver, in gene therapy studies for neonatal hemophilia B mice. Ad-E4-122aT exhibited significantly higher transduction efficiencies than a conventional Ad vector in neonatal mice. In neonatal hemophilia B mice, a single neonatal injection of Ad-E4-122aT expressing human FIX (hFIX) (Ad-E4-122aT-AHAFIX) maintained more than 6% of the normal plasma hFIX activity levels for approximately 100 days. Sequential administration of Ad-E4-122aT-AHAFIX resulted in more than 100% of the plasma hFIX activity levels for more than 100 days and rescued the bleeding phenotypes of hemophilia B mice. In addition, immunotolerance to hFIX was induced by Ad-E4-122aT-AHAFIX administration in neonatal hemophilia B mice. These results indicated that Ad-E4-122aT is a promising gene delivery vector for neonatal or infant gene therapy for hemophilia B.

RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

International Nuclear Information System (INIS)

Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

2006-01-01

RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

A set of vectors for introduction of antibiotic resistance genes by in vitro Cre-mediated recombination

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Vassetzky Yegor S

2008-12-01

Full Text Available Abstract Background Introduction of new antibiotic resistance genes in the plasmids of interest is a frequent task in molecular cloning practice. Classical approaches involving digestion with restriction endonucleases and ligation are time-consuming. Findings We have created a set of insertion vectors (pINS carrying genes that provide resistance to various antibiotics (puromycin, blasticidin and G418 and containing a loxP site. Each vector (pINS-Puro, pINS-Blast or pINS-Neo contains either a chloramphenicol or a kanamycin resistance gene and is unable to replicate in most E. coli strains as it contains a conditional R6Kγ replication origin. Introduction of the antibiotic resistance genes into the vector of interest is achieved by Cre-mediated recombination between the replication-incompetent pINS and a replication-competent target vector. The recombination mix is then transformed into E. coli and selected by the resistance marker (kanamycin or chloramphenicol present in pINS, which allows to recover the recombinant plasmids with 100% efficiency. Conclusion Here we propose a simple strategy that allows to introduce various antibiotic-resistance genes into any plasmid containing a replication origin, an ampicillin resistance gene and a loxP site.

Transcriptional Silencing of Retroviral Vectors

DEFF Research Database (Denmark)

Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

1996-01-01

. Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t......RNA primer for reverse transcription may have a major influence on transcriptional silencing. Alterations of these elements of the vector backbone as well as the use of internal promoter elements from housekeeping genes may contribute to reduce transcriptional silencing. The use of cell culture and animal...

The loss of episodic memories in retrograde amnesia: single-case and group studies.

OpenAIRE

Kopelman, M D; Kapur, N

2001-01-01

Retrograde amnesia in neurological disorders is a perplexing and fascinating research topic. The severity of retrograde amnesia is not well correlated with that of anterograde amnesia, and there can be disproportionate impairments of either. Within retrograde amnesia, there are various dissociations which have been claimed-for example, between the more autobiographical (episodic) and more semantic components of memory. However, the associations of different types of retrograde amnesia are als...

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

Science.gov (United States)

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

2015-01-01

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

Retrograde Transvenous Ethanol Embolization of High-flow Peripheral Arteriovenous Malformations

International Nuclear Information System (INIS)

Linden, Edwin van der; Baalen, Jary M. van; Pattynama, Peter M. T.

2012-01-01

Purpose: To report the clinical efficiency and complications in patients treated with retrograde transvenous ethanol embolization of high-flow peripheral arteriovenous malformations (AVMs). Retrograde transvenous ethanol embolization of high-flow AVMs is a technique that can be used to treat AVMs with a dominant outflow vein whenever conventional interventional procedures have proved insufficient. Methods: This is a retrospective study of the clinical effectiveness and complications of retrograde embolization in five patients who had previously undergone multiple arterial embolization procedures without clinical success. Results: Clinical outcomes were good in all patients but were achieved at the cost of serious, although transient, complications in three patients. Conclusion: Retrograde transvenous ethanol embolization is a highly effective therapy for high-flow AVMs. However, because of the high complication rate, it should be reserved as a last resort, to be used after conventional treatment options have failed.

The “SAFARI” Technique Using Retrograde Access Via Peroneal Artery Access

International Nuclear Information System (INIS)

Zhuang, Kun Da; Tan, Seck Guan; Tay, Kiang Hiong

2012-01-01

The “SAFARI” technique or subintimal arterial flossing with antegrade–retrograde intervention is a method for recanalisation of chronic total occlusions (CTOs) when subintimal angioplasty fails. Retrograde access is usually obtained via the popliteal, distal anterior tibial artery (ATA)/dorsalis pedis (DP), or distal posterior tibial artery (PTA). Distal access via the peroneal artery has not been described and has a risk of continued bleeding, leading to compartment syndrome due to its deep location. We describe our experience in two patients with retrograde access via the peroneal artery and the use of balloon-assisted hemostasis for these retrograde punctures. This approach may potentially give more options for endovascular interventions in lower limb CTOs.

Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

Directory of Open Access Journals (Sweden)

Balaji Balakrishnan

Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

Vaccines for viral and parasitic diseases produced with baculovirus vectors

NARCIS (Netherlands)

Oers, van M.M.

2006-01-01

The baculovirus¿insect cell expression system is an approved system for the production of viral antigens with vaccine potential for humans and animals and has been used for production of subunit vaccines against parasitic diseases as well. Many candidate subunit vaccines have been expressed in this

Brown dwarfs in retrogradely precessing cataclysmic variables?

Directory of Open Access Journals (Sweden)

Martin E.L.

2011-07-01

Full Text Available We compare Smoothed Particle Hydrodynamic simulations of retrogradely precessing accretion disks that have a white dwarf primary and a main sequence secondary with observational data and with theory on retrograde precession via tidal torques like those by the Moon and the Sun on the Earth [1, 2]. Assuming the primary does not accrete much of the mass lost from the secondary, we identify the theoretical low mass star/brown dwarf boundary. We find no observational candidates in our study that could qualify as brown dwarfs.

Fluoroscopically guided pyeloureteral interventions using a retrograde perurethral approach

International Nuclear Information System (INIS)

Amendola, M.A.; Banner, M.P.; Pollack, H.M.; Gordon, R.L.; Van Arsdalen, K.N.

1987-01-01

Employing standard interventional equipment, fluoroscopy, and partially or completely inserted ureteral catheters for access, the authors performed 168 perurethral interventional procedures since 1985. Procedures have included insertion of double (n = 42) or single pigtail stents (n = 47), advancement of retrograde ureteral catheters with or without displacement of a ureteral stone to the renal pelvis (n = 42), urothelial biopsy (n = 30), balloon dilation of ureteral structures (n = 3), ureteral stone extraction (n = 1), and conversion of retrograde to antegrade catheters for balloon dilation of ureteropelvic junction strictures (n = 3). This retrograde approach often obviates the need for antegrade interventional procedures (including percutaneous nephrostomy and ureteral stenting), ureteroscopy, or surgery. Indications, techniques, pitfalls, and complications are illustrated

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

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Vidya P Nair

2016-04-01

Full Text Available Hepatitis E virus (HEV causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4. Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp, X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1 and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient

Porcine semen as a vector for transmission of viral pathogens.

Science.gov (United States)

Maes, Dominiek; Van Soom, Ann; Appeltant, Ruth; Arsenakis, Ioannis; Nauwynck, Hans

2016-01-01

Different viruses have been detected in porcine semen. Some of them are on the list of the World Organization for Animal Health (OIE), and consequently, these pathogens are of socioeconomic and/or public health importance and are of major importance in the international trade of animals and animal products. Artificial insemination (AI) is one of the most commonly used assisted reproductive technologies in pig production worldwide. This extensive use has enabled pig producers to benefit from superior genetics at a lower cost compared to natural breeding. However, the broad distribution of processed semen doses for field AI has increased the risk of widespread transmission of swine viral pathogens. Contamination of semen can be due to infections of the boar or can occur during semen collection, processing, and storage. It can result in reduced semen quality, embryonic mortality, endometritis, and systemic infection and/or disease in the recipient female. The presence of viral pathogens in semen can be assessed by demonstration of viable virus, nucleic acid of virus, or indirectly by measuring serum antibodies in the boar. The best way to prevent disease transmission via the semen is to assure that the boars in AI centers are free from the disease, to enforce very strict biosecurity protocols, and to perform routine health monitoring of boars. Prevention of viral semen contamination should be the primary focus because it is easier to prevent contamination than to eliminate viruses once present in semen. Nevertheless, research and development of novel semen processing treatments such as single-layer centrifugation is ongoing and may allow in the future to decontaminate semen. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhancing poxvirus vectors vaccine immunogenicity.

Science.gov (United States)

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.

Prostatic urethra malformation associated with retrograde ejaculation: a case report.

Science.gov (United States)

Zhao, Kai; Zhang, Jianzhong; Xu, Aiming; Zhang, Cheng; Wang, Zengjun

2016-12-21

Retrograde ejaculation can have anatomical, neurogenic, or pharmacological causes. Among these factors, malformation of the prostatic urethra is an uncommon cause. We describe a 29-year-old Han Chinese man with absence of his verumontanum combined with ejaculatory duct cysts, and no other cause for ejaculatory dysfunction. His verumontanum was replaced by a deep groove adjacent to his bladder neck, which could significantly influence bladder neck contraction. In addition, the large cysts in the ejaculatory duct could obstruct the anterior outlet of his prostatic urethra and prevent seminal fluid flow in an anterograde direction. There are few reports of retrograde ejaculation associated with congenital malformations of the posterior urethra. Malformations associated with bladder neck laxity and increased tone of the prostatic urethral outlet can contribute to retrograde ejaculation. Malformation of the prostatic urethra is an uncommon cause of retrograde ejaculation, and can be difficult to treat.

Retrograde Melting and Internal Liquid Gettering in Silicon

Energy Technology Data Exchange (ETDEWEB)

Hudelson, Steve; Newman, Bonna K.; Bernardis, Sarah; Fenning, David P.; Bertoni, Mariana I.; Marcus, Matthew A.; Fakra, Sirine C.; Lai, Barry; Buonassisi, Tonio

2011-07-01

Retrograde melting (melting upon cooling) is observed in silicon doped with 3d transition metals, via synchrotron-based temperature-dependent X-ray microprobe measurements. Liquid metal-silicon droplets formed via retrograde melting act as efficient sinks for metal impurities dissolved within the silicon matrix. Cooling results in decomposition of the homogeneous liquid phase into solid multiple-metal alloy precipitates. These phenomena represent a novel pathway for engineering impurities in semiconductor-based systems.

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

Science.gov (United States)

Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

2014-11-01

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Dynein is the motor for retrograde axonal transport of organelles

International Nuclear Information System (INIS)

Schnapp, B.J.; Reese, T.S.

1989-01-01

Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport

Disruption of Ethylene Responses by Turnip mosaic virus Mediates Suppression of Plant Defense against the Green Peach Aphid Vector.

Science.gov (United States)

Casteel, Clare L; De Alwis, Manori; Bak, Aurélie; Dong, Haili; Whitham, Steven A; Jander, Georg

2015-09-01

Plants employ diverse responses mediated by phytohormones to defend themselves against pathogens and herbivores. Adapted pathogens and herbivores often manipulate these responses to their benefit. Previously, we demonstrated that Turnip mosaic virus (TuMV) infection suppresses callose deposition, an important plant defense induced in response to feeding by its aphid vector, the green peach aphid (Myzus persicae), and increases aphid fecundity compared with uninfected control plants. Further, we determined that production of a single TuMV protein, Nuclear Inclusion a-Protease (NIa-Pro) domain, was responsible for changes in host plant physiology and increased green peach aphid reproduction. To characterize the underlying molecular mechanisms of this phenomenon, we examined the role of three phytohormone signaling pathways, jasmonic acid, salicylic acid, and ethylene (ET), in TuMV-infected Arabidopsis (Arabidopsis thaliana), with or without aphid herbivory. Experiments with Arabidopsis mutants ethylene insensitive2 and ethylene response1, and chemical inhibitors of ET synthesis and perception (aminoethoxyvinyl-glycine and 1-methylcyclopropene, respectively), show that the ET signaling pathway is required for TuMV-mediated suppression of Arabidopsis resistance to the green peach aphid. Additionally, transgenic expression of NIa-Pro in Arabidopsis alters ET responses and suppresses aphid-induced callose formation in an ET-dependent manner. Thus, disruption of ET responses in plants is an additional function of NIa-Pro, a highly conserved potyvirus protein. Virus-induced changes in ET responses may mediate vector-plant interactions more broadly and thus represent a conserved mechanism for increasing transmission by insect vectors across generations. © 2015 American Society of Plant Biologists. All Rights Reserved.

The Influence of Green Viral Communications on Green Purchase Intentions: The Mediating Role of Consumers’ Susceptibility to Interpersonal Influences

Directory of Open Access Journals (Sweden)

Sheng-Hsiung Chang

2015-04-01

Full Text Available This paper aims to incorporate the diffusion of innovation theory and conformity theory to explain consumers’ green purchase intentions. To this end, a conceptual model has been proposed and subjected to empirical verification with the use of a survey method. Using a sample of Taiwanese consumers who had the actual purchase experience of green detergents, this study employed structural equation modeling to verify the hypothesis proposed. The empirical results suggested that green viral communication was positively related to normative interpersonal influence, informational interpersonal influence and green purchase intention. Informational interpersonal influence also had a positive impact on green purchase intention. However, the relationship between consumer’s normative interpersonal influence and green purchase intention was not supported. Thus, this study concludes that green marketers must strengthen their green viral communications skills to enhance consumers’ purchase intentions. In addition, this study also contributes to the literature by stating that consumers’ susceptibility to informational interpersonal relationships is an important mediator in the green viral communication and green purchase intentions relationship. This study discusses implications of the findings and research limitations at the end of the paper.

The formation of retrograde planetary orbits by close stellar encounters

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Ford E. B.

2011-02-01

Full Text Available We consider the growing number of observations of the RossiterMcLaughlin effect in transiting planets, which seem to suggest that ~30% of transiting planets are in highly inclined or retrograde orbits. We consider the dense cluster environment in which stars are born and investigate whether perturbations from passing stars can drive planetary systems into retrograde configurations. We find that fly-bys can result in significantly more inclination excitation than might naively be expected from impulse approximations, leading to several percent of stellar systems possessing planets in retrograde orbits.

Using Kinesthetic Activities to Teach Ptolemaic and Copernican Retrograde Motion

Science.gov (United States)

Richards, Ted

2012-06-01

This paper describes a method for teaching planetary retrograde motion, and the Ptolemaic and Copernican accounts of retrograde motion, by means of a series kinesthetic learning activities (KLAs). In the KLAs described, the students literally walk through the motions of the planets in both systems. A retrospective statistical analysis shows that students who participated in these activities performed better on examination questions pertaining to retrograde motion than students who did not. Potential explanations for this result, including the breaking of classroom routine, the effect of body movement on conceptual memory, and egocentric spatial proprioception, are considered.

Colonic perforation following endoscopic retrograde ...

African Journals Online (AJOL)

We highlight a potentially lethal complication of acute severe pancreatitis that may not be suspected in severely ill patients. A 41-year-old woman developed acute severe pancreatitis following endoscopic retrograde cholangiopancreatography (ERCP) for suspected choledocholithiasis. When her condition deteriorated ...

Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.

Science.gov (United States)

Huo, Wenying; Zhao, Guannan; Yin, Jinggang; Ouyang, Xuan; Wang, Yinan; Yang, Chuanhe; Wang, Baojing; Dong, Peixin; Wang, Zhixiang; Watari, Hidemichi; Chaum, Edward; Pfeffer, Lawrence M; Yue, Junming

2017-01-01

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

Evaluating the dosimetric effect of treatment-induced changes in virally mediated head and neck cancer patients

International Nuclear Information System (INIS)

Brown, Elizabeth; Owen, Rebecca; Mengersen, Kerrie; Harden, Fiona; Porceddu, Sandro

2013-01-01

Patients with virally mediated head and neck cancer (VMHNC) often present with advanced nodal disease that is highly radioresponsive as demonstrated by tumour and nodal regression during treatment. The resultant changes may impact on the planned dose distribution and so adversely affect the therapeutic ratio. The aim of this study was to evaluate the dosimetric effect of treatment-induced anatomical changes in VMHNC patients who had undergone a replan. Thirteen patients with virally mediated oropharyngeal or nasopharyngeal cancer who presented for definitive radiotherapy between 2005 and 2010 and who had a replan generated were investigated. The dosimetric effect of anatomical changes was quantified by comparing dose–volume histograms (DVH) of primary and nodal gross target volumes and organs at risk (OAR), including spinal cord and parotid glands, from the original plan and a comparison plan. Eleven three-dimensional conformal radiation therapy (3DCRT) and two intensity modulated radiation therapy (IMRT) plans were evaluated. Dose to the spinal cord and brainstem increased by 4.1% and 2.6%, respectively. Mean dose to the parotid glands also increased by 3.5%. In contrast, the dose received by 98% of the primary and nodal gross tumour volumes decreased by 0.15% and 0.3%, respectively, when comparing the initial treatment plan to the comparison plan. In this study, treatment-induced anatomical changes had the greatest impact on OAR dose with negligible effect on the dose to nodal gross tumour volumes. In the era of IMRT, accounting for treatment-induced anatomical changes is important as focus is placed on minimizing the acute and long-term side effects of treatment

Reverse transcriptase directs viral evolution in a deep ocean methane seep

Science.gov (United States)

Paul, B. G.; Bagby, S. C.

2013-12-01

Deep ocean methane seeps are sites of intense microbial activity, with complex communities fueled by aerobic and anaerobic methanotrophy. Methane consumption in these communities has a substantial impact on the global carbon cycle, yet little is known about their evolutionary history or their likely evolutionary trajectories in a warming ocean. As in other marine systems, viral predation and virally mediated horizontal gene transfer are expected to be major drivers of evolutionary change in these communities; however, the host cells' resistance to cultivation has impeded direct study of the viral population. We conducted a metagenomic study of viruses in the anoxic sediments of a deep methane seep in the Santa Monica Basin in the Southern California Bight. We retrieved 1660 partial viral genomes, tentatively assigning 1232 to bacterial hosts and 428 to archaea. One abundant viral genome, likely hosted by Clostridia species present in the sediment, was found to encode a diversity-generating retroelement (DGR), a module for reverse transcriptase-mediated directed mutagenesis of a distal tail fiber protein. While DGRs have previously been described in the viruses of human pathogens, where diversification of viral tail fibers permits infection of a range of host cell types, to our knowledge this is the first description of such an element in a marine virus. By providing a mechanism for massively broadening potential host range, the presence of DGRs in these systems may have a major impact on the prevalence of virally mediated horizontal gene transfer, and even on the phylogenetic distances across which genes are moved.

Mal de Río Cuarto Virus Infection Triggers the Production of Distinctive Viral-Derived siRNA Profiles in Wheat and Its Planthopper Vector.

Science.gov (United States)

de Haro, Luis A; Dumón, Analía D; Mattio, María F; Argüello Caro, Evangelina Beatriz; Llauger, Gabriela; Zavallo, Diego; Blanc, Hervé; Mongelli, Vanesa C; Truol, Graciela; Saleh, María-Carla; Asurmendi, Sebastián; Del Vas, Mariana

2017-01-01

Plant reoviruses are able to multiply in gramineae plants and delphacid vectors encountering different defense strategies with unique features. This study aims to comparatively assess alterations of small RNA (sRNA) populations in both hosts upon virus infection. For this purpose, we characterized the sRNA profiles of wheat and planthopper vectors infected by Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae ) and quantified virus genome segments by quantitative reverse transcription PCR We provide evidence that plant and insect silencing machineries differentially recognize the viral genome, thus giving rise to distinct profiles of virus-derived small interfering RNAs (vsiRNAs). In plants, most of the virus genome segments were targeted preferentially within their upstream sequences and vsiRNAs mapped with higher density to the smaller genome segments than to the medium or larger ones. This tendency, however, was not observed in insects. In both hosts, vsiRNAs were equally derived from sense and antisense RNA strands and the differences in vsiRNAs accumulation did not correlate with mRNAs accumulation. We also established that the piwi-interacting RNA (piRNA) pathway was active in the delphacid vector but, contrary to what is observed in virus-infected mosquitoes, virus-specific piRNAs were not detected. This work contributes to the understanding of the silencing response in insect and plant hosts.

Endoscopic retrograde cholangiopancreatography and endoscopic ...

African Journals Online (AJOL)

An approach to suspected gallstone pancreatitis'based on endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic sphincterotomy (ES) was adopted in 1976 and was followed in 29 patients. ERCp became the routine method of early biliary tract assessment when gallstone pancreatitis was suspected on ...

Replication-competent human adenovirus 11p vectors can propagate in Vero cells

International Nuclear Information System (INIS)

Gokumakulapalle, Madhuri; Mei, Ya-Fang

2016-01-01

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

Replication-competent human adenovirus 11p vectors can propagate in Vero cells

Energy Technology Data Exchange (ETDEWEB)

Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

2016-08-15

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

Mast cells in viral infections

Directory of Open Access Journals (Sweden)

Piotr Witczak

2012-04-01

Full Text Available  There are some premises suggesting that mast cells are involved in the mechanisms of anti-virus defense and in viral disease pathomechanisms. Mast cells are particularly numerous at the portals of infections and thus may have immediate and easy contact with the external environment and invading pathogens. These cells express receptors responsible for recognition of virus-derived PAMP molecules, mainly Toll-like receptors (TLR3, TLR7/8 and TLR9, but also RIG-I-like and NOD-like molecules. Furthermore, mast cells generate various mediators, cytokines and chemokines which modulate the intensity of inflammation and regulate the course of innate and adaptive anti-viral immunity. Indirect evidence for the role of mast cells in viral infections is also provided by clinical observations and results of animal studies. Currently, more and more data indicate that mast cells can be infected by some viruses (dengue virus, adenoviruses, hantaviruses, cytomegaloviruses, reoviruses, HIV-1 virus. It is also demonstrated that mast cells can release pre formed mediators as well as synthesize de novo eicosanoids in response to stimulation by viruses. Several data indicate that virus-stimulated mast cells secrete cytokines and chemokines, including interferons as well as chemokines with a key role in NK and Tc lymphocyte influx. Moreover, some information indicates that mast cell stimulation via TLR3, TLR7/8 and TLR9 can affect their adhesion to extracellular matrix proteins and chemotaxis, and influence expression of some membrane molecules. Critical analysis of current data leads to the conclusion that it is not yet possible to make definitive statements about the role of mast cells in innate and acquired defense mechanisms developing in the course of viral infection and/or pathomechanisms of viral diseases.

Importance of variants in cerebrovascular anatomy for potential retrograde embolization in cryptogenic stroke

International Nuclear Information System (INIS)

Markl, Michael; Semaan, Edouard; Carr, James; Collins, Jeremy; Stromberg, LeRoy; Prabhakaran, Shyam

2017-01-01

To test the hypothesis that variants in cerebrovascular anatomy will affect the number of patients demonstrating a plausible retrograde embolization mechanism from plaques in the descending aorta (DAo). Thirty-five patients (aged 63 ± 17 years) with cryptogenic stroke underwent 4D flow MRI for the assessment of aortic 3D blood flow and MR angiography for the evaluation of circle of Willis, posterior circulation, and aortic arch architecture. In patients with proven DAo plaque, retrograde embolization was considered a potential mechanism if retrograde flow extended from the DAo to a supra-aortic vessel supplying the cerebral infarct territory. Retrograde embolization with matching cerebral infarct territory was detected in six (17%) patients. Circle of Willis and aortic arch variant anatomy was found in 60% of patients, leading to reclassification of retrograde embolization risk as present in three (9%) additional patients, for a total 26% of cryptogenic stroke patients. 4D flow MRI demonstrated 26% concordance with infarct location on imaging with retrograde diastolic flow into the feeding vessels of the affected cerebral area, identifying a potential etiology for cryptogenic stroke. Our findings further demonstrate the importance of cerebrovascular anatomy when determining concordance of retrograde flow pathways with vascular stroke territory from DAo plaques. (orig.)

Importance of variants in cerebrovascular anatomy for potential retrograde embolization in cryptogenic stroke

Energy Technology Data Exchange (ETDEWEB)

Markl, Michael [Northwestern University, Department of Radiology, Feinberg School of Medicine, Chicago, IL (United States); Northwestern University, Department of Biomedical Engineering, McCormick School of Engineering, Chicago, IL (United States); Semaan, Edouard; Carr, James; Collins, Jeremy [Northwestern University, Department of Radiology, Feinberg School of Medicine, Chicago, IL (United States); Stromberg, LeRoy [Northwestern University, Department of Neurology, Feinberg School of Medicine, Chicago, IL (United States); Edward Hospital, Department of Radiology, Naperville, IL (United States); Prabhakaran, Shyam [Northwestern University, Department of Neurology, Feinberg School of Medicine, Chicago, IL (United States)

2017-10-15

To test the hypothesis that variants in cerebrovascular anatomy will affect the number of patients demonstrating a plausible retrograde embolization mechanism from plaques in the descending aorta (DAo). Thirty-five patients (aged 63 ± 17 years) with cryptogenic stroke underwent 4D flow MRI for the assessment of aortic 3D blood flow and MR angiography for the evaluation of circle of Willis, posterior circulation, and aortic arch architecture. In patients with proven DAo plaque, retrograde embolization was considered a potential mechanism if retrograde flow extended from the DAo to a supra-aortic vessel supplying the cerebral infarct territory. Retrograde embolization with matching cerebral infarct territory was detected in six (17%) patients. Circle of Willis and aortic arch variant anatomy was found in 60% of patients, leading to reclassification of retrograde embolization risk as present in three (9%) additional patients, for a total 26% of cryptogenic stroke patients. 4D flow MRI demonstrated 26% concordance with infarct location on imaging with retrograde diastolic flow into the feeding vessels of the affected cerebral area, identifying a potential etiology for cryptogenic stroke. Our findings further demonstrate the importance of cerebrovascular anatomy when determining concordance of retrograde flow pathways with vascular stroke territory from DAo plaques. (orig.)

Synthesis, and Characterization, and Evaluation of Cellular Effects of the FOL-PEG-g-PEI-GAL Nanoparticles as a Potential Non-Viral Vector for Gene Delivery

International Nuclear Information System (INIS)

Ghiamkazemi, S.; Amanzadeh, A.; Dinarvand, R.; Rafiee-Tehrani, M.; Amini, M.; Ghiamkazemi, S.; Dinarvand, R.; Rafiee-Tehrani, M.; Ghiamkazemi, S.; Rafiee-Tehrani, M.; Amanzadeh, A.; Rafiee-Tehrani, M.

2010-01-01

In this manuscript, we synthesized the potential non viral vector for gene delivery with proper transfection efficiency and low cytotoxicity. Polyethylenimine (PEI) is a well-known cationic polymer which has high positive surface charge for condensing plasmid DNA. However; it is highly cytotoxic in many cell lines because of the high surface charge, non-biodegradability and non-biocompatibility. To enhance PEI biodegradability, the graft copolymer PEG-g-PEI was synthesized. To target cancer liver cells, two targeting ligands folic acid and galactose (lactobionic acid) which are over expressed on human hepatocyte carcinoma were attached to graft copolymer and FOL-PEG-g-PEI-GAL copolymer was synthesized. Composition of this grafted copolymer was characterized using 1H-NMR and FTIR spectra. The molecular weight and zeta potential of this copolymer was compared to PEI. The particle size and zeta potential of FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratio were measured using dynamic light scattering (DLS). Cytotoxicity of the copolymer was also studied in cultured HepG2 human hepatoblastoma cell line. The FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratios exhibited no cytotoxicity in HepG2 cell line compared to PEI 25K as a control. The novel copolymer showed enhanced biodegradability in physiological conditions in compared with PEI and targeted cultured HepG2 cells. More importantly, significant transfection efficiency was exhibited in cancer liver cells. Together, our results showed that FOL-PEG-g-PEI-GAL nanoparticles could be considered as a useful non-viral vector for targeted gene delivery.

Selective retrograde labeling of cholinergic neurons with [3H]choline

International Nuclear Information System (INIS)

Bagnoli, P.; Beaudet, A.; Stella, M.; Cuenod, M.

1981-01-01

Evidence is presented which is consistent with a specific retrograde labeling of cholinergic neurons following [ 3 H]choline application in their zone of termination. [ 3 H]Choline injection in the rat hippocampus leads to perikaryal retrograde labeling in the ipsilateral medial septal nuclease and nucleus of the diagonal band, thus delineating an established cholinergic pathway, while only diffuse presumably anterograde labeling was observed in the lateral septum, the entorhinal cortex, and the opposite hippocampus. After [ 3 H]choline injection in the pigeon visual Wulst, only the ipsilateral thalamic relay, of all inputs, showed similar perikaryal retrograde labeling, an observation supporting the suggestion that at least some thalamo-Wulst neurons are cholinergic

Using Kinesthetic Activities to Teach Ptolemaic and Copernican Retrograde Motion

Science.gov (United States)

Richards, Ted

2012-01-01

This paper describes a method for teaching planetary retrograde motion, and the Ptolemaic and Copernican accounts of retrograde motion, by means of a series kinesthetic learning activities (KLAs). In the KLAs described, the students literally walk through the motions of the planets in both systems. A retrospective statistical analysis shows that…

A Healthy Live Birth Following ICSI with Retrograde Ejaculated Sperm

African Journals Online (AJOL)

AJRH Managing Editor

Retrograde ejaculation, sometimes called dry orgasm, refers to the medical condition when semen enters the urinary bladder. (retrograde) instead of emerging through the penis after orgasm (antegrade). In some instances, a very minute quantity of antegrade semen appears in the ejaculate and may or may not be devoid of ...

Differential Cotton leaf crumple virus-VIGS-mediated gene silencing and viral genome localization in different Gossypium hirsutum genetic backgrounds

KAUST Repository

Idris, Ali

2010-12-01

A Cotton leaf crumple virus (CLCrV)-based gene silencing vector containing a fragment of the Gossypium hirsutum Magnesium chelatase subunit I was used to establish endogenous gene silencing in cotton of varied genetic backgrounds. Biolistic inoculation resulted in systemic and persistent photo-bleaching of the leaves and bolls of the seven cultivars tested, however, the intensity of silencing was variable. CLCrV-VIGS-mediated expression of green fluorescent protein was used to monitor the in planta distribution of the vector, indicating successful phloem invasion in all cultivars tested. Acala SJ-1, one of the cotton cultivars, was identified as a particularly optimal candidate for CLCrV-VIGS-based cotton reverse-genetics. © 2010 Elsevier Ltd.

The 'SAFARI' Technique Using Retrograde Access Via Peroneal Artery Access

Energy Technology Data Exchange (ETDEWEB)

Zhuang, Kun Da, E-mail: zkunda@gmail.com [Singapore General Hospital, Interventional Radiology Centre (Singapore); Tan, Seck Guan [Singapore General Hospital, Department of General Surgery (Singapore); Tay, Kiang Hiong [Singapore General Hospital, Interventional Radiology Centre (Singapore)

2012-08-15

The 'SAFARI' technique or subintimal arterial flossing with antegrade-retrograde intervention is a method for recanalisation of chronic total occlusions (CTOs) when subintimal angioplasty fails. Retrograde access is usually obtained via the popliteal, distal anterior tibial artery (ATA)/dorsalis pedis (DP), or distal posterior tibial artery (PTA). Distal access via the peroneal artery has not been described and has a risk of continued bleeding, leading to compartment syndrome due to its deep location. We describe our experience in two patients with retrograde access via the peroneal artery and the use of balloon-assisted hemostasis for these retrograde punctures. This approach may potentially give more options for endovascular interventions in lower limb CTOs.

Vector choice determines immunogenicity and potency of genetic vaccines against Angola Marburg virus in nonhuman primates

NARCIS (Netherlands)

Geisbert, Thomas W.; Bailey, Michael; Geisbert, Joan B.; Asiedu, Clement; Roederer, Mario; Grazia-Pau, Maria; Custers, Jerome; Jahrling, Peter; Goudsmit, Jaap; Koup, Richard; Sullivan, Nancy J.

2010-01-01

The immunogenicity and durability of genetic vaccines are influenced by the composition of gene inserts and choice of delivery vector. DNA vectors are a promising vaccine approach showing efficacy when combined in prime-boost regimens with recombinant protein or viral vectors, but they have shown

Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP-1.

Science.gov (United States)

Matalon, Omri; Ben-Shmuel, Aviad; Kivelevitz, Jessica; Sabag, Batel; Fried, Sophia; Joseph, Noah; Noy, Elad; Biber, Guy; Barda-Saad, Mira

2018-03-01

Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between β-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems. © 2018 The Authors.

An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer.

Science.gov (United States)

Morse, Michael A; Hobeika, Amy C; Osada, Takuya; Berglund, Peter; Hubby, Bolyn; Negri, Sarah; Niedzwiecki, Donna; Devi, Gayathri R; Burnett, Bruce K; Clay, Timothy M; Smith, Jonathan; Lyerly, H Kim

2010-09-01

Therapeutic anticancer vaccines are designed to boost patients' immune responses to tumors. One approach is to use a viral vector to deliver antigen to in situ DCs, which then activate tumor-specific T cell and antibody responses. However, vector-specific neutralizing antibodies and suppressive cell populations such as Tregs remain great challenges to the efficacy of this approach. We report here that an alphavirus vector, packaged in virus-like replicon particles (VRP) and capable of efficiently infecting DCs, could be repeatedly administered to patients with metastatic cancer expressing the tumor antigen carcinoembryonic antigen (CEA) and that it overcame high titers of neutralizing antibodies and elevated Treg levels to induce clinically relevant CEA-specific T cell and antibody responses. The CEA-specific antibodies mediated antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. In addition, patients with CEA-specific T cell responses exhibited longer overall survival. These data suggest that VRP-based vectors can overcome the presence of neutralizing antibodies to break tolerance to self antigen and may be clinically useful for immunotherapy in the setting of tumor-induced immunosuppression.

Retrograde amnesia after electroconvulsive therapy: a temporary effect?

Science.gov (United States)

Meeter, Martijn; Murre, Jaap M J; Janssen, Steve M J; Birkenhager, Tom; van den Broek, W W

2011-07-01

Although electroconvulsive therapy (ECT) is generally considered effective against depression, it remains controversial because of its association with retrograde memory loss. Here, we assessed memory after ECT in circumstances most likely to yield strong retrograde amnesia. A cohort of patients undergoing ECT for major depression was tested before and after ECT, and again at 3-months follow-up. Included were 21 patients scheduled to undergo bilateral ECT for severe major depression and 135 controls matched for gender, age, education, and media consumption. Two memory tests were used: a verbal learning test to assess anterograde memory function, and a remote memory test that assessed memory for news during the course of one year. Before ECT the patients' scores were lower than those of controls. They were lower again after treatment, suggesting retrograde amnesia. At follow-up, however, memory for events before treatment had returned to the pre-ECT level. Memory for events in the months after treatment was as good as that of controls. The sample size in this study was not large. Moreover, memory impairment did not correlate with level of depression, which may be due to restriction of range. Our results are consistent with the possibility that ECT as currently practiced does not cause significant lasting retrograde amnesia, but that amnesia is mostly temporary and related to the period of impairment immediately following ECT. Copyright © 2011 Elsevier B.V. All rights reserved.

Production and purification of non replicative canine adenovirus type 2 derived vectors.

Science.gov (United States)

Szelechowski, Marion; Bergeron, Corinne; Gonzalez-Dunia, Daniel; Klonjkowski, Bernard

2013-12-03

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10(10) infectious particles per ml and can be directly administrated in vivo.

Viral vectors encoding endomorphins and serine histogranin attenuate neuropathic pain symptoms after spinal cord injury in rats.

Science.gov (United States)

Nasirinezhad, Farinaz; Gajavelli, Shyam; Priddy, Blake; Jergova, Stanislava; Zadina, James; Sagen, Jacqueline

2015-01-07

The treatment of spinal cord injury (SCI)-induced neuropathic pain presents a challenging healthcare problem. The lack of available robust pharmacological treatments underscores the need for novel therapeutic methods and approaches. Due to the complex character of neuropathic pain following SCI, therapies targeting multiple mechanisms may be a better choice for obtaining sufficient long-term pain relief. Previous studies in our lab showed analgesic effects using combinations of an NMDA antagonist peptide [Ser1]histogranin (SHG), and the mu-opioid peptides endomorphins (EMs), in several pain models. As an alternative to drug therapy, this study evaluated the analgesic potential of these peptides when delivered via gene therapy. Lentiviruses encoding SHG and EM-1 and EM-2 were intraspinally injected, either singly or in combination, into rats with clip compression SCI 2 weeks following injury. Treated animals showed significant reduction in mechanical and thermal hypersensitivity, compared to control groups injected with GFP vector only. The antinociceptive effects of individually injected components were modest, but the combination of EMs and SHG produced robust and sustained antinociception. The onset of the analgesic effects was observed between 1-5 weeks post-injection and sustained without decrement for at least 7 weeks. No adverse effects on locomotor function were observed. The involvement of SHG and EMs in the observed antinociception was confirmed by pharmacologic inhibition using intrathecal injection of either the opioid antagonist naloxone or an anti-SHG antibody. Immunohistochemical analysis showed the presence of SHG and EMs in the spinal cord of treated animals, and immunodot-blot analysis of CSF confirmed the presence of these peptides in injected animals. In a separate group of rats, delayed injection of viral vectors was performed in order to mimic a more likely clinical scenario. Comparable and sustained antinociceptive effects were observed in

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

Science.gov (United States)

Langouet-Astrie, Christophe J; Yang, Zhiyong; Polisetti, Sraavya M; Welsbie, Derek S; Hauswirth, William W; Zack, Donald J; Merbs, Shannath L; Enke, Raymond A

2016-10-01

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Optimal Timing for Laparoscopic Cholecystectomy After Endoscopic Retrograde Cholangiopancreatography: A Systematic Review.

Science.gov (United States)

Friis, C; Rothman, J P; Burcharth, J; Rosenberg, J

2018-06-01

Endoscopic retrograde cholangiopancreatography followed by laparoscopic cholecystectomy is often used as definitive treatment for common bile duct stones. The aim of this study was to investigate the optimal time interval between endoscopic retrograde cholangiopancreatography and laparoscopic cholecystectomy. PubMed and Embase were searched for studies comparing different time delays between endoscopic retrograde cholangiopancreatography and laparoscopic cholecystectomy. Observational studies and randomized controlled trials were included. Primary outcome was conversion rate from laparoscopic to open cholecystectomy and secondary outcomes were complications, mortality, operating time, and length of stay. A total of 14 studies with a total of 1930 patients were included. The pooled estimate revealed an increase from a 4.2% conversion rate when laparoscopic cholecystectomy was performed within 24 h of endoscopic retrograde cholangiopancreatography to 7.6% for 24-72 h delay to 12.3% when performed within 2 weeks, to 12.3% for 2-6 weeks, and to a 14% conversion rate when operation was delayed more than 6 weeks. According to this systematic review, it is preferable to perform cholecystectomy within 24 h of endoscopic retrograde cholangiopancreatography to reduce conversion rate. Early laparoscopic cholecystectomy does not increase mortality, perioperative complications, or length of stay and on the contrary it reduces the risk of reoccurrence and progression of disease in the delay between endoscopic retrograde cholangiopancreatography and laparoscopic cholecystectomy.

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

Science.gov (United States)

Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

2015-01-01

The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. PMID:26157166

Retrograde contrast radiography of the distal portions of the intestinal tract in foals

International Nuclear Information System (INIS)

Fischer, A.T.; Yarbrough, T.Y.

1995-01-01

A technique for retrograde contrast radiography of the distal portions of the intestinal tract of foals was developed and then performed in 25 foals (1 to 30 days old) with colic. Retrograde contrast radiography was shown to be sensitive (100%) and specific (100%) for evaluating obstruction of the small colon or transverse colon. It was slightly less sensitive (86%) and specific (83%) for evaluation of the entire large colon, particularly in older foals. Retrograde contrast radiography provided increased diagnostic capability, compared with that for noncontrast radiography. Retrograde contrast radiography can provide valuable information when evaluating foals with colic and should be part of the diagnostic evaluation

Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using an Insulated Lentiviral Vector

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Swati Singh

2017-03-01

Full Text Available Wiskott-Aldrich syndrome (WAS is a life-threatening immunodeficiency caused by mutations within the WAS gene. Viral gene therapy to restore WAS protein (WASp expression in hematopoietic cells of patients with WAS has the potential to improve outcomes relative to the current standard of care, allogeneic bone marrow transplantation. However, the development of viral vectors that are both safe and effective has been problematic. While use of viral transcriptional promoters may increase the risk of insertional mutagenesis, cellular promoters may not achieve WASp expression levels necessary for optimal therapeutic effect. Here we evaluate a self-inactivating (SIN lentiviral vector combining a chromatin insulator upstream of a viral MND (MPSV LTR, NCR deleted, dl587 PBS promoter driving WASp expression. Used as a gene therapeutic in Was−/− mice, this vector resulted in stable WASp+ cells in all hematopoietic lineages and rescue of T and B cell defects with a low number of viral integrations per cell, without evidence of insertional mutagenesis in serial bone marrow transplants. In a gene transfer experiment in non-human primates, the insulated MND promoter (driving GFP expression demonstrated long-term polyclonal engraftment of GFP+ cells. These observations demonstrate that the insulated MND promoter safely and efficiently reconstitutes clinically effective WASp expression and should be considered for future WAS therapy.

Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.

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Fisher-Adams, G; Wong, K K; Podsakoff, G; Forman, S J; Chatterjee, S

1996-07-15

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

Multi-resistance strategy for viral diseases and short hairpin RNA verification method in pigs

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Jong-nam Oh

2018-04-01

Full Text Available Objective Foot and mouth disease (FMD and porcine reproductive and respiratory syndrome (PRRS are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV and PRRS virus (PRRSV, the present study introduced two genetic modification techniques to porcine cells. Methods First, cluster of differentiation 163 (CD163, the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7 gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. Results shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. Conclusion We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.

A novel fluorescent retrograde neural tracer: cholera toxin B conjugated carbon dots

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Zhou, Nan; Hao, Zeyu; Zhao, Xiaohuan; Maharjan, Suraj; Zhu, Shoujun; Song, Yubin; Yang, Bai; Lu, Laijin

2015-09-01

The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde tracers.The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde

siRNA-Mediated Silencing of doublesex during Female Development of the Dengue Vector Mosquito Aedes aegypti.

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Keshava Mysore

2015-11-01

Full Text Available The development of sex-specific traits, including the female-specific ability to bite humans and vector disease, is critical for vector mosquito reproduction and pathogen transmission. Doublesex (Dsx, a terminal transcription factor in the sex determination pathway, is known to regulate sex-specific gene expression during development of the dengue fever vector mosquito Aedes aegypti. Here, the effects of developmental siRNA-mediated dsx silencing were assessed in adult females. Targeting of dsx during A. aegypti development resulted in decreased female wing size, a correlate for body size, which is typically larger in females. siRNA-mediated targeting of dsx also resulted in decreased length of the adult female proboscis. Although dsx silencing did not impact female membrane blood feeding or mating behavior in the laboratory, decreased fecundity and fertility correlated with decreased ovary length, ovariole length, and ovariole number in dsx knockdown females. Dsx silencing also resulted in disruption of olfactory system development, as evidenced by reduced length of the female antenna and maxillary palp and the sensilla present on these structures, as well as disrupted odorant receptor expression. Female lifespan, a critical component of the ability of A. aegypti to transmit pathogens, was also significantly reduced in adult females following developmental targeting of dsx. The results of this investigation demonstrate that silencing of dsx during A. aegypti development disrupts multiple sex-specific morphological, physiological, and behavioral traits of adult females, a number of which are directly or indirectly linked to mosquito reproduction and pathogen transmission. Moreover, the olfactory phenotypes observed connect Dsx to development of the olfactory system, suggesting that A. aegypti will be an excellent system in which to further assess the developmental genetics of sex-specific chemosensation.

The Yeast Retrograde Response as a Model of Intracellular Signaling of Mitochondrial Dysfunction

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S. Michal eJazwinski

2012-05-01

Full Text Available Mitochondrial dysfunction activates intracellular signaling pathways that impact yeast longevity, and the best known of these pathways is the retrograde response. More recently, similar responses have been discerned in other systems, from invertebrates to human cells. However, the identity of the signal transducers is either unknown or apparently diverse, contrasting with the well-established signaling module of the yeast retrograde response. On the other hand, it has become equally clear that several other pathways and processes interact with the retrograde response, embedding it in a network responsive to a variety of cellular states. An examination of this network supports the notion that the master regulator NFkB aggregated a variety of mitochondria-related cellular responses at some point in evolution and has become the retrograde transcription factor. This has significant consequences for how we view some of the deficits associated with aging, such as inflammation. The support for NFkB as the retrograde response transcription factor is not only based on functional analyses. It is bolstered by the fact that NFkB can regulate Myc-Max, which is activated in human cells with dysfunctional mitochondria and impacts cellular metabolism. Myc-Max is homologous to the yeast retrograde response transcription factor Rtg1-Rtg3. Further research will be needed to disentangle the pro-aging from the anti-aging effects of NFkB. Interestingly, this is also a challenge for the complete understanding of the yeast retrograde response.

Effects of inulin with different degree of polymerization on gelatinization and retrogradation of wheat starch.

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Luo, Denglin; Li, Yun; Xu, Baocheng; Ren, Guangyue; Li, Peiyan; Li, Xuan; Han, Sihai; Liu, Jianxue

2017-08-15

The effects of three types of inulin, including FS (DP≤10), FI (DP of 2-60) and FXL (DP≥23), on the gelatinization and retrogradation characteristics of wheat starch were investigated. As the concentration of inulin added into starch increased, the gelatinization temperature increased whereas the breakdown value decreased, and the value of setback first decreased and then increased slightly. The three types of inulin with lower concentrations (inulin showed a significant suppression of starch retrogradation in the addition range of 5-7.5%. They can all inhibit amylose retrogradation, but accelerate amylopectin retrogradation. Inulin with lower DP has stronger effects on the starch retrogradation. Generally, the three types of inulin can all retard the retrogradation performance of wheat starch to some extent in the long-term storage. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bioreducible poly(amido amine)s for non-viral gene delivery

NARCIS (Netherlands)

Lin, C.

2008-01-01

This thesis describes the design and development of bioreducible poly(amido amine)s as non-viral vectors for gene delivery in vitro and in vivo. The structural influences of these polymers on their physico-chemical properties and gene delivery properties, transfection capability and cytotoxicity in

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

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Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

2014-08-03

Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

The Insect Microbiome Modulates Vector Competence for Arboviruses

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Natapong Jupatanakul

2014-11-01

Full Text Available Diseases caused by arthropod-borne viruses (arboviruses, such as Dengue, West Nile, and Chikungunya, constitute a major global health burden and are increasing in incidence and geographic range. The natural microbiota of insect vectors influences various aspects of host biology, such as nutrition, reproduction, metabolism, and immunity, and recent studies have highlighted the ability of insect-associated bacteria to reduce vector competence for arboviruses and other pathogens. This reduction can occur through mechanisms, such as immune response activation, resource competition, or the production of anti-viral molecules. Studying the interactions between insect vectors and their microbiota is an important step toward developing alternative strategies for arbovirus transmission control.

Retrograde or antegrade double-pigtail stent placement for malignant ureteric obstruction?

International Nuclear Information System (INIS)

Uthappa, M.C.; Cowan, N.C.

2005-01-01

AIM: To determine the optimum approach for double-pigtail stent placement in malignant ureteric obstruction. PATIENTS AND METHODS: Retrograde stent placement was attempted in a consecutive series of patients presenting with malignant ureteric obstruction. If retrograde stent placement was unsuccessful, percutaneous nephrostomy was performed immediately followed by elective antegrade stent placement. Identical digital C-arm fluoroscopy for image-guidance and conditions for anaesthesia and analgesia were employed for both retrograde and antegrade procedures. Identical 8 Fr (20-26 cm) double-pigtail hydrophilic coated stents were used for each approach. RESULTS: Retrograde placement was attempted in 50 ureters in 30 patients {19 male, 11 female, average age 61.4 yr (range 29-90 yr)} over a 24-month period. The success rate for retrograde ureteric stent placement was 50% (n=25/50). Technical failures were due to failure to identify the ureteric orifice (n=22), failure to cross the stricture (n=1), failure to pass the stent (n=1) and failure to pass a 4 Fr catheter (n=1). Antegrade placement was attempted in 25 ureters with a success rate of 96% (n=24/25). Failure in the one case was due to inability to cross an upper third stricture secondary to pyeloureteritis cystica. CONCLUSION: It is suggested that retrograde route should be the initial approach if imaging shows no involvement of ureteric orifice (UO), when nephrostomy is technically very difficult or in cases of solitary kidney. The antegrade route is preferred if imaging shows tumour occlusion of the UO or if there is a tight stricture very close to the uretero-vesical junction (UVJ) making purchase within the ureter difficult for crossing the stricture

Effects of low molecular sugars on the retrogradation of tapioca starch gels during storage.

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Xiaoyu Zhang

Full Text Available The effects of low molecular sugars (sucrose, glucose and trehalose on the retrogradation of tapioca starch (TS gels stored at 4°C for different periods were examined with different methods. Decrease in melting enthalpy (ΔHmelt were obtained through differential scanning calorimetry analysis. Analysis of decrease in crystallization rate constant (k and increase in semi-crystallization time (τ1/2 results obtained from retrogradation kinetics indicated that low molecular sugars could retard the retrogradation of TS gels and further revealed trehalose as the best inhibitor among the sugars used in this study. Fourier transform infrared (FTIR analysis indicated that the intensity ratio of 1047 to 1022 cm-1 was increased with the addition of sugars in the order of trehalose > sucrose > glucose. Decrease in hardness parameters and increase in springiness parameters obtained from texture profile analysis (TPA analysis also indicated that low molecular sugars could retard the retrogradation of TS gels. The results of FTIR and TPA showed a consistent sugar effect on starch retrogradation with those of DSC and retrogradation kinetics analysis.

Complications of bladder distension during retrograde urethrography.

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Barsanti, J A; Crowell, W; Losonsky, J; Talkington, F D

1981-05-01

A severe, ulcerative cystitis that resulted in macroscopic hematuria occurred in 8 of 20 healthy dogs undergoing a series of diagnostic tests. Four of the remaining 12 dogs had mild bladder lesions consisting of submucosal edema and hemorrhage. Nine of the 20 dogs developed urinary tract infection after the procedures. These complications seemed associated with the radiographic technique of retrograde urethrography performed when the urinary bladder was distended. To test this hypothesis, retrograde urethrography was performed on 5 additional dogs. With the bladder undistended, no complications occurred. However, distention of these same dogs' bladders for 1 minute or less with sterile lactated Ringer's solution administered through a Foley catheter in the penile urethra resulted in a macroscopic hematuria in all 5 dogs which persisted for 24 hours. A microscopic hematuria continued for 5 days. One dog developed a bacterial urinary tract infection. A severe fibrinopurulent cystitis was present at necropsy of 2 dogs 2 days after distention. The morphologic changes in the bladder gradually diminished over 7 days, but mild submucosal edema and hemorrhage were still present when 2 dogs were necropsied, 7 days after distention. These studies indicated that retrograde urethrography in dogs may be complicated by hemorrhagic cystitis and urinary tract infection if performed with urinary bladder distention.

Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis)

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Stading, Benjamin; Osorio, Jorge E.; Velasco-Villa, Andres; Smotherman, Michael; Kingstad-Bakke, Brock; Rocke, Tonie E.

2016-01-01

Bats (Order Chiroptera) are an abundant group of mammals with tremendous ecological value as insectivores and plant dispersers, but their role as reservoirs of zoonotic diseases has received more attention in the last decade. With the goal of managing disease in free-ranging bats, we tested modified vaccinia Ankara (MVA) and raccoon poxvirus (RCN) as potential vaccine vectors in the Brazilian Free-tailed bat (Tadarida brasiliensis), using biophotonic in vivo imaging and immunogenicity studies. Animals were administered recombinant poxviral vectors expressing the luciferase gene (MVA-luc, RCN-luc) through oronasal (ON) or intramuscular (IM) routes and subsequently monitored for bioluminescent signal indicative of viral infection. No clinical illness was noted after exposure to any of the vectors, and limited luciferase expression was observed. Higher and longer levels of expression were observed with the RCN-luc construct. When given IM, luciferase expression was limited to the site of injection, while ON exposure led to initial expression in the oral cavity, often followed by secondary replication at another location, likely the gastric mucosa or gastric associated lymphatic tissue. Viral DNA was detected in oral swabs up to 7 and 9 days post infection (dpi) for MVA and RCN, respectively. While no live virus was detected in oral swabs from MVA-infected bats, titers up to 3.88 x 104 PFU/ml were recovered from oral swabs of RCN-infected bats. Viral DNA was also detected in fecal samples from two bats inoculated IM with RCN, but no live virus was recovered. Finally, we examined the immunogenicity of a RCN based rabies vaccine (RCN-G) following ON administration. Significant rabies neutralizing antibody titers were detected in the serum of immunized bats using the rapid fluorescence focus inhibition test (RFFIT). These studies highlight the safety and immunogenicity of attenuated poxviruses and their potential use as vaccine vectors in bats.

Design and Potential of Non-Integrating Lentiviral Vectors

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Aaron Shaw

2014-01-01

Full Text Available Lentiviral vectors have demonstrated promising results in clinical trials that target cells of the hematopoietic system. For these applications, they are the vectors of choice since they provide stable integration into cells that will undergo extensive expansion in vivo. Unfortunately, integration can have unintended consequences including dysregulated cell growth. Therefore, lentiviral vectors that do not integrate are predicted to have a safer profile compared to integrating vectors and should be considered for applications where transient expression is required or for sustained episomal expression such as in quiescent cells. In this review, the system for generating lentiviral vectors will be described and used to illustrate how alterations in the viral integrase or vector Long Terminal Repeats have been used to generate vectors that lack the ability to integrate. In addition to their safety advantages, these non-integrating lentiviral vectors can be used when persistent expression would have adverse consequences. Vectors are currently in development for use in vaccinations, cancer therapy, site-directed gene insertions, gene disruption strategies, and cell reprogramming. Preclinical work will be described that illustrates the potential of this unique vector system in human gene therapy.

Neuron-specific RNA interference using lentiviral vectors

DEFF Research Database (Denmark)

Nielsen, Troels Tolstrup; Marion, Ingrid van; Hasholt, Lis

2009-01-01

BACKGROUND: Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise...... transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding...... the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. METHODS: In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain. RESULTS: We...

The evolution of heart gene delivery vectors

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Wasala, Nalinda B.; Shin, Jin-Hong; Duan, Dongsheng

2012-01-01

Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. PMID:21837689

Charge collection control using retrograde well tested by proton microprobe irradiation

International Nuclear Information System (INIS)

Sayama, Hirokazu; Takai, Mikio; Kimura, Hiroshi; Ohno, Yoshikazu; Satoh, Shinichi; Sonoda, Kenichirou; Katani, Norihiko.

1993-01-01

Soft error reduction by high-energy ion-implanted layers has been investigated by novel evaluation techniques using high-energy proton microprobes. A retrograde well formed by 160 and 700 keV boron ion implantation could completely suppress soft errors induced by the proton microprobes at 400 keV. The proton-induced current revealed the charge collection efficiency for the retrograde well structure. The collected charge for the retrograde well in the soft-error mapping was proved to be lower than the critical charge of the measured DRAMs(dynamic random-access memories). Complementary use of soft-error mapping and ion-induced-current measurement could clarify well structures immune against soft errors. (author)

Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

DEFF Research Database (Denmark)

Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun

2011-01-01

The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

Adeno-associated viral vector transduction of human mesenchymal stem cells

DEFF Research Database (Denmark)

Stender, Stefan; Murphy, Mary; O'Brien, Tim

2007-01-01

Mesenchymal stem cells (MSCs) have received considerable attention in the emerging field of regenerative medicine. One aspect of MSC research focuses on genetically modifying the cells with the aim of enhancing their regenerative potential. Adeno-associated virus (AAV) holds promise as a vector...

Investigation of the Usability of Retrograded Flour in Meatball Production as A Structure Enhancer.

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Dinçer, Elif Aykin; Büyükkurt, Özlem Kiliç; Candal, Cihadiye; Bilgiç, Büşra Fatma; Erbaş, Mustafa

2018-02-01

This study aimed to research the possibilities of using retrograded flour produced in the laboratory environment in meatballs and the characteristics of these meatballs. In the use of retrograded flour to produce meatballs, it was ensured that the meatball properties, with respect to chemical, physical and sensorial aspects, were comparable to those of meatballs produced with bread (traditional) and rusk flour (commercial). The cooking loss of meatballs produced with using retrograded flour was similar to that of commercial meatballs. Doses of retrograded flour from 5% to 20% led to a significant decrease in cooking loss, from 21.95% to 6.19%, and in the diameter of meatballs, from 18.60% to 12.74%, but to an increase in the thickness of meatballs, from 28.82% to 41.39% compared to the control. The increase of a * and b * values was shown in that the meatballs were browned on cooking with increasing retrograded flour doses because of non-enzymatic reactions. The springiness of the traditional meatballs was significantly higher than that of the other meatballs. This might have been due to the bread crumbs having a naturally springy structure. Moreover, the addition of retrograded flour in the meatballs significantly ( p meatballs with respect to textural properties. Accordingly, it is considered that the use of 10% retrograded flour is ideal to improve the sensorial values of meatballs and the properties of their structure.

An indigenous economic technique of positive pressure retrograde urethrography in female patients

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H Singh

2001-01-01

Full Text Available Usually double balloon catheter is required forpositive pressure retrograde urethrography in females. We describe a technique of positive pressure retrograde urethrography using Foley catheter and rubber stopper, inexpensive and could be adopted in any hospital or radiological suite.

Deterioration of cholestasis after endoscopic retrograde cholangiography in advanced primary sclerosing cholangitis

NARCIS (Netherlands)

Beuers, U.; Spengler, U.; Sackmann, M.; Paumgartner, G.; Sauerbruch, T.

1992-01-01

Complications of endoscopic retrograde cholangiography specific to patients with primary sclerosing cholangitis have not yet been reported. We observed transient rises of serum bilirubin after diagnostic endoscopic retrograde cholangiography in five of 15 patients and persistent rises in three of 15

Emerging Vector-Borne Diseases.

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Huntington, Mark K; Allison, Jay; Nair, Dilip

2016-10-01

Several mosquito-borne viral infections have recently emerged in North America; West Nile virus is the most common in the United States. Although West Nile virus generally causes a self-limited, flulike febrile illness, a serious neuroinvasive form may occur. Dengue is the most common vector-borne viral disease worldwide, and it has been a significant public health threat in the United States since 2009. Known as breakbone fever for its severe myalgias and arthralgias, dengue may cause a hemorrhagic syndrome. Chikungunya also causes flulike febrile illness and disabling arthralgias. Although meningoencephalitis may occur with chikungunya, bleeding is uncommon. Symptoms of Zika virus infection are similar to those of dengue, but milder. Zika virus increases the risk of fetal brain abnormalities, including microcephaly, if a pregnant woman is infected. Zika virus is spread through Aedes albopictus mosquito bites, is transmitted sexually, and may rarely spread nonsexually from person to person. Diagnosis of these vectorborne infections is clinical and serologic, and treatment is supportive. Other, well-established vector-borne diseases are also important. Ehrlichiosis is a tick-borne bacterial disease that presents as a nonspecific syndrome of fever, headache, malaise, and myalgias. It is diagnosed via blood smear testing, with confirmatory serology. Ehrlichiosis is treated with doxycycline. Rickettsial infections are transmitted by fleas, mites, and ticks, and severity ranges from mild to life threatening. Rocky Mountain spotted fever, the most significant rickettsial infection, is primarily a clinical diagnosis that presents as fever, headache, myalgias, petechial rash, and tick exposure. Doxycycline is effective for rickettsial infections if administered promptly. Vector avoidance strategies are critical to the prevention of all of these infections.

Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging

NARCIS (Netherlands)

Pfeifer, A.; Kessler, T.; Yang, M.; Baranov, E.; Kootstra, N.; Cheresh, D. A.; Hoffman, R. M.; Verma, I. M.

2001-01-01

Viral vectors based on lentiviruses, such as the human immunodeficiency virus, are able to transduce a broad spectrum of nondividing cells in vivo. This ability of lentiviral vectors makes them an attractive vehicle for gene transfer into the liver. In order to determine the requirements for

The Polerovirus Minor Capsid Protein Determines Vector Specificity and Intestinal Tropism in the Aphid

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Brault, Véronique; Périgon, Sophie; Reinbold, Catherine; Erdinger, Monique; Scheidecker, Danièle; Herrbach, Etienne; Richards, Ken; Ziegler-Graff, Véronique

2005-01-01

Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD. PMID:16014930

IL-10 mediated by herpes simplex virus vector reduces neuropathic pain induced by HIV gp120 combined with ddC in rats.

Science.gov (United States)

Zheng, Wenwen; Huang, Wan; Liu, Shue; Levitt, Roy C; Candiotti, Keith A; Lubarsky, David A; Hao, Shuanglin

2014-07-30

HIV-associated sensory neuropathy affects over 50% of HIV patients and is a common peripheral nerve complication of HIV infection and highly active antiretroviral therapy (HAART). Evidence shows that painful HIV sensory neuropathy is influenced by neuroinflammatory events that include the proinflammatory molecules, MAP Kinase, tumor necrosis factor-α (TNFα), stromal cell-derived factor 1-α (SDF1α), and C-X-C chemokine receptor type 4 (CXCR4). However, the exact mechanisms of painful HIV sensory neuropathy are not known, which hinders our ability to develop effective treatments. In this study, we investigated whether inhibition of proinflammatory factors reduces the HIV-associated neuropathic pain state. Neuropathic pain was induced by peripheral HIV coat protein gp120 combined with 2',3'-dideoxycytidine (ddC, one of the nucleoside reverse transcriptase inhibitors (NRTIs)). Mechanical threshold was tested using von Frey filament fibers. Non-replicating herpes simplex virus (HSV) vectors expressing interleukin 10 (IL10) were inoculated into the hindpaws of rats. The expression of TNFα, SDF1α, and CXCR4 in the lumbar spinal cord and L4/5 dorsal root ganglia (DRG) was examined using western blots. IL-10 expression mediated by the HSV vectors resulted in a significant elevation of mechanical threshold. The anti-allodynic effect of IL-10 expression mediated by the HSV vectors lasted more than 3 weeks. The area under the effect-time curves (AUC) in mechanical threshold in rats inoculated with the HSV vectors expressing IL-10, was increased compared with the control vectors, indicating antinociceptive effect of the IL-10 vectors. The HSV vectors expressing IL-10 also concomitantly reversed the upregulation of p-p38, TNFα, SDF1α, and CXCR4 induced by gp120 in the lumbar spinal dorsal horn and/or the DRG at 2 and/or 4 weeks. The blocking of the signaling of these proinflammatory molecules is able to reduce HIV-related neuropathic pain, which provide a novel

Retrograde Tibiopedal Access as a Bail-Out Procedure for Endovascular Intervention Complications

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Ahmed Amro

2016-01-01

Full Text Available Introduction. Retrograde pedal access has been well described in the literature as a secondary approach for limb salvage in critical limb ischemia (CLI patients. In this manuscript we are presenting a case where retrograde tibiopedal access has been used as a bail-out procedure for the management of superficial femoral artery (SFA intervention complications. Procedure/Technique. After development of a perforation while trying to cross the totally occluded mid SFA using the conventional CFA access, we were able to cross the mid SFA lesion after accessing the posterior tibial artery in a retrograde fashion and delivered a self-expanding stent which created a flap that sealed the perforation without the need for covered stent. Conclusion. Retrograde tibiopedal access is a safe and effective approach for delivery of stents from the distal approach and so can be used as a bail-out technique for SFA perforation.

Novel human central nervous system 3D in vitro models: useful tools for evaluation of viral vector-mediated gene delivery

OpenAIRE

Pinto, Ana Catarina Pereira, 1989-

2012-01-01

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2012 A prevenção e tratamento de doenças neurodegenerativas, como a doença de Parkinson, estão ainda longe de se tornarem realidade. Embora as estratégias farmacológicas convencionais se tenham revelado pouco eficazes, resultados preliminares indicam que a terapia génica poderá ter grande potencial. Os vectores adenovirais baseados no serotipo canino 2 (CAV-2) transduzem preferencialm...

Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery.

Science.gov (United States)

Volak, Adrienn; LeRoy, Stanley G; Natasan, Jeya Shree; Park, David J; Cheah, Pike See; Maus, Andreas; Fitzpatrick, Zachary; Hudry, Eloise; Pinkham, Kelsey; Gandhi, Sheetal; Hyman, Bradley T; Mu, Dakai; GuhaSarkar, Dwijit; Stemmer-Rachamimov, Anat O; Sena-Esteves, Miguel; Badr, Christian E; Maguire, Casey A

2018-05-16

The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.

Virus infection mediates the effects of elevated CO2 on plants and vectors

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Trębicki, Piotr; Vandegeer, Rebecca K.; Bosque-Pérez, Nilsa A.; Powell, Kevin S.; Dader, Beatriz; Freeman, Angela J.; Yen, Alan L.; Fitzgerald, Glenn J.; Luck, Jo E.

2016-03-01

Atmospheric carbon dioxide (CO2) concentration has increased significantly and is projected to double by 2100. To increase current food production levels, understanding how pests and diseases respond to future climate driven by increasing CO2 is imperative. We investigated the effects of elevated CO2 (eCO2) on the interactions among wheat (cv. Yitpi), Barley yellow dwarf virus and an important pest and virus vector, the bird cherry-oat aphid (Rhopalosiphum padi), by examining aphid life history, feeding behavior and plant physiology and biochemistry. Our results showed for the first time that virus infection can mediate effects of eCO2 on plants and pathogen vectors. Changes in plant N concentration influenced aphid life history and behavior, and N concentration was affected by virus infection under eCO2. We observed a reduction in aphid population size and increased feeding damage on noninfected plants under eCO2 but no changes to population and feeding on virus-infected plants irrespective of CO2 treatment. We expect potentially lower future aphid populations on noninfected plants but no change or increased aphid populations on virus-infected plants therefore subsequent virus spread. Our findings underscore the complexity of interactions between plants, insects and viruses under future climate with implications for plant disease epidemiology and crop production.

An improved ternary vector system for Agrobacterium-mediated rapid maize transformation.

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Anand, Ajith; Bass, Steven H; Wu, Emily; Wang, Ning; McBride, Kevin E; Annaluru, Narayana; Miller, Michael; Hua, Mo; Jones, Todd J

2018-05-01

A simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation. The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86-103%) raw transformation frequency in an elite maize inbred.

Viral Pseudo Enzymes Activate RIG-I via Deamidation to Evade Cytokine Production

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He, Shanping; Zhao, Jun; Song, Shanshan; He, Xiaojing; Minassian, Arlet; Zhou, Yu; Zhang, Junjie; Brulois, Kevin; Wang, Yuqi; Cabo, Jackson; Zandi, Ebrahim; Liang, Chengyu; Jung, Jae U; Zhang, Xuewu; Feng, Pinghui

2015-01-01

SUMMARY RIG-I is a pattern recognition receptor that senses viral RNA and is crucial for host innate immune defense. Here we describe a mechanism of RIG-I activation through amidotransferase-mediated deamidation. We show that viral homologues of phosphoribosylformyglycinamide synthase (PFAS), although lacking intrinsic enzyme activity, recruit cellular PFAS to deamidate and activate RIG-I. Accordingly, depletion and biochemical inhibition of PFAS impair RIG-I deamidation and concomitant activation. Purified PFAS and viral homologue thereof deamidate RIG-I in vitro. Ultimately, herpesvirus hijacks activated RIG-I to avoid antiviral cytokine production; loss of RIG-I or inhibition of RIG-I deamidation results in elevated cytokine production. Together, these findings demonstrate a surprising mechanism of RIG-I activation that is mediated by an enzyme. PMID:25752576

On the electrodynamic explanation of the retrograde motion of the electric arc

International Nuclear Information System (INIS)

Hong, J.S.; Allen, J.E.

1992-01-01

The retrograde motion of the cathode spot in a transverse magnetic field is one of the more intriguing phenomena of the electric arc. Although the phenomenon has been known for nearly ninety years since its discovery by Stark and has stimulated numerous investigations which result in many models giving explanation from different points of view, there is still no theory that can account both qualitatively and quantitatively for all the observations. Most of the explanations of the retrograde motion involve the study of cathode processes to give the preferential formation of new cathode spots along the retrograde direction. One line of explanation, which is rather different from the others, is based on electrodynamics. In this approach the retrograde motion is treated as an electrodynamic event. The present paper develops the theory suggested by Robson and von Engel. A more complete model is proposed and studied in detail by means of electromagnetic field theory. The results obtained not only show that the retrograde motion can be explained by the electrodynamics, but also confirm that the average current density on the cathode spot must be around the order of 10 12 A/m 2 . Recent studies of spot current density have shown values of this order. (author) 22 refs., 4 figs., 1 tab

TRANSIT TIMING VARIATIONS FOR INCLINED AND RETROGRADE EXOPLANETARY SYSTEMS

International Nuclear Information System (INIS)

Payne, Matthew J.; Ford, Eric B.; Veras, Dimitri

2010-01-01

We perform numerical calculations of the expected transit timing variations (TTVs) induced on a hot-Jupiter by an Earth-mass perturber. Motivated by the recent discoveries of retrograde transiting planets, we concentrate on an investigation of the effect of varying relative planetary inclinations, up to and including completely retrograde systems. We find that planets in low-order (e.g., 2:1) mean-motion resonances (MMRs) retain approximately constant TTV amplitudes for 0 deg. 170 deg. Systems in higher order MMRs (e.g., 5:1) increase in TTV amplitude as inclinations increase toward 45 deg., becoming approximately constant for 45 deg. 135 deg. Planets away from resonance slowly decrease in TTV amplitude as inclinations increase from 0 deg. to 180 deg., whereas planets adjacent to resonances can exhibit a huge range of variability in TTV amplitude as a function of both eccentricity and inclination. For highly retrograde systems (135 deg. < i ≤ 180 deg.), TTV signals will be undetectable across almost the entirety of parameter space, with the exceptions occurring when the perturber has high eccentricity or is very close to an MMR. This high inclination decrease in TTV amplitude (on and away from resonance) is important for the analysis of the known retrograde and multi-planet transiting systems, as inclination effects need to be considered if TTVs are to be used to exclude the presence of any putative planetary companions: absence of evidence is not evidence of absence.

Treatment of lower extremity arterial occlusive through retrograde access

International Nuclear Information System (INIS)

Liu Xueqiang; Guo Pingfan; Zhang Jinchi; Cai Fanggang

2012-01-01

Objective: To explore the clinical significance of retrograde access for the interventional treatment of lower extremity arterial occlusive diseases when the occluded segment of lower extremity artery could not be reached through antegrade access. Methods: Twenty-seven cases (male 17, female 10; age range 32-89 years) were retrospectively investigated, including 18 with lower limb arteriosclerosis obliterans, 7 with diabetic foot and 2 with thromboangiitis obliterans. According to the Fontaine staging, 6 cases were classified as Fontaine Ⅱ, 11 were classified as Fontaine Ⅲ and 10 were classified as Fontaine Ⅳ. All cases underwent endovascular operation through antegrade access first with an attempt to cross the occlusive segment, but in vain. So retrograde access was tried via puncture of pedis dorsalis or posterior tibial artery or exposure of lateral branches of posterior tibial artery, peroneal artery or dorsal artery by open surgery,which followed by Percutaneous transluminal angiography and (or) stenting. Results: The operation through retrograde access was successful in all cases with obvious improvement of ischemic symptoms. Hematoma at the puncture site occurred in 3 patients, and paresthesia of toes occurred in 1 after dorsalis pedis arteriotomy. No severe perioperative complication occurred. The average ankle brachial index increased from 0.37 ± 0.11 preoperatively to 0.85 ± 0.12 postoperatively. Conclusions: Retrograde access could be used as an alternative strategy in lower extremity arterial occlusive diseases when the occluded segment could not reach through antegrade access. (authors)

Incorporating double copies of a chromatin insulator into lentiviral vectors results in less viral integrants

DEFF Research Database (Denmark)

Nielsen, Troels T; Jakobsson, Johan; Rosenqvist, Nina

2009-01-01

BACKGROUND: Lentiviral vectors hold great promise as gene transfer vectors in gene therapeutic settings. However, problems related to the risk of insertional mutagenesis, transgene silencing and positional effects have stalled the use of such vectors in the clinic. Chromatin insulators are boundary...

Mucosal vaccination with heterologous viral vectored vaccine targeting subdominant SIV accessory antigens strongly inhibits early viral replication

DEFF Research Database (Denmark)

Xu, Huanbin; Andersson, Anne-Marie Carola; Ragonnaud, Emeline

2017-01-01

Conventional HIV T cell vaccine strategies have not been successful in containing acute peak viremia, nor in providing long-term control. We immunized rhesus macaques intramuscularly and rectally using a heterologous adenovirus vectored SIV vaccine regimen encoding normally weakly immunogenic tat...

Highly-Immunogenic Virally-Vectored T-cell Vaccines Cannot Overcome Subversion of the T-cell Response by HCV during Chronic Infection

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Leo Swadling

2016-08-01

Full Text Available An effective therapeutic vaccine for the treatment of chronic hepatitis C virus (HCV infection, as an adjunct to newly developed directly-acting antivirals (DAA, or for the prevention of reinfection, would significantly reduce the global burden of disease associated with chronic HCV infection. A recombinant chimpanzee adenoviral (ChAd3 vector and a modified vaccinia Ankara (MVA, encoding the non-structural proteins of HCV (NSmut, used in a heterologous prime/boost regimen induced multi-specific, high-magnitude, durable HCV-specific CD4+ and CD8+ T-cell responses in healthy volunteers, and was more immunogenic than a heterologous Ad regimen. We now assess the immunogenicity of this vaccine regimen in HCV infected patients (including patients with a low viral load suppressed with interferon/ribavirin therapy, determine T-cell cross-reactivity to endogenous virus, and compare immunogenicity with that observed previously in both healthy volunteers and in HCV infected patients vaccinated with the heterologous Ad regimen. Vaccination of HCV infected patients with ChAd3-NSmut/MVA-NSmut was well tolerated. Vaccine-induced HCV-specific T-cell responses were detected in 8/12 patients; however, CD4+ T-cell responses were rarely detected, and the overall magnitude of HCV-specific T-cell responses was markedly reduced when compared to vaccinated healthy volunteers. Furthermore, HCV-specific cells had a distinct partially-functional phenotype (lower expression of activation markers, granzyme B, and TNFα production, weaker in vitro proliferation, and higher Tim3 expression, with comparable Tbet and Eomes expression compared to healthy volunteers. Robust anti-vector T-cells and antibodies were induced, showing that there is no global defect in immunity. The level of viremia at the time of vaccination did not correlate with the magnitude of the vaccine-induced T-cell response. Full-length, next-generation sequencing of the circulating virus demonstrated that T

Construction of non-viral vector (mPEG5k-PCL1.2k)1.4-g-PEI10k and its gene delivery efficacy in vitro

OpenAIRE

Wei HUANG; Ming LV; Zhong-gao GAO; Ming-ji JIN; Fei-fei YANG; Yu-li WANG

2011-01-01

Objective To construct(mPEG5k-PCL1.2k)1.4-g-PEI10k,a copolymer designed as delivery vector for non-viral gene therapy,and explore its cytotoxicity and efficacy in delivery of plasmid DNA(pDNA).Methods The copolymer,mPEG5k-PCL1.2k-OH,was prepared by ring-opening polymerization and then followed by a conversion of hydroxyl terminal(-OH) into N-hydroxysuccinimide(NHS) to prepare mPEG5k-PCL1.2k-NHS.One of the branches,PEI10k,was then reacted with mPEG5k-PCL1.2k-NHS to synthesize a ternary copolym...

Viral vector-mediated overexpression of estrogen receptor-alpha in striatum enhances the estradiol-induced motor activity in female rats and estradiol-modulated GABA release.

Science.gov (United States)

Schultz, Kristin N; von Esenwein, Silke A; Hu, Ming; Bennett, Amy L; Kennedy, Robert T; Musatov, Sergei; Toran-Allerand, C Dominique; Kaplitt, Michael G; Young, Larry J; Becker, Jill B

2009-02-11

Classical estrogen receptor-signaling mechanisms involve estradiol binding to intracellular nuclear receptors [estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta)] to promote changes in protein expression. Estradiol can also exert effects within seconds to minutes, however, a timescale incongruent with genomic signaling. In the brain, estradiol rapidly potentiates stimulated dopamine release in the striatum of female rats and enhances spontaneous rotational behavior. Furthermore, estradiol rapidly attenuates the K(+)-evoked increase of GABA in dialysate. We hypothesize that these rapid effects of estradiol in the striatum are mediated by ERalpha located on the membrane of medium spiny GABAergic neurons. This experiment examined whether overexpression of ERalpha in the striatum would enhance the effect of estradiol on rotational behavior and the K(+)-evoked increase in GABA in dialysate. Ovariectomized female rats were tested for rotational behavior or underwent microdialysis experiments after unilateral intrastriatal injections of a recombinant adeno-associated virus (AAV) containing the human ERalpha cDNA (AAV.ERalpha) into the striatum; controls received either the same vector into areas outside the striatum or an AAV containing the human alkaline phosphatase gene into the striatum (AAV.ALP). Animals that received AAV.ERalpha in the striatum exhibited significantly greater estradiol-induced contralateral rotations compared with controls and exhibited behavioral sensitization of contralateral rotations induced by a low-dose of amphetamine. ERalpha overexpression also enhanced the inhibitory effect of estradiol on K(+)-evoked GABA release suggesting that disinhibition of dopamine release from terminals in the striatum resulted in the enhanced rotational behavior.

Evidence of differential HLA class I-mediated viral evolution in functional and accessory/regulatory genes of HIV-1.

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Zabrina L Brumme

2007-07-01

Full Text Available Despite the formidable mutational capacity and sequence diversity of HIV-1, evidence suggests that viral evolution in response to specific selective pressures follows generally predictable mutational pathways. Population-based analyses of clinically derived HIV sequences may be used to identify immune escape mutations in viral genes; however, prior attempts to identify such mutations have been complicated by the inability to discriminate active immune selection from virus founder effects. Furthermore, the association between mutations arising under in vivo immune selection and disease progression for highly variable pathogens such as HIV-1 remains incompletely understood. We applied a viral lineage-corrected analytical method to investigate HLA class I-associated sequence imprinting in HIV protease, reverse transcriptase (RT, Vpr, and Nef in a large cohort of chronically infected, antiretrovirally naïve individuals. A total of 478 unique HLA-associated polymorphisms were observed and organized into a series of "escape maps," which identify known and putative cytotoxic T lymphocyte (CTL epitopes under selection pressure in vivo. Our data indicate that pathways to immune escape are predictable based on host HLA class I profile, and that epitope anchor residues are not the preferred sites of CTL escape. Results reveal differential contributions of immune imprinting to viral gene diversity, with Nef exhibiting far greater evidence for HLA class I-mediated selection compared to other genes. Moreover, these data reveal a significant, dose-dependent inverse correlation between HLA-associated polymorphisms and HIV disease stage as estimated by CD4(+ T cell count. Identification of specific sites and patterns of HLA-associated polymorphisms across HIV protease, RT, Vpr, and Nef illuminates regions of the genes encoding these products under active immune selection pressure in vivo. The high density of HLA-associated polymorphisms in Nef compared to other

Introduction of optical reporter gene into cancer and immune cells using lentiviral vector

International Nuclear Information System (INIS)

Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung; Kim, Yeon Soo

2004-01-01

For some applications such as gene therapy or reporter gene imaging, a gene has to be introduced into the organism of interest. Adenoviral vectors are capable of transducing both replicating and non-dividing cells. The adenoviral vectors do not integrate their DNA into host DNA, but do lead to an immune response. Lentiviruses belong to the retrovirus family and are capable of infecting both dividing and non-dividing cells. The human immunodeficiency virus (HIV) is an example of a lentavirus. A disabled HIV virus has been developed and could be used for in vivo gene delivery. A portion of the viral genome which encodes for accessory proteins canbe deleted without affecting production of the vector and efficiency of infection. Lentiviral delivery into various rodent tissues shows sustained expression of the transgene of up to six months. Furthermore, there seems to be little or no immune response with these vectors. These lentiviral vectors hold significant promise for in vivo gene delivery. We constructed lentiviral vector encoding firefly luciferase (Fluc) and eGFP. Fluc-eGFP fusion gene was inserted into multiple cloning sites of pLentiM1.3 vector. Reporter gene (Fluc-eGFP) was designed to be driven by murine CMV promoter with enhanced efficacy of transgene expression as compared to human CMV promoter. We transfected pLenti1.3-Fluc into human cervix cancer cell line (HeLa) and murine T lymphocytes. We also constructed adenovirus encoding Fluc and transfected to HeLa and T cells. This LentiM1.3-Fluc was transfected into HeLa cells and murine T lymphocytes in vitro, showing consistent expression of eGFP under the fluorescence microscopy from the 2nd day of transfection. Firefly luciferase reporter gene was not expressed in immune cells when it is mediated by adenovirus. Lentivirus was validated as a useful vector for both immune and cancer cells

Insights into the internalization and retrograde trafficking of Dengue 2 virus in BHK-21 cells.

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Nidhi Shrivastava

Full Text Available BACKGROUND: Dengue virus (DENV enters cells via endocytosis, traffics to perinuclear (PN region, the site of morphogenesis and exits by exocytosis. This study aims to understand the role of dynamin II, endosomes, microtubules (MT and dynein in the early events of DENV replication. FINDINGS: Using double immunoflourescence labelling of DENV-2 infected BHK-21 cells it was observed that the surface envelope (E protein of the virion associated with dynamin II from 0-30 min post infection (p.i.. The sphincter like array of dynamin II supported its pinchase-like activity. The association with endosomes was observed from 0 min at cell periphery to 30 min in the perinuclear (PN region, suggesting that internalization continued for 30 min. Association of E protein with alpha-tubulin was observed from 8 h indicating that it was the newly translated protein that trafficked on the MT. Dynein was found to associate with the E protein from 4 h in the cytoplasm to 48 h in the PN region and dissociate at 72 h. Association of E protein with dynein was confirmed by immunoprecipitation. Overexpression of dynamitin, which disrupts the dynein complex, resulted in loss of trafficking of viral E and core proteins. The findings corroborated with the growth kinetics assessed by quantitation of viral RNA in infected BHK-21 cells. The detection of E protein at 4 h-8 h correlated with detectable increase in viral RNA from 8 h. The detection of high concentrations of E protein in the PN region at 24-48 h coincided with release of virus into the supernatant starting from 36 h p.i. The dissociation of dynein from E protein by 72 h was coincident with maximum release of virus, hinting at a possible negative feedback for viral protein translation. CONCLUSION: The study shows for the first time the association of dynamin II with DENV-2 during entry and dynein dependent retrograde trafficking of DENV proteins on microtubules.

Retrograde Jejuno-Jejunal Intussusception after Total Gastrectomy

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Akira Yoneda

2008-08-01

Full Text Available An eighty-year-old female was transferred to the hospital after experiencing abdominal pain and nausea. She had had a history of total gastrectomy for gastric cancer 14 years previously. Abdominal X-ray revealed a localized expansion of the small bowel. Computed tomography revealed a mass with a lamellar structure in a concentric circle. With a tentative diagnosis of small bowel obstruction due to intussusception, she underwent emergency operation. Laparotomy revealed a retrograde jejuno-jejunal intussusception. Bowel resection was performed due to the severe ischemic damage. All reported intussusception cases after total gastrectomy displayed retrograde characteristics and could occur both during the early and late period after surgery. It is important to consider the possibility of intussusception for patients presenting with acute abdomen who have previously undergone gastric resection.

AAVPG: A vigilant vector where transgene expression is induced by p53

Energy Technology Data Exchange (ETDEWEB)

Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.; Strauss, Bryan E., E-mail: bstrauss@usp.br

2013-12-15

Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 fold increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.

Interaction of measles virus vectors with Auger electron emitting radioisotopes

International Nuclear Information System (INIS)

Dingli, David; Peng, K.-W.; Harvey, Mary E.; Vongpunsawad, Sompong; Bergert, Elizabeth R.; Kyle, Robert A.; Cattaneo, Roberto; Morris, John C.; Russell, Stephen J.

2005-01-01

A recombinant measles virus (MV) expressing the sodium iodide symporter (NIS) is being considered for therapy of advanced multiple myeloma. Auger electrons selectively damage cells in which the isotope decays. We hypothesized that the Auger electron emitting isotope 125 I can be used to control viral proliferation. MV was engineered to express both carcinoembryonic antigen and NIS (MV-NICE). Cells were infected with MV-NICE and exposed to 125 I with appropriate controls. MV-NICE replication in vitro is inhibited by the selective uptake of 125 I by cells expressing NIS. Auger electron damage is partly mediated by free radicals and abrogated by glutathione. In myeloma xenografts, control of MV-NICE with 125 I was not possible under the conditions of the experiment. MV-NICE does not replicate faster in the presence of radiation. Auger electron emitting isotopes effectively stop propagation of MV vectors expressing NIS in vitro. Additional work is necessary to translate these observations in vivo

Viral Interference and Persistence in Mosquito-Borne Flaviviruses

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Juan Santiago Salas-Benito

2015-01-01

Full Text Available Mosquito-borne flaviviruses are important pathogens for humans, and the detection of two or more flaviviruses cocirculating in the same geographic area has often been reported. However, the epidemiological impact remains to be determined. Mosquito-borne flaviviruses are primarily transmitted through Aedes and Culex mosquitoes; these viruses establish a life-long or persistent infection without apparent pathological effects. This establishment requires a balance between virus replication and the antiviral host response. Viral interference is a phenomenon whereby one virus inhibits the replication of other viruses, and this condition is frequently associated with persistent infections. Viral interference and persistent infection are determined by several factors, such as defective interfering particles, competition for cellular factors required for translation/replication, and the host antiviral response. The interaction between two flaviviruses typically results in viral interference, indicating that these viruses share common features during the replicative cycle in the vector. The potential mechanisms involved in these processes are reviewed here.

Adenovirus-encoding virus-associated RNAs suppress HDGF gene expression to support efficient viral replication.

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Saki Kondo

Full Text Available Non-coding small RNAs are involved in many physiological responses including viral life cycles. Adenovirus-encoding small RNAs, known as virus-associated RNAs (VA RNAs, are transcribed throughout the replication process in the host cells, and their transcript levels depend on the copy numbers of the viral genome. Therefore, VA RNAs are abundant in infected cells after genome replication, i.e. during the late phase of viral infection. Their function during the late phase is the inhibition of interferon-inducible protein kinase R (PKR activity to prevent antiviral responses; recently, mivaRNAs, the microRNAs processed from VA RNAs, have been reported to inhibit cellular gene expression. Although VA RNA transcription starts during the early phase, little is known about its function. The reason may be because much smaller amount of VA RNAs are transcribed during the early phase than the late phase. In this study, we applied replication-deficient adenovirus vectors (AdVs and novel AdVs lacking VA RNA genes to analyze the expression changes in cellular genes mediated by VA RNAs using microarray analysis. AdVs are suitable to examine the function of VA RNAs during the early phase, since they constitutively express VA RNAs but do not replicate except in 293 cells. We found that the expression level of hepatoma-derived growth factor (HDGF significantly decreased in response to the VA RNAs under replication-deficient condition, and this suppression was also observed during the early phase under replication-competent conditions. The suppression was independent of mivaRNA-induced downregulation, suggesting that the function of VA RNAs during the early phase differs from that during the late phase. Notably, overexpression of HDGF inhibited AdV growth. This is the first report to show the function, in part, of VA RNAs during the early phase that may be contribute to efficient viral growth.

Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation

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Ichim Christine V

2011-08-01

Full Text Available Abstract Background Viral vectors provide a method of stably introducing exogenous DNA into cells that are not easily transfectable allowing for the ectopic expression or silencing of genes for therapeutic or experimental purposes. However, some cell types, in particular bone marrow cells, dendritic cells and neurons are difficult to transduce with viral vectors. Successful transduction of such cells requires preparation of highly concentrated viral stocks, which permit a high virus concentration and multiplicity of infection (MOI during transduction. Pseudotyping with the vesicular stomatitis virus G (VSV-G envelope protein is common practice for both lentiviral and retroviral vectors. The VSV-G glycoprotein adds physical stability to retroviral particles, allowing concentration of virus by high-speed ultracentrifugation. Here we describe a method report for concentration of virus from large volumes of culture supernatant by means of successive rounds of ultracentrifugation into the same ultracentrifuge tube. Method Stable retrovirus producer cell lines were generated and large volumes of virus-containing supernatant were produced. We then tested the transduction ability of virus following varying rounds of concentration by ultra-centrifugation. In a second series of experiments lentivirus-containing supernatant was produced by transient transfection of 297T/17 cells and again we tested the transduction ability of virus following multiple rounds of ultra-centrifugation. Results We report being able to centrifuge VSV-G coated retrovirus for as many as four rounds of ultracentrifugation while observing an additive increase in viral titer. Even after four rounds of ultracentrifugation we did not reach a plateau in viral titer relative to viral supernatant concentrated to indicate that we had reached the maximum tolerated centrifugation time, implying that it may be possible to centrifuge VSV-G coated retrovirus even further should it be necessary

Non-viral gene delivery strategies for gene therapy: a “ménage à trois” among nucleic acids, materials, and the biological environment

International Nuclear Information System (INIS)

Pezzoli, Daniele; Candiani, Gabriele

2013-01-01

Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription–translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral delivery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic application is still rather limited as high transfection efficiencies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the landscape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors.

Focal retrograde amnesia: voxel-based morphometry findings in a case without MRI lesions.

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Bernhard Sehm

Full Text Available Focal retrograde amnesia (FRA is a rare neurocognitive disorder presenting with an isolated loss of retrograde memory. In the absence of detectable brain lesions, a differentiation of FRA from psychogenic causes is difficult. Here we report a case study of persisting FRA after an epileptic seizure. A thorough neuropsychological assessment confirmed severe retrograde memory deficits while anterograde memory abilities were completely normal. Neurological and psychiatric examination were unremarkable and high-resolution MRI showed no neuroradiologically apparent lesion. However, voxel-based morphometry (VBM-comparing the MRI to an education-, age-and sex-matched control group (n = 20 disclosed distinct gray matter decreases in left temporopolar cortex and a region between right posterior parahippocampal and lingual cortex. Although the results of VBM-based comparisons between a single case and a healthy control group are generally susceptible to differences unrelated to the specific symptoms of the case, we believe that our data suggest a causal role of the cortical areas detected since the retrograde memory deficit is the preeminent neuropsychological difference between patient and controls. This was paralleled by grey matter differences in central nodes of the retrograde memory network. We therefore suggest that these subtle alterations represent structural correlates of the focal retrograde amnesia in our patient. Beyond the implications for the diagnosis and etiology of FRA, our results advocate the use of VBM in conditions that do not show abnormalities in clinical radiological assessment, but show distinct neuropsychological deficits.

Contribution of viral recombinants to the study of the immune response against the Epstein-Barr virus.

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Delecluse, Henri-Jacques; Feederle, Regina; Behrends, Uta; Mautner, Josef

2008-12-01

Over the past two decades, Epstein-Barr virus (EBV) mutants have become valuable tools for the analysis of viral functions. Several experimental strategies are currently used to generate recombinant mutant genomes that carry alterations in one or several viral genes. The probably most versatile approach utilizes bacterial artificial chromosomes (BAC) carrying parts or the whole EBV genome, which permits extensive genetic manipulations in Escherichia coli cells. The 'mini-EBVs', for example, which contain roughly half of the wild type viral information, efficiently transform primary B cells and have been used as gene vectors for foreign antigens. After expression in lymphoblastoid cell lines (LCLs), these antigens are efficiently presented on MHC molecules and recognized by antigen-specific T cells. These vectors, however, cannot undergo lytic replication and require a helper cell line for efficient replication and DNA packaging. Further experimental systems include the complete viral genome cloned onto a BAC. These mutants can typically be complemented by expression plasmids, some of which are expressed on EBV-derived vectors and can be propagated without requirement of a helper cell line. Over the last years, these viral recombinants have been utilized increasingly to analyse different aspects of the immune response against EBV. Immunological applications are manifold and steadily growing and include crude screening of T cell clones for their specificity towards latent versus lytic antigens, or more detailed analyses in which the exact specificity of T cells is determined using EBV mutants that lack a single viral antigen. Other applications include detailed analysis of protein domains important for immune recognition, e.g. Gly-Ala repeats in the EBV nuclear antigen 1 (EBNA1) protein, expansion of T cell clones directed against virion structures using virus-like particles and phenotypic analysis of virus mutants defective in infection. Future developments might

Causal Mediation Analysis of Survival Outcome with Multiple Mediators.

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Huang, Yen-Tsung; Yang, Hwai-I

2017-05-01

Mediation analyses have been a popular approach to investigate the effect of an exposure on an outcome through a mediator. Mediation models with multiple mediators have been proposed for continuous and dichotomous outcomes. However, development of multimediator models for survival outcomes is still limited. We present methods for multimediator analyses using three survival models: Aalen additive hazard models, Cox proportional hazard models, and semiparametric probit models. Effects through mediators can be characterized by path-specific effects, for which definitions and identifiability assumptions are provided. We derive closed-form expressions for path-specific effects for the three models, which are intuitively interpreted using a causal diagram. Mediation analyses using Cox models under the rare-outcome assumption and Aalen additive hazard models consider effects on log hazard ratio and hazard difference, respectively; analyses using semiparametric probit models consider effects on difference in transformed survival time and survival probability. The three models were applied to a hepatitis study where we investigated effects of hepatitis C on liver cancer incidence mediated through baseline and/or follow-up hepatitis B viral load. The three methods show consistent results on respective effect scales, which suggest an adverse estimated effect of hepatitis C on liver cancer not mediated through hepatitis B, and a protective estimated effect mediated through the baseline (and possibly follow-up) of hepatitis B viral load. Causal mediation analyses of survival outcome with multiple mediators are developed for additive hazard and proportional hazard and probit models with utility demonstrated in a hepatitis study.

An influenza viral vector Brucella abortus vaccine induces good cross-protection against Brucella melitensis infection in pregnant heifers.

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Tabynov, Kaissar; Ryskeldinova, Sholpan; Sansyzbay, Abylai

2015-07-17

Brucella melitensis can be transmitted and cause disease in cattle herds as a result of inadequate management of mixed livestock farms. Ideally, vaccines against Brucella abortus for cattle should also provide cross-protection against B. melitensis. Previously we created a novel influenza viral vector B. abortus (Flu-BA) vaccine expressing the Brucella ribosomal proteins L7/L12 or Omp16. This study demonstrated Flu-BA vaccine with adjuvant Montanide Gel01 provided 100% protection against abortion in vaccinated pregnant heifers and good cross-protection of the heifers and their calves or fetuses (90-100%) after challenge with B. melitensis 16M; the level of protection provided by Flu-BA was comparable to the commercial vaccine B. abortus S19. In terms of the index of infection and colonization of Brucella in tissues, both vaccines demonstrated significant (P=0.02 to P<0.0001) protection against B. melitensis 16M infection compared to the negative control group (PBS+Montanide Gel01). Thus, we conclude the Flu-BA vaccine provides cross-protection against B. melitensis infection in pregnant heifers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.

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Jiang, Xiaoou; Yu, Han; Teo, Cui Rong; Tan, Genim Siu Xian; Goh, Sok Chin; Patel, Parasvi; Chua, Yiqiang Kevin; Hameed, Nasirah Banu Sahul; Bertoletti, Antonio; Patzel, Volker

2016-09-01

Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.

Dysplastic hepatocytes develop nuclear inclusions in a mouse model of viral hepatitis.

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Priyanka Thakur

Full Text Available Viral hepatitis resulting in chronic liver disease is an important clinical challenge and insight into the cellular processes that drive pathogenesis will be critical in order to develop new diagnostic and therapeutic options. Nuclear inclusions in viral and non-viral hepatitis are well documented and have diagnostic significance in some disease contexts. However, the origins and functional consequences of these nuclear inclusions remain elusive. To date the clinical observation of nuclear inclusions in viral and non-viral hepatitis has not been explored at depth in murine models of liver disease. Herein, we report that in a transgenic model of hepatitis B surface antigen mediated hepatitis, murine hepatocytes exhibit nuclear inclusions. Cells bearing nuclear inclusions were more likely to express markers of cell proliferation. We also established a correlation between these inclusions and oxidative stress. N-acetyl cysteine treatment effectively reduced oxidative stress levels, relieved endoplasmic reticulum (ER stress, and the number of nuclear inclusions we observed in the transgenic mice. Our results suggest that the presence of nuclear inclusions in hepatocytes correlates with oxidative stress and cellular proliferation in a model of antigen mediated hepatitis.

Hepatitis C Virus: Viral Quasispecies and Genotypes.

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Tsukiyama-Kohara, Kyoko; Kohara, Michinori

2017-12-22

Hepatitis C virus (HCV) mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs), which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

Retrograde cholangiopancreatography in the diagnosis of biliary and pancreatic duct diseases

International Nuclear Information System (INIS)

Vasil'ev, Yu.D.; Sedletskaya, T.N.

1980-01-01

Results of retrograde cannulation with the aid of flexible fibroduodenoscopes with subsequent introduction of a contrast substance into biliary and pancreatic ducts are presented. The investigation is carried out on 120 patients with different diseases of hepatopancreatoduodenal zone. The standard technique of X-ray examination has been applied permitting to obtain the most exhaustive information. Using retrograde cholangiopancreatography revealed have been choledocholithiasis, deformation of biliary ducts after surgical intervention, pancreatic cyst, tumor of the main pancreatic duct etc. Results of investigation of biliary and pancreatic ducts using retrograde cannulation are reaffirmed with the data of operations on biliary tract in 72 patients. Intraoperational cholangiography has been carried out on 36 of them during operation. An attempt to cannulate big duodenal papilla in 12 patients proved to be ineffective. No complications have been observed during examination

A new retrograde transillumination technique for videolaryngoscopic tracheal intubation

DEFF Research Database (Denmark)

Biro, P; Fried, E; Schlaepfer, M

2018-01-01

This single-centre, prospective trial was designed to assess the efficacy of a new retrograde transillumination device called the 'Infrared Red Intubation System' (IRRIS) to aid videolaryngoscopic tracheal intubation. We included 40 adult patients, who were undergoing elective urological surgery......-10])), credibility (10 (8-10 [5-10])) and ease of use (10 (9-10 [8-10])). Tracheal intubation with the system lasted 26 (16-32 [6-89]) s. No alternative technique of securing the airway was necessary. The lowest SpO2 during intubation was 98 (97-99 [91-100])%. We conclude that this method of retrograde...

Development of a gene silencing DNA vector derived from a broad host range geminivirus

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Hancock Leandria C

2009-07-01

Full Text Available Abstract Background Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems. Results The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV, named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS, transketolase, the sulfur allele of magnesium chelatase (ChlI, and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant. Conclusion The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.

Foamy Virus Biology and Its Application for Vector Development

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Axel Rethwilm

2011-05-01

Full Text Available Spuma- or foamy viruses (FV, endemic in most non-human primates, cats, cattle and horses, comprise a special type of retrovirus that has developed a replication strategy combining features of both retroviruses and hepadnaviruses. Unique features of FVs include an apparent apathogenicity in natural hosts as well as zoonotically infected humans, a reverse transcription of the packaged viral RNA genome late during viral replication resulting in an infectious DNA genome in released FV particles and a special particle release strategy depending capsid and glycoprotein coexpression and specific interaction between both components. In addition, particular features with respect to the integration profile into the host genomic DNA discriminate FV from orthoretroviruses. It appears that some inherent properties of FV vectors set them favorably apart from orthoretroviral vectors and ask for additional basic research on the viruses as well as on the application in Gene Therapy. This review will summarize the current knowledge of FV biology and the development as a gene transfer system.

Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route.

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Carey, John B; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V S; Draper, Simon J; Moore, Anne C

2014-08-21

Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP1₄₂, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP1₄₂ also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP1₄₂ using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies.

Microneedle-mediated immunization of an adenovirus-based malaria vaccine enhances antigen-specific antibody immunity and reduces anti-vector responses compared to the intradermal route

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Carey, John B.; Vrdoljak, Anto; O'Mahony, Conor; Hill, Adrian V. S.; Draper, Simon J.; Moore, Anne C.

2014-01-01

Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP142, to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP142 also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP142 using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies. PMID:25142082

Virus infection mediates the effects of elevated CO2 on plants and vectors

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Trębicki, Piotr; Vandegeer, Rebecca K.; Bosque-Pérez, Nilsa A.; Powell, Kevin S.; Dader, Beatriz; Freeman, Angela J.; Yen, Alan L.; Fitzgerald, Glenn J.; Luck, Jo E.

2016-01-01

Atmospheric carbon dioxide (CO2) concentration has increased significantly and is projected to double by 2100. To increase current food production levels, understanding how pests and diseases respond to future climate driven by increasing CO2 is imperative. We investigated the effects of elevated CO2 (eCO2) on the interactions among wheat (cv. Yitpi), Barley yellow dwarf virus and an important pest and virus vector, the bird cherry-oat aphid (Rhopalosiphum padi), by examining aphid life history, feeding behavior and plant physiology and biochemistry. Our results showed for the first time that virus infection can mediate effects of eCO2 on plants and pathogen vectors. Changes in plant N concentration influenced aphid life history and behavior, and N concentration was affected by virus infection under eCO2. We observed a reduction in aphid population size and increased feeding damage on noninfected plants under eCO2 but no changes to population and feeding on virus-infected plants irrespective of CO2 treatment. We expect potentially lower future aphid populations on noninfected plants but no change or increased aphid populations on virus-infected plants therefore subsequent virus spread. Our findings underscore the complexity of interactions between plants, insects and viruses under future climate with implications for plant disease epidemiology and crop production. PMID:26941044

Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV in cattle

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Loy John Dustin

2013-01-01

Full Text Available Abstract Background Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen. Findings Replicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection. Conclusions Replicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.

Recycling Endosomes and Viral Infection.

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Vale-Costa, Sílvia; Amorim, Maria João

2016-03-08

Many viruses exploit specific arms of the endomembrane system. The unique composition of each arm prompts the development of remarkably specific interactions between viruses and sub-organelles. This review focuses on the viral-host interactions occurring on the endocytic recycling compartment (ERC), and mediated by its regulatory Ras-related in brain (Rab) GTPase Rab11. This protein regulates trafficking from the ERC and the trans-Golgi network to the plasma membrane. Such transport comprises intricate networks of proteins/lipids operating sequentially from the membrane of origin up to the cell surface. Rab11 is also emerging as a critical factor in an increasing number of infections by major animal viruses, including pathogens that provoke human disease. Understanding the interplay between the ERC and viruses is a milestone in human health. Rab11 has been associated with several steps of the viral lifecycles by unclear processes that use sophisticated diversified host machinery. For this reason, we first explore the state-of-the-art on processes regulating membrane composition and trafficking. Subsequently, this review outlines viral interactions with the ERC, highlighting current knowledge on viral-host binding partners. Finally, using examples from the few mechanistic studies available we emphasize how ERC functions are adjusted during infection to remodel cytoskeleton dynamics, innate immunity and membrane composition.

Neuropeptide Y Y1 receptor hippocampal overexpression via viral vectors is associated with modest anxiolytic-like and proconvulsant effects in mice

DEFF Research Database (Denmark)

Olesen, Mikkel V; Christiansen, Søren Hofman Oliveira; Gøtzsche, Casper René

2012-01-01

overexpression was found to be associated with modest anxiolytic-like effect in the open field and elevated plus maze tests, but no effect was seen on depression-like behavior using the tail suspension and forced swim tests. However, the rAAV-Y1 vector modestly aggravated kainate-induced seizures. These data...... in the hippocampus of adult mice and tested the animals in anxiety- and depression-like behavior. Hippocampal Y1 receptors have been suggested to mediate seizure-promoting effect, so the effects of rAAV-induced Y1 receptor overexpression were also tested in kainate-induced seizures. Y1 receptor transgene...

Improvement of oncolytic adenovirus vectors through genetic capsid modifications

NARCIS (Netherlands)

Vrij, Jeroen de

2012-01-01

Recombinant viral vectors hold great promise in the field of cancer gene therapy. While a plethora of viruses is being evaluated as oncolytic agents, human adenoviruses of serotype 5 (HAdV-5) are among the most popular of viruses to be developed. Although clinical studies have demonstrated safety of

Antegrade Ureteral Stenting is a Good Alternative for the Retrograde Approach.

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van der Meer, Rutger W; Weltings, Saskia; van Erkel, Arian R; Roshani, Hossain; Elzevier, Henk W; van Dijk, Lukas C; van Overhagen, Hans

2017-07-01

Double J (JJ) stents for treating obstructive ureteral pathology are generally inserted through a retrograde route with cystoscopic guidance. Antegrade percutaneous insertion using fluoroscopy can be performed alternatively but is less known. Indications, success rate and complications of antegrade ureteral stenting were evaluated. Data of consecutive patients in which antegrade ureteral stenting was performed were retrospectively analysed using the radiology information system and patient records. Patient characteristics, details of the antegrade JJ stent insertion procedure and registered complications were collected. Furthermore, it was investigated if prior to the antegrade procedure a retrograde attempt for JJ stent insertion was performed. Total 130 attempts for antegrade JJ stent insertion were performed in 100 patients. A percutaneous nephrostomy catheter had already been placed in the majority of kidneys (n = 109) for initial treatment of hydronephrosis. Most prevelant indication for a JJ stent was obstructive ureteral pathology due to malignancy (n = 63). A JJ stent was successfully inserted in 125 of 130 procedures. In 21 cases, previous retrograde ureteral stenting had failed but, subsequent antegrade ureteral stenting was successful. There were 8 procedure related complications; 6 infections, 1 false tract and 1 malposition. Antegrade percutaneous insertion of a JJ stent is a good alternative for retrograde insertion.

Radiation therapy with concurrent retrograde superselective intra-arterial chemotherapy for gingival carcinoma

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Mukai, Y.; Hata, M.; Koike, I.; Inoue, T. [Yokohama City University Graduate School of Medicine, Department of Radiology, Kanazawa-ku, Yokohama, Kanagawa (Japan); Mitsudo, K.; Koizumi, T.; Oguri, S.; Kioi, M.; Tohnai, I. [Yokohama City University Graduate School of Medicine, Department of Oral and Maxillofacial Surgery, Yokohama, Kanagawa (Japan); Omura, M. [Shonankamakura General Hospital, Department of Radiation Oncology, Kamakura, Kanagawa (Japan)

2014-02-15

The aim of this study was to review the efficacy and toxicity of radiation therapy with concurrent retrograde superselective intra-arterial chemotherapy in the treatment of gingival carcinoma. In all, 34 patients (21 men and 13 women) with squamous cell carcinoma of the gingiva underwent radiation therapy with concurrent retrograde superselective intra-arterial chemotherapy. Treatment consisted of daily external irradiation and concurrent retrograde superselective intra-arterial infusion with cisplatin and docetaxel. A median total dose of 60 Gy in 30 fractions was delivered to tumors. Of the 34 patients, 29 (85 %) achieved a complete response (CR) and 5 had residual tumors. Of the 29 patients with a CR, 2 had local recurrences and 1 had distant metastasis 1-15 months after treatment. Twenty-six of the 36 patients had survived at a median follow-up time of 36 months (range 12-79 months); 4 died of cancer and 4 died of non-cancer-related causes. At both 3 and 5 years after treatment, the overall survival rates were 79 % and the cause-specific survival rates were 85 %. Osteoradionecrosis of the mandibular bone only developed in 1 patient after treatment. Radiation therapy with concurrent retrograde superselective intra-arterial chemotherapy was effective and safe in the treatment of gingival carcinoma. This treatment may be a promising curative and organ-preserving treatment option for gingival carcinoma. (orig.) [German] Das Ziel dieser Studie war die Ueberpruefung der Effizienz und Toxizitaet einer Strahlenbehandlung des Gingivakarzinoms mit gleichzeitiger retrograder, superselektiver intraarterieller Chemotherapie. Insgesamt 34 Patienten (21 Maenner und 13 Frauen) mit Zahnfleischplattenzellkarzinom erhielten eine Strahlenbehandlung mit gleichzeitiger retrograder, superselektiver intraarterieller Chemotherapie. Die Behandlung umfasste eine taegliche externe Bestrahlung mit gleichzeitiger retrograder, superselektiver intraarterieller Infusion von Cisplatin und

Reprogramming of murine macrophages through TLR2 confers viral resistance via TRAF3-mediated, enhanced interferon production.

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Darren J Perkins

Full Text Available The cell surface/endosomal Toll-like Receptors (TLRs are instrumental in initiating immune responses to both bacteria and viruses. With the exception of TLR2, all TLRs and cytosolic RIG-I-like receptors (RLRs with known virus-derived ligands induce type I interferons (IFNs in macrophages or dendritic cells. Herein, we report that prior ligation of TLR2, an event previously shown to induce "homo" or "hetero" tolerance, strongly "primes" macrophages for increased Type I IFN production in response to subsequent TLR/RLR signaling. This occurs by increasing activation of the transcription factor, IFN Regulatory Factor-3 (IRF-3 that, in turn, leads to enhanced induction of IFN-β, while expression of other pro-inflammatory genes are suppressed (tolerized. In vitro or in vivo "priming" of murine macrophages with TLR2 ligands increase virus-mediated IFN induction and resistance to infection. This priming effect of TLR2 is mediated by the selective upregulation of the K63 ubiquitin ligase, TRAF3. Thus, we provide a mechanistic explanation for the observed antiviral actions of MyD88-dependent TLR2 and further define the role of TRAF3 in viral innate immunity.

Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers.

NARCIS (Netherlands)

Yang, X.; Walboomers, X.F.; Dolder, J. van den; Yang, F.; Bian, Z.; Fan, M.; Jansen, J.A.

2008-01-01

Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and

Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

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Stacia L. Phillips

2016-01-01

Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

Generation of transgene-free induced pluripotent stem cells with non-viral methods.

Science.gov (United States)

Wang, Tao; Zhao, Hua-shan; Zhang, Qiu-ling; Xu, Chang-lin; Liu, Chang-bai

2013-03-01

Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.

Hepatitis C Virus: Viral Quasispecies and Genotypes

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Kyoko Tsukiyama-Kohara

2017-12-01

Full Text Available Hepatitis C virus (HCV mainly replicates in the cytoplasm, where it easily establishes persistent infection, resulting in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Due to its high rate of mutation, HCV forms viral quasispecies, categorized based on the highly variable regions in the envelope protein and nonstructural 5A protein. HCV possesses seven major genotypes, among which genotype 1 is the most prevalent globally. The distribution of HCV genotypes varies based on geography, and each genotype has a different sensitivity to interferon treatment. Recently-developed direct-acting antivirals (DAAs, which target viral proteases or polymerases, mediate drastically better antiviral effects than previous therapeutics. Although treatment with DAAs has led to the development of drug-resistant HCV mutants, the most recently approved DAAs show improved pan-genomic activity, with a higher barrier to viral resistance.

Viral Vector Mediated Over-Expression of Estrogen Receptor–α in Striatum Enhances the Estradiol-induced Motor Activity in Female Rats and Estradiol Modulated GABA Release

Science.gov (United States)

Schultz, Kristin N.; von Esenwein, Silke A.; Hu, Ming; Bennett, Amy L.; Kennedy, Robert T.; Musatov, Sergei; Toran-Allerand, C. Dominique; Kaplitt, Michael G.; Young, Larry J.; Becker, Jill B.

2009-01-01

Classical estrogen receptor signaling mechanisms involve estradiol binding to intracellular nuclear receptors (estrogen receptor-α (ERα) and estrogen receptor-β (ERβ)) to promote changes in protein expression. Estradiol can also exert effects within seconds to minutes, however, a timescale incongruent with genomic signaling. In the brain, estradiol rapidly potentiates stimulated dopamine release in the striatum of female rats and enhances spontaneous rotational behavior. Furthermore, estradiol rapidly attenuates the K+- evoked increase of GABA in dialysate. We hypothesize that these rapid effects of estradiol in the striatum are mediated by ERα located on the membrane of medium spiny GABAergic neurons. This experiment examined whether over-expression of ERα in the striatum would enhance the effect of estradiol on rotational behavior and the K+- evoked increase in GABA in dialysate. Ovariectomized female rats were tested for rotational behavior or underwent microdialysis experiments after unilateral intrastriatal injections of a recombinant adeno-associated virus (AAV) containing the human ERα cDNA (AAV.ERα) into the striatum; controls received either the same vector into areas outside the striatum or an AAV containing the human alkaline phosphatase gene into the striatum (AAV.ALP). Animals that received AAV.ERα in the striatum exhibited significantly greater estradiol-induced contralateral rotations compared to controls and exhibited behavioral sensitization of contralateral rotations induced by a low dose of amphetamine. ERα over-expression also enhanced the inhibitory effect of estradiol on K+- evoked GABA release suggesting that disinhibition of dopamine release from terminals in the striatum resulted in the enhanced rotational behavior. PMID:19211896

Real-time visualization and quantification of retrograde cardioplegia delivery using near infrared fluorescent imaging.

Science.gov (United States)

Rangaraj, Aravind T; Ghanta, Ravi K; Umakanthan, Ramanan; Soltesz, Edward G; Laurence, Rita G; Fox, John; Cohn, Lawrence H; Bolman, R M; Frangioni, John V; Chen, Frederick Y

2008-01-01

Homogeneous delivery of cardioplegia is essential for myocardial protection during cardiac surgery. Presently, there exist no established methods to quantitatively assess cardioplegia distribution intraoperatively and determine when retrograde cardioplegia is required. In this study, we evaluate the feasibility of near infrared (NIR) imaging for real-time visualization of cardioplegia distribution in a porcine model. A portable, intraoperative, real-time NIR imaging system was utilized. NIR fluorescent cardioplegia solution was developed by incorporating indocyanine green (ICG) into crystalloid cardioplegia solution. Real-time NIR imaging was performed while the fluorescent cardioplegia solution was infused via the retrograde route in five ex vivo normal porcine hearts and in five ex vivo porcine hearts status post left anterior descending (LAD) coronary artery ligation. Horizontal cross-sections of the hearts were obtained at proximal, middle, and distal LAD levels. Videodensitometry was performed to quantify distribution of fluorophore content. The progressive distribution of cardioplegia was clearly visualized with NIR imaging. Complete visualization of retrograde distribution occurred within 4 minutes of infusion. Videodensitometry revealed retrograde cardioplegia, primarily distributed to the left ventricle (LV) and anterior septum. In hearts with LAD ligation, antegrade cardioplegia did not distribute to the anterior LV. This deficiency was compensated for with retrograde cardioplegia supplementation. Incorporation of ICG into cardioplegia allows real-time visualization of cardioplegia delivery via NIR imaging. This technology may prove useful in guiding intraoperative decisions pertaining to when retrograde cardioplegia is mandated.

Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.

Science.gov (United States)

Asenjo, Ana; Villanueva, Nieves

2016-01-04

The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Anxiolytic-like effects after vector-mediated overexpression of neuropeptide Y in the amygdala and hippocampus of mice

DEFF Research Database (Denmark)

Christiansen, Søren Hofman Oliveira; Olesen, Mikkel Vestergaard; Gøtzsche, Casper René

2014-01-01

, injections of rAAV-NPY caused significant anxiolytic-like effect in the open field, elevated plus maze, and light-dark transition tests. In the hippocampus, rAAV-NPY treatment was associated with anxiolytic-like effect only in the elevated plus maze. No additive effect was observed after combined r....... Using a recombinant adeno-associated viral (rAAV) vector, we addressed this idea by testing effects on anxiolytic- and depression-like behaviours in adult mice after overexpression of NPY transgene in the amygdala and/or hippocampus, two brain regions implicated in emotional behaviours. In the amygdala......AAV-NPY injection into both the amygdala and hippocampus where anxiolytic-like effect was found in the elevated plus maze and light-dark transition tests. Antidepressant-like effects were not detected in any of the rAAV-NPY injected groups. Immobility was even increased in the tail suspension and forced swim tests...

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

Science.gov (United States)

Prang, N; Wolf, H; Schwarzmann, F

1999-12-01

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo. Copyright 1999 Wiley-Liss, Inc.

Right retrograde brachial cerebral angiography with simultaneous compression of the left carotid artery

International Nuclear Information System (INIS)

Ericson, K.; Mosskin, M.

1981-01-01

Right retrograde brachial angiography with simultaneous compression of the left common carotid artery was performed in 12 patients, invariably resulting in filling of the right vertebral and the basilar artery. In all but one patient, the right carotid artery and its branches were also filled. Retrograde filling of the left internal carotid artery occurred in 8 patients. Furthermore, retrograde filling of the intracranial part of the left vertebral artery was obtained in 5 of 12 patients. A complete four-vessel cranial angiography was thus obtained in one third of the patients. The method may be considered as a safe and valuable adjunct to other angiographic techniques. (Auth.)

A metagenomic survey of viral abundance and diversity in mosquitoes from Hubei province.

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Chenyan Shi

Full Text Available Mosquitoes as one of the most common but important vectors have the potential to transmit or acquire a lot of viruses through biting, however viral flora in mosquitoes and its impact on mosquito-borne disease transmission has not been well investigated and evaluated. In this study, the metagenomic techniquehas been successfully employed in analyzing the abundance and diversity of viral community in three mosquito samples from Hubei, China. Among 92,304 reads produced through a run with 454 GS FLX system, 39% have high similarities with viral sequences belonging to identified bacterial, fungal, animal, plant and insect viruses, and 0.02% were classed into unidentified viral sequences, demonstrating high abundance and diversity of viruses in mosquitoes. Furthermore, two novel viruses in subfamily Densovirinae and family Dicistroviridae were identified, and six torque tenosus virus1 in family Anelloviridae, three porcine parvoviruses in subfamily Parvovirinae and a Culex tritaeniorhynchus rhabdovirus in Family Rhabdoviridae were preliminarily characterized. The viral metagenomic analysis offered us a deep insight into the viral population of mosquito which played an important role in viral initiative or passive transmission and evolution during the process.

A Metagenomic Survey of Viral Abundance and Diversity in Mosquitoes from Hubei Province

Science.gov (United States)

Shi, Chenyan; Liu, Yi; Hu, Xiaomin; Xiong, Jinfeng; Zhang, Bo; Yuan, Zhiming

2015-01-01

Mosquitoes as one of the most common but important vectors have the potential to transmit or acquire a lot of viruses through biting, however viral flora in mosquitoes and its impact on mosquito-borne disease transmission has not been well investigated and evaluated. In this study, the metagenomic techniquehas been successfully employed in analyzing the abundance and diversity of viral community in three mosquito samples from Hubei, China. Among 92,304 reads produced through a run with 454 GS FLX system, 39% have high similarities with viral sequences belonging to identified bacterial, fungal, animal, plant and insect viruses, and 0.02% were classed into unidentified viral sequences, demonstrating high abundance and diversity of viruses in mosquitoes. Furthermore, two novel viruses in subfamily Densovirinae and family Dicistroviridae were identified, and six torque tenosus virus1 in family Anelloviridae, three porcine parvoviruses in subfamily Parvovirinae and a Culex tritaeniorhynchus rhabdovirus in Family Rhabdoviridae were preliminarily characterized. The viral metagenomic analysis offered us a deep insight into the viral population of mosquito which played an important role in viral initiative or passive transmission and evolution during the process. PMID:26030271

The aryl hydrocarbon receptor is a modulator of anti-viral immunity

Science.gov (United States)

Head, Jennifer L.; Lawrence, B. Paige

2009-01-01

Although immune modulation by AhR ligands has been studied for many years, the impact of AhR activation on host defenses against viral infection has not, until recently, garnered much attention. The development of novel reagents and model systems, new information regarding antiviral immunity, and a growing appreciation for the global health threat posed by viruses have invigorated interest in understanding how environmental signals affect susceptibility to and pathological consequences of viral infection. Using influenza A virus as a model of respiratory viral infection, recent studies show that AhR activation cues signaling events in both leukocytes and non-immune cells. Functional alterations include suppressed lymphocyte responses and increased inflammation in the infected lung. AhR-mediated events within and extrinsic to hematopoietic cells has been investigated using bone marrow chimeras, which show that AhR alters different elements of the immune response by affecting different tissue targets. In particular, suppressed CD8+ T cell responses are due to deregulated events within leukocytes themselves, whereas increased neutrophil recruitment to and IFN-γ levels in the lung result from AhR-regulated events extrinsic to bone marrow-derived cells. This latter discovery suggests that epithelial and endothelial cells are overlooked targets of AhR-mediated changes in immune function. Further support that AhR influences host cell responses to viral infection are provided by several studies demonstrating that AhR interacts directly with viral proteins and affects viral latency. While AhR clearly modulates host responses to viral infection, we still have much to understand about the complex interactions between immune cells, viruses, and the host environment. PMID:19027719

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

Science.gov (United States)

Mei, Yu; Zhang, Chunquan; Kernodle, Bliss M; Hill, John H; Whitham, Steven A

2016-06-01

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. © 2016 American Society of Plant Biologists. All Rights Reserved.

Profound loss of general knowledge in retrograde amnesia: Evidence from an amnesic artist

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Emma eGregory

2014-05-01

Full Text Available Studies of retrograde amnesia have focused on autobiographical memory, with fewer studies examining how non-autobiographical memory is affected. Those that have done so have focused primarily on memory for famous people and public events—relatively limited aspects of memory that are tied to learning during specific times of life and do not deeply tap into the rich and extensive knowledge structures that are developed over a lifetime. To assess whether retrograde amnesia can also cause impairments to other forms of general world knowledge, we explored losses across a broad range of knowledge domains in a newly-identified amnesic. LSJ is a professional artist, amateur musician and history buff with extensive bilateral medial temporal and left anterior temporal damage. We examined LSJ's knowledge across a range of everyday domains (e.g., sports and domains for which she had premorbid expertise (e.g., famous paintings. Across all domains tested, LSJ showed losses of knowledge at a level of breadth and depth never before documented in retrograde amnesia. These results show that retrograde amnesia can involve broad and deep deficits across a range of general world knowledge domains. Thus, losses that have already been well-documented (famous people and public events may severely underestimate the nature of human knowledge impairment that can occur in retrograde amnesia.

Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

Science.gov (United States)

Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

2014-09-01

Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. © 2014 Wiley Periodicals, Inc.

Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

International Nuclear Information System (INIS)

Kennedy, Edward M.; Cullen, Bryan R.

2015-01-01

CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

Energy Technology Data Exchange (ETDEWEB)

Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

2015-05-15

CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

Synchronous Retrograde and Micturating Cysto Urethrography A ...

African Journals Online (AJOL)

Background: Retrograde Urethrography (RUG) combined with Micturating cystourethrography (MCUG) is imaging method of choice for studying the urethra and its 1-9 abnormalities . Though there are many modern imaging modalities that are also useful but these are not available in most developing countries. Even the ...

Effective genetic modification and differentiation of hMSCs upon controlled release of rAAV vectors using alginate/poloxamer composite systems.

Science.gov (United States)

Díaz-Rodríguez, P; Rey-Rico, A; Madry, H; Landin, M; Cucchiarini, M

2015-12-30

Viral vectors are common tools in gene therapy to deliver foreign therapeutic sequences in a specific target population via their natural cellular entry mechanisms. Incorporating such vectors in implantable systems may provide strong alternatives to conventional gene transfer procedures. The goal of the present study was to generate different hydrogel structures based on alginate (AlgPH155) and poloxamer PF127 as new systems to encapsulate and release recombinant adeno-associated viral (rAAV) vectors. Inclusion of rAAV in such polymeric capsules revealed an influence of the hydrogel composition and crosslinking temperature upon the vector release profiles, with alginate (AlgPH155) structures showing the fastest release profiles early on while over time vector release was more effective from AlgPH155+PF127 [H] capsules crosslinked at a high temperature (50°C). Systems prepared at room temperature (AlgPH155+PF127 [C]) allowed instead to achieve a more controlled release profile. When tested for their ability to target human mesenchymal stem cells, the different systems led to high transduction efficiencies over time and to gene expression levels in the range of those achieved upon direct vector application, especially when using AlgPH155+PF127 [H]. No detrimental effects were reported on either cell viability or on the potential for chondrogenic differentiation. Inclusion of PF127 in the capsules was also capable of delaying undesirable hypertrophic cell differentiation. These findings are of promising value for the further development of viral vector controlled release strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

Retrograde prostatic urethroplasty with a balloon catheter

International Nuclear Information System (INIS)

Castaneda, F.; Reddy, P.; Hulbert, J.; Letourneau, J.G.; Hunter, D.W.; Castaneda-Zuniga, W.R.; Amplatz, K.

1987-01-01

Twenty-five patients with prostatism and documented BPH who were candidates for transurethral resection of the prostate were dilated for 10 minutes with 25-mm urethroplasty balloons using a retrograde transurethral approach. The procedure was performed under local anesthesia using 2% viscous lidocaine on an outpatient basis. A mild discomfort was experienced by all patients with a moderate urgency sensation. Mild transient hematuria was present in all, which cleared in 4 to 6 hours. Dysuria usually lasted for 72 hours. Significant improvement has been seen in the relief of symptoms in patients without middle-lobe hypertrophy as documented by uroflow studies, voiding cystourethrograms, and retrograde urethrograms. In patients with middle-lobe hypertrophy, moderate improvement in uroflow studies was observed, which correlated well with symptomatic improvement. Rectal US and MR studies have shown no evidence of intraprostatic or periprostatic abnormalities. No complications have been encountered so far. The longest current follow-up is 20 months, with a mean of 10 months

A Robust and Efficient Numerical Method for RNA-Mediated Viral Dynamics

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Vladimir Reinharz

2017-10-01

Full Text Available The multiscale model of hepatitis C virus (HCV dynamics, which includes intracellular viral RNA (vRNA replication, has been formulated in recent years in order to provide a new conceptual framework for understanding the mechanism of action of a variety of agents for the treatment of HCV. We present a robust and efficient numerical method that belongs to the family of adaptive stepsize methods and is implicit, a Rosenbrock type method that is highly suited to solve this problem. We provide a Graphical User Interface that applies this method and is useful for simulating viral dynamics during treatment with anti-HCV agents that act against HCV on the molecular level.

Retrograde flow in the dural sinuses detected by three-dimensional time-of-flight MR angiography

International Nuclear Information System (INIS)

Uchino, Akira; Nomiyama, Keita; Takase, Yukinori; Nakazono, Takahiko; Tominaga, Yukiko; Imaizumi, Takeshi; Kudo, Sho

2007-01-01

Retrograde flow in the left dural sinuses is sometimes detected by three-dimensional time-of-flight (3D-TOF) magnetic resonance (MR) angiography. The purpose of this study was to evaluate the incidence of this phenomenon and its characteristic features on 3D-TOF MR angiograms. We retrospectively reviewed cranial MR angiography images of 1,078 patients examined at our institution. All images were obtained by the 3D-TOF technique with one of two 1.5-T scanners. Maximum intensity projection (MIP) images in the horizontal rotation view were displayed stereoscopically. We reviewed the source images, inferosuperior MIP images, and horizontal MIP images and identified retrograde flow in the dural sinuses. We found retrograde flow in the dural sinuses of 67 patients on the source images from 3D-TOF MR angiography; the incidence was 6.2%. In 47 of the 67 patients, retrograde flow was identified in the left inferior petrosal sinus, in 13, it was seen in the left sigmoid sinus, and in 6, it was seen in the left inferior petrosal and left sigmoid sinuses. The remaining patient had retrograde flow in the left inferior petrosal and left and right sigmoid sinuses. The mean age of the patients with retrograde flow was slightly greater than that of the patients without this phenomenon (70 years vs 63 years). Retrograde flow in the dural sinuses frequently occurs on the left side in middle-aged and elderly patients during 3D-TOF MR angiography performed with the patient in the supine position. This phenomenon should not be misdiagnosed as a dural arteriovenous fistula. (orig.)

A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants.

Science.gov (United States)

Kelloniemi, Jani; Mäkinen, Kristiina; Valkonen, Jari P T

2006-05-01

Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.

Eicosanoids and Respiratory Viral Infection: Coordinators of Inflammation and Potential Therapeutic Targets

Directory of Open Access Journals (Sweden)

Mary K. McCarthy

2012-01-01

Full Text Available Viruses are frequent causes of respiratory infection, and viral respiratory infections are significant causes of hospitalization, morbidity, and sometimes mortality in a variety of patient populations. Lung inflammation induced by infection with common respiratory pathogens such as influenza and respiratory syncytial virus is accompanied by increased lung production of prostaglandins and leukotrienes, lipid mediators with a wide range of effects on host immune function. Deficiency or pharmacologic inhibition of prostaglandin and leukotriene production often results in a dampened inflammatory response to acute infection with a respiratory virus. These mediators may, therefore, serve as appealing therapeutic targets for disease caused by respiratory viral infection.

Biomechanical Strength of Retrograde Fixation in Proximal Third Scaphoid Fractures.

Science.gov (United States)

Daly, Charles A; Boden, Allison L; Hutton, William C; Gottschalk, Michael B

2018-04-01

Current techniques for fixation of proximal pole scaphoid fractures utilize antegrade fixation via a dorsal approach endangering the delicate vascular supply of the dorsal scaphoid. Volar and dorsal approaches demonstrate equivalent clinical outcomes in scaphoid wrist fractures, but no study has evaluated the biomechanical strength for fractures of the proximal pole. This study compares biomechanical strength of antegrade and retrograde fixation for fractures of the proximal pole of the scaphoid. A simulated proximal pole scaphoid fracture was produced in 22 matched cadaveric scaphoids, which were then assigned randomly to either antegrade or retrograde fixation with a cannulated headless compression screw. Cyclic loading and load to failure testing were performed and screw length, number of cycles, and maximum load sustained were recorded. There were no significant differences in average screw length (25.5 mm vs 25.6 mm, P = .934), average number of cyclic loading cycles (3738 vs 3847, P = .552), average load to failure (348 N vs 371 N, P = .357), and number of catastrophic failures observed between the antegrade and retrograde fixation groups (3 in each). Practical equivalence between the 2 groups was calculated and the 2 groups were demonstrated to be practically equivalent (upper threshold P = .010). For this model of proximal pole scaphoid wrist fractures, antegrade and retrograde screw configuration have been proven to be equivalent in terms of biomechanical strength. With further clinical study, we hope surgeons will be able to make their decision for fixation technique based on approaches to bone grafting, concern for tenuous blood supply, and surgeon preference without fear of poor biomechanical properties.

Improving Mycobacterium bovis Bacillus Calmette-Guèrin as a Vaccine Delivery Vector for Viral Antigens by Incorporation of Glycolipid Activators of NKT Cells

OpenAIRE

Venkataswamy, Manjunatha M.; Ng, Tony W.; Kharkwal, Shalu S.; Carreño, Leandro J.; Johnson, Alison J.; Kunnath-Velayudhan, Shajo; Liu, Zheng; Bittman, Robert; Jervis, Peter J.; Cox, Liam R.; Besra, Gurdyal S.; Wen, Xiangshu; Yuan, Weiming; Tsuji, Moriya; Li, Xiangming

2014-01-01

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approac...

Supplementary Material for: CRISPR/Cas9-mediated viral interference in plants

KAUST Repository

Ali, Zahir; Abulfaraj, Aala A.; Idris, Ali; Ali, Shakila; Tashkandi, Manal; Mahfouz, Magdy

2015-01-01

Abstract Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

Management of Endovascular Aortic Aneurysm Complications via Retrograde Catheterization Through the Distal Stent-Graft Landing Zone.

Science.gov (United States)

Zhang, Xicheng; Sun, Yuan; Chen, Zhaolei; Jing, Yuanhu; Xu, Miao

2017-08-01

A retrograde technique through the gap between the distal stent landing zone and the iliac artery wall has been applied to treat type II endoleak after endovascular aortic aneurysm repair (EVAR). In this study, we tried to investigate its efficacy in the management of type III endoleak and intraoperative accidental events. We reported 2 complications of EVAR that were difficult to treat with conventional methods. One patient had a sustained type III endoleak after EVAR, and the right renal artery was accidentally sealed by a graft stent in the other patient during the operation. Both complications were managed by the retrograde technique from the distal stent landing zone. In the first case, the endoleak was easily embolized by the retrograde catheterization technique, and in the second case, a stent was implanted in the right renal artery using the retrograde technique to restore blood flow. In some EVAR cases, the technique of retrograde catheterization through the distal stent-graft landing zone is feasible, safe, and easy to perform.

Retrograde CTO-PCI of Native Coronary Arteries Via Left Internal Mammary Artery Grafts: Insights From a Multicenter U.S. Registry.

Science.gov (United States)

Tajti, Peter; Karatasakis, Aris; Karmpaliotis, Dimitri; Alaswad, Khaldoon; Jaffer, Farouc A; Yeh, Robert W; Patel, Mitul; Mahmud, Ehtisham; Choi, James W; Doing, Anthony H; Toma, Catalin; Uretsky, Barry; Garcia, Santiago; Moses, Jeffrey W; Parikh, Manish; Kirtane, Ajay; Ali, Ziad A; Hatem, Raja; Karacsonyi, Judit; Danek, Barbara A; Rangan, Bavana V; Banerjee, Subhash; Ungi, Imre; Brilakis, Emmanouil S

2018-03-01

Retrograde percutaneous coronary intervention (PCI) of native coronary artery chronic total occlusion (CTO) via left internal mammary artery (LIMA) graft has received limited study. We compared the clinical and procedural characteristics and outcomes of retrograde CTO-PCI through LIMA grafts vs other conduits in a contemporary multicenter CTO registry. The LIMA was used as the collateral channel in 20 of 990 retrograde CTO-PCIs (2.02%) performed at 18 United States centers. The mean age of the study patients was 69 ± 7 years and 95% were men. The most common CTO target vessel was the right coronary artery (55%). The mean J-CTO score in the LIMA group was high (3.45 ± 0.76). The technical success rates were 70% for retrograde PCI via LIMA graft vs 81.05% for retrograde via other conduits (P=.25), while procedural success rates were 70% for retrograde PCI via LIMA graft and 78.19% for retrograde via other conduits (P=.41). The incidence of major in-hospital complications was also similar between the LIMA and non-LIMA retrograde groups (5% vs 6%; P>.99). Use of guide-catheter extensions (40% vs 28%; P=.22), intravascular ultrasound (45% vs 31%; P=.20), and left ventricular assist devices (24% vs 10%; P=.08) was numerically higher in retrograde CTO-PCIs via LIMA grafts. Retrograde CTO-PCI is infrequently performed via LIMA grafts and is associated with similar success and major in-hospital complication rates as retrograde CTO-PCI performed via other conduits.

Tailored HIV-1 vectors for genetic modification of primary human dendritic cells and monocytes.

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Durand, Stéphanie; Nguyen, Xuan-Nhi; Turpin, Jocelyn; Cordeil, Stephanie; Nazaret, Nicolas; Croze, Séverine; Mahieux, Renaud; Lachuer, Joël; Legras-Lachuer, Catherine; Cimarelli, Andrea

2013-01-01

Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.

Impact of Emulsifiers Addition on the Retrogradation of Rice Gels during Low-Temperature Storage

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Zhe Yang

2017-01-01

Full Text Available Rice and its products are widely consumed in Asian countries; however, starch retrogradation decreases the quality and shortens the shelf-life of rice foods particularly at low temperature. In this study sucrose ester (SE, glycerol monostearate (GMS, and sodium stearoyl lactylate (SSL were added to rice flour and corresponding rice gels. Then, gelatinization properties, retrogradation characteristics, texture, and water content of these rice gels were investigated at 4°C and −20°C storage, respectively. The results demonstrated that the rice gels with 0.2% GMS had the lowest retrogradation index (ΔHr/ΔHg (11.84% and hardness (1359 g at 4°C for a 10 d period, which was significantly lower in comparison to control and the other two emulsifiers (P<0.05. Adhesiveness and water content were increased compared to the other samples. Furthermore, the retrogradation of rice gels stored at 4°C was comparatively rapid compared to gels stored at −20°C. Gel samples stored at −20°C were still acceptable for more than 15 days. Thus it was revealed that GMS has the potential to retard starch retrogradation and produce high-quality rice products in preservation.

Proteomic Analysis of Interaction between a Plant Virus and Its Vector Insect Reveals New Functions of Hemipteran Cuticular Protein.

Science.gov (United States)

Liu, Wenwen; Gray, Stewart; Huo, Yan; Li, Li; Wei, Taiyun; Wang, Xifeng

2015-08-01

Numerous viruses can be transmitted by their corresponding vector insects; however, the molecular mechanisms enabling virus transmission by vector insects have been poorly understood, especially the identity of vector components interacting with the virus. Here, we used the yeast two-hybrid system to study proteomic interactions of a plant virus (Rice stripe virus, RSV, genus Tenuivirus) with its vector insect, small brown planthopper (Laodelphax striatellus). Sixty-six proteins of L. striatellus that interacted with the nucleocapsid protein (pc3) of RSV were identified. A virus-insect interaction network, constructed for pc3 and 29 protein homologs of Drosophila melanogaster, suggested that nine proteins might directly interact with pc3. Of the 66 proteins, five (atlasin, a novel cuticular protein, jagunal, NAC domain protein, and vitellogenin) were most likely to be involved in viral movement, replication, and transovarial transmission. This work also provides evidence that the novel cuticular protein, CPR1, from L. striatellus is essential for RSV transmission by its vector insect. CPR1 binds the nucleocapsid protein (pc3) of RSV both in vivo and in vitro and colocalizes with RSV in the hemocytes of L. striatellus. Knockdown of CPR1 transcription using RNA interference resulted in a decrease in the concentration of RSV in the hemolymph, salivary glands and in viral transmission efficiency. These data suggest that CPR1 binds RSV in the insect and stabilizes the viral concentration in the hemolymph, perhaps to protect the virus or to help move the virus to the salivary tissues. Our studies provide direct experimental evidence that viruses can use existing vector proteins to aid their survival in the hemolymph. Identifying these putative vector molecules should lead to a better understanding of the interactions between viruses and vector insects. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Growth Inhibition of Breast Cancer in Rat by AAV Mediated Angiostatin Gene

Institute of Scientific and Technical Information of China (English)

LI Ran; CHEN Hong; REN Chang-shan

2007-01-01

Objective: To observe growth inhibition effect of adeno-associated viral vectors (AAV) mediated angiostatin (ANG) gene on implanted breast cancer in rat and its mechanism. Methods: Gene transfer technique was used to transfer AAV-ANG to the tumor. Growth curves were drawn to observe the growth of breast cancer implanted in rat, and immunohistochemical method was used to detect the effects of angiostatin on microvesel density (MVD) of breast cancer implanted in rat. Results: Angiostatin inhibited the growth of breast cancer implanted in rat and decreased the microvessel density of tumor. Conclusion: Expression of an angiostatin transgene can suppress the growth of breast cancer implanted in rat through the inhibition of the growth of microvessels, surggesting that angiostatin gene transfer technique may be effective against breast cancer.

Irrelevant, Incidental and Core Features in the Retrograde Amnesia Associated with Korsakoff’s Psychosis: A Review

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P. R. Meudell

1992-01-01

Full Text Available A brief review of the literature on retrograde amnesia in Korsakoff's syndrome is presented. Various explanations of the phenomenon are discussed including the notions that it results from the effects of “state-dependency”, that it occurs as a result of a progressive learning problem and that it arises through a failure in contextual processing. None of these hypotheses can satisfactorily account for the length and temporal gradient of alcoholic amnesics retrograde amnesia. Although some evidence points towards the hypothesis that anterograde and retrograde amnesia might result from separate and independent impairments, this view is presently unproven and leaves open what causes the form and duration of Korsakoffs retrograde amnesia.

On the observed excess of retrograde orbits among long-period comets

International Nuclear Information System (INIS)

Fernandez, J.A.

1981-01-01

The distribution of orbital inclinations of the observed long-period comets is analysed. An excess of retrograde orbits is found which increases with the perihelion distance, except for the range 1.1 10 3 A U) has the same behaviour as the total sample. It is thus suggested that the excess of retrograde orbits among long-period comets is related to an already existent excess among the incoming new comets (i.e. comets driven into the planetary region by stellar perturbations). Using theoretical considerations and a numerical model it is proposed that an important fraction of the so-called new comets are actually repeating passages through the planetary region. Nearly a half of the new comets with q > 2 A U may be repeating passages. An important consequence of the presence of comets repeating passages among the new ones is the production of an excess of retrograde orbits in the whole sample. (author)

An integrated approach to elucidate the intra-viral and viral-cellular protein interaction networks of a gamma-herpesvirus.

Directory of Open Access Journals (Sweden)

Shaoying Lee

2011-10-01

Full Text Available Genome-wide yeast two-hybrid (Y2H screens were conducted to elucidate the molecular functions of open reading frames (ORFs encoded by murine γ-herpesvirus 68 (MHV-68. A library of 84 MHV-68 genes and gene fragments was generated in a Gateway entry plasmid and transferred to Y2H vectors. All possible pair-wise interactions between viral proteins were tested in the Y2H assay, resulting in the identification of 23 intra-viral protein-protein interactions (PPIs. Seventy percent of the interactions between viral proteins were confirmed by co-immunoprecipitation experiments. To systematically investigate virus-cellular protein interactions, the MHV-68 Y2H constructs were screened against a cellular cDNA library, yielding 243 viral-cellular PPIs involving 197 distinct cellar proteins. Network analyses indicated that cellular proteins targeted by MHV-68 had more partners in the cellular PPI network and were located closer to each other than expected by chance. Taking advantage of this observation, we scored the cellular proteins based on their network distances from other MHV-68-interacting proteins and segregated them into high (Y2H-HP and low priority/not-scored (Y2H-LP/NS groups. Significantly more genes from Y2H-HP altered MHV-68 replication when their expression was inhibited with siRNAs (53% of genes from Y2H-HP, 21% of genes from Y2H-LP/NS, and 16% of genes randomly chosen from the human PPI network; p<0.05. Enriched Gene Ontology (GO terms in the Y2H-HP group included regulation of apoptosis, protein kinase cascade, post-translational protein modification, transcription from RNA polymerase II promoter, and IκB kinase/NFκB cascade. Functional validation assays indicated that PCBP1, which interacted with MHV-68 ORF34, may be involved in regulating late virus gene expression in a manner consistent with the effects of its viral interacting partner. Our study integrated Y2H screening with multiple functional validation approaches to create �

Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta.

Science.gov (United States)

Eskelin, Katri; Hafrén, Anders; Rantalainen, Kimmo I; Mäkinen, Kristiina

2011-09-01

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.

Potyviral VPg Enhances Viral RNA Translation and Inhibits Reporter mRNA Translation In Planta▿

Science.gov (United States)

Eskelin, Katri; Hafrén, Anders; Rantalainen, Kimmo I.; Mäkinen, Kristiina

2011-01-01

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5′ untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation. PMID:21697470

Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

International Nuclear Information System (INIS)

Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

2012-01-01

Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

Psychogenic amnesia: syndromes, outcome, and patterns of retrograde amnesia.

Science.gov (United States)

Harrison, Neil A; Johnston, Kate; Corno, Federica; Casey, Sarah J; Friedner, Kimberley; Humphreys, Kate; Jaldow, Eli J; Pitkanen, Mervi; Kopelman, Michael D

2017-09-01

There are very few case series of patients with acute psychogenic memory loss (also known as dissociative/functional amnesia), and still fewer studies of outcome, or comparisons with neurological memory-disordered patients. Consequently, the literature on psychogenic amnesia is somewhat fragmented and offers little prognostic value for individual patients. In the present study, we reviewed the case records and neuropsychological findings in 53 psychogenic amnesia cases (ratio of 3:1, males:females), in comparison with 21 consecutively recruited neurological memory-disordered patients and 14 healthy control subjects. In particular, we examined the pattern of retrograde amnesia on an assessment of autobiographical memory (the Autobiographical Memory Interview). We found that our patients with psychogenic memory loss fell into four distinct groups, which we categorized as: (i) fugue state; (ii) fugue-to-focal retrograde amnesia; (iii) psychogenic focal retrograde amnesia following a minor neurological episode; and (iv) patients with gaps in their memories. While neurological cases were characterized by relevant neurological symptoms, a history of a past head injury was actually more common in our psychogenic cases (P = 0.012), perhaps reflecting a 'learning episode' predisposing to later psychological amnesia. As anticipated, loss of the sense of personal identity was confined to the psychogenic group. However, clinical depression, family/relationship problems, financial/employment problems, and failure to recognize the family were also statistically more common in that group. The pattern of autobiographical memory loss differed between the psychogenic groups: fugue cases showed a severe and uniform loss of memories for both facts and events across all time periods, whereas the two focal retrograde amnesia groups showed a 'reversed' temporal gradient with relative sparing of recent memories. After 3-6 months, the fugue patients had improved to normal scores for facts

Geographic range of vector-borne infections and their vectors: the role of African wildlife.

Science.gov (United States)

van Vuuren, M; Penzhorn, B L

2015-04-01

The role of African wildlife in the occurrence of vector-borne infections in domestic animals has gained renewed interest as emerging and re-emerging infections occur worldwide at an increasing rate. In Africa, biodiversity conservation and the expansion of livestock production have increased the risk of transmitting vector-borne infections between wildlife and livestock. The indigenous African pathogens with transboundary potential, such as Rift Valley fever virus, African horse sickness virus, bluetongue virus, lumpy skin disease virus, African swine fever virus, and blood-borne parasites have received the most attention. There is no evidence for persistent vector-borne viral infections in African wildlife. For some viral infections, wildlife may act as a reservoir through the inter-epidemic circulation of viruses with mild or subclinical manifestations. Wildlife may also act as introductory or transporting hosts when moved to new regions, e.g. for lumpy skin disease virus, Rift Valley fever virus and West Nile virus. Wildlife may also act as amplifying hosts when exposed to viruses in the early part of the warm season when vectors are active, with spillover to domestic animals later in the season, e.g. with bluetongue and African horse sickness. Some tick species found on domestic animals are more abundant on wildlife hosts; some depend on wildlife hosts to complete their life cycle. Since the endemic stability of a disease depends on a sufficiently large tick population to ensure that domestic animals become infected at an early age, the presence of wildlife hosts that augment tick numbers may be beneficial. Many wild ungulate species are reservoirs of Anaplasma spp., while the role of wildlife in the epidemiology of heartwater (Ehrlichia ruminantium infection) has not been elucidated. Wild ungulates are not usually reservoirs of piroplasms that affect livestock; however, there are two exceptions: zebra, which are reservoirs of Babesia caballi and Theileria

Determining putative vectors of the Bogia Coconut Syndrome phytoplasma using loop-mediated isothermal amplification of single-insect feeding media

Science.gov (United States)

Lu, Hengyu; Wilson, Bree A. L.; Ash, Gavin J.; Woruba, Sharon B.; Fletcher, Murray J.; You, Minsheng; Yang, Guang; Gurr, Geoff M.

2016-01-01

Phytoplasmas are insect vectored mollicutes responsible for disease in many economically important crops. Determining which insect species are vectors of a given phytoplasma is important for managing disease but is methodologically challenging because disease-free plants need to be exposed to large numbers of insects, often over many months. A relatively new method to detect likely transmission involves molecular testing for phytoplasma DNA in sucrose solution that insects have fed upon. In this study we combined this feeding medium method with a loop-mediated isothermal amplification (LAMP) assay to study 627 insect specimens of 11 Hemiptera taxa sampled from sites in Papua New Guinea affected by Bogia coconut syndrome (BCS). The LAMP assay detected phytoplasma DNA from the feeding solution and head tissue of insects from six taxa belonging to four families: Derbidae, Lophopidae, Flatidae and Ricaniidae. Two other taxa yielded positives only from the heads and the remainder tested negative. These results demonstrate the utility of combining single-insect feeding medium tests with LAMP assays to identify putative vectors that can be the subject of transmission tests and to better understand phytoplasma pathosystems. PMID:27786249

Targeting of breast metastases using a viral gene vector with tumour-selective transcription.

LENUS (Irish Health Repository)

Rajendran, Simon

2012-01-31

BACKGROUND: Adeno-associated virus (AAV) vectors have significant potential as gene delivery vectors for cancer gene therapy. However, broad AAV2 tissue tropism results in nonspecific gene expression. MATERIALS AND METHODS: We investigated use of the C-X-C chemokine receptor type 4 (CXCR4) promoter to restrict AAV expression to tumour cells, in subcutaneous MCF-7 xenograft mouse models of breast cancer and in patient samples, using bioluminescent imaging and flow cytometric analysis. RESULTS: Higher transgene expression levels were observed in subcutaneous MCF-7 tumours relative to normal tissue (muscle) using the CXCR4 promoter, unlike a ubiquitously expressing Cytomegalovirus promoter construct, with preferential AAVCXCR4 expression in epithelial tumour and CXCR4-positive cells. Transgene expression following intravenously administered AAVCXCR4 in a model of liver metastasis was detected specifically in livers of tumour bearing mice. Ex vivo analysis using patient samples also demonstrated higher AAVCXCR4 expression in tumour compared with normal liver tissue. CONCLUSION: This study demonstrates for the first time, the potential for systemic administration of AAV2 vector for tumour-selective gene therapy.

Reconsidering the nature and mode of action of metabolite retrograde signals from the chloroplast

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Gonzalo Martín Estavillo

2013-01-01

Full Text Available Plant organelles produce retrograde signals to alter nuclear gene expression in order to coordinate their biogenesis, maintain homeostasis or optimize their performance under adverse conditions. Many signals of different chemical nature have been described in the past decades, including chlorophyll intermediates, reactive oxygen species and adenosine derivatives. While the effects of retrograde signalling on gene expression are well understood, the initiation and transport of the signals and their mode of action have either not been resolved, or are a matter of speculation. Moreover, retrograde signalling should be consider as part of a broader cellular network, instead of as separate pathways, required to adjust to changing physiologically relevant conditions. Here we summarize current plastid retrograde signalling models in plants, with a focus on new signalling pathways, SAL1-PAP, MEcPP and β- cyclocitral, and outline missing links or future areas of research that we believe need to be addressed to have a better understanding of plant intracellular signalling networks.

Sonourethrography compared to retrograde urethrography

International Nuclear Information System (INIS)

Kim, Jong Chul; Chang, Nam Sik; Park, Cheong Hee; Rhee, Byung Chul; Kong, Jae Chul; Park, Jong Yoon

1989-01-01

A total of 15 patients with suspected urethral stricture or fistula underwent conventional retrograde urethrography and following sonourethrography with saline infusion or voiding against Eschmann penile clamp, in Gyeongsang and Chungnam National University Hospital from July, 1989 to June, 1989. The sonographic findings were as diagnostic as the roentgen findings in 12 patients. When the length of the strictures assessed by each imaging modality was compared to measurement at open urothroplasty of 2 patients, sonourethrography was consistently more accurate. Urethroscopy was done in all cases. Sonourethrography using distension technique of the urethra enabled classification of the degree of spongiofibrosis, thus provided the guidance of direct vision internal urethrotomy in 9 patients. In 2 patients, the sonourethrogram identified periurethral tumor and urethral polyp which were not definitely analysed on the retrograde urethrogram. In the patient of posttraumatic postoperative urethrorectal fistula, residual fistuous tract was seen on both examinations. In 1 patient of stricture with severe periurethral scar, urethral stricture recurred after graft. No patient reported significant discomfort during the sonourethrogram. The sonourethrogram provided valuable, dynamic. 3 dimensional information about the luminal and extraluminal anatomy and pathology of the anterior urethra. The new method of sonourethrogram allows for the appropriate decision to be made easier for optimal treatment of urethal stricture, etc, and can be used as a follow up study

Lymphocytes Negatively Regulate NK Cell Activity via Qa-1b following Viral Infection

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Haifeng C. Xu

2017-11-01

Full Text Available NK cells can reduce anti-viral T cell immunity during chronic viral infections, including infection with the lymphocytic choriomeningitis virus (LCMV. However, regulating factors that maintain the equilibrium between productive T cell and NK cell immunity are poorly understood. Here, we show that a large viral load resulted in inhibition of NK cell activation, which correlated with increased expression of Qa-1b, a ligand for inhibitory NK cell receptors. Qa-1b was predominantly upregulated on B cells following LCMV infection, and this upregulation was dependent on type I interferons. Absence of Qa-1b resulted in increased NK cell-mediated regulation of anti-viral T cells following viral infection. Consequently, anti-viral T cell immunity was reduced in Qa-1b- and NKG2A-deficient mice, resulting in increased viral replication and immunopathology. NK cell depletion restored anti-viral immunity and virus control in the absence of Qa-1b. Taken together, our findings indicate that lymphocytes limit NK cell activity during viral infection in order to promote anti-viral T cell immunity.

Impact of mass generation for spin-1 mediator simplified models

International Nuclear Information System (INIS)

Bell, Nicole F.; Cai, Yi; Leane, Rebecca K.

2017-01-01

In the simplified dark matter models commonly studied, the mass generation mechanism for the dark fields is not typically specified. We demonstrate that the dark matter interaction types, and hence the annihilation processes relevant for relic density and indirect detection, are strongly dictated by the mass generation mechanism chosen for the dark sector particles, and the requirement of gauge invariance. We focus on the class of models in which fermionic dark matter couples to a spin-1 vector or axial-vector mediator. However, in order to generate dark sector mass terms, it is necessary in most cases to introduce a dark Higgs field and thus a spin-0 scalar mediator will also be present. In the case that all the dark sector fields gain masses via coupling to a single dark sector Higgs field, it is mandatory that the axial-vector coupling of the spin-1 mediator to the dark matter is non-zero; the vector coupling may also be present depending on the charge assignments. For all other mass generation options, only pure vector couplings between the spin-1 mediator and the dark matter are allowed. If these coupling restrictions are not obeyed, unphysical results may be obtained such as a violation of unitarity at high energies. These two-mediator scenarios lead to important phenomenology that does not arise in single mediator models. We survey two-mediator dark matter models which contain both vector and scalar mediators, and explore their relic density and indirect detection phenomenology.

Liver parenchumography following endoscopic retrograde cholangiopancreatography (ERCP)

International Nuclear Information System (INIS)

Revert, A.; Arana, E.; Pertejo, V.; Berenguer, M.; Masip, M.J.

1998-01-01

Focal liver opacification during endoscopic retrograde cholangiography (ERCP) is an uncommon complication caused by excessive pressure during contrast injection. In this situation, ERCP must be interrupted and the position of the cannula checked. We recommend that these images be excluded from the diagnosis of tumor or cystic cavities. 4 refs

Retrograde cystography US. A new ultrasound technique for the diagnosis and staging of vesicoureteral reflux

International Nuclear Information System (INIS)

Farina, R.; Arena, C.; Pennisi, F.; Di Benedetto, V.; Politi, G.; Di Benedetto, A.

1999-01-01

The authors investigated the accuracy of a new US (ultrasound) investigation technique, called retrograde cystography US, in the early diagnosis and staging of vesicoureteral reflux. 5 patients, aged 3 months to 10 years, suffering from hydronephrosis and/or pyelonephritis, were examined using retrograde cystography US followed by conventional retrograde cystography. Retrograde cystography US consists in the transcatheter introduction of a contrast agent into the bladder and a subsequent color Doppler examination to show or exclude the presence of reflux. Superpubic scanning of bladder, ureters and pyelocaliceal cavity was performed after echo contrast agent introduction to assess the reflux grade. US was performed with an Esaote AU 590 asynchronous scanner with a 3.5 MHz convex probe. The total agreement of conventional and US findings seems to confirm the importance of the US method for the diagnosis and staging of vesicoureteral reflux [it

Rift Valley fever potential mosquito vectors and their infection status ...

African Journals Online (AJOL)

Background: Rift Valley fever (RVF) is a mosquito-borne viral zoonotic disease. Rift Valley fever virus (RVFV) has been isolated from more than 40 species of mosquitoes from eight genera. This study was conducted to determine the abundance of potential mosquito vectors and their RVFV infection status in Ngorongoro ...

Dating of retrograde metamorphism in Western Carpathians by K-Ar analysis of muscovites

International Nuclear Information System (INIS)

Cambel, B.; Korikovskij, S.P.; Krasivskaya, I.S.; Arakelyants, M.M.

1986-01-01

Using the K-Ar isotope dating method of muscovites it was found that many retrogradely metamorphosed rocks are the results of Variscan retrograde metamorphism and are not pre-Cambrian or Alpine metamorphites (diaphthorites). Samples for dating were taken from the Western Carpathian crystalline formation. The content of radiogenic argon was determined by mass spectrometry using the method of isotope dilution. (M.D.)

Endoskopisk ultralydvejledt rendezvouskolangiografi ved mislykket endoskopisk retrograd kolangiopankreatikografi

DEFF Research Database (Denmark)

Boman, Pia Snedker; Perdawid, Sharafaden Karim; Lykkegaard, John

2012-01-01

In this case report we describe an alternative method of cholangiography. Endoscopic retrograde cholangiopancreatography (ERCP) was not successful in a patient with choledocolithiasis. A combined endoscopic ultrasound (EUS) and ERCP procedure was performed and a stent was inserted in the common...

A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein.

Science.gov (United States)

Majer, Eszter; Navarro, José-Antonio; Daròs, José-Antonio

2015-09-01

Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA.

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Matthew G Cottingham

2008-02-01

Full Text Available The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20. The genomic BAC clone was 'rescued' back to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA, now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+ T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced specific CD8(+ T cell responses to one of two endogenous viral epitopes (from the E2 and F2 proteins, in accordance with published work (Staib et al., 2005, J. Gen. Virol.86, 1997-2006. In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+ T cells against an epitope from Plasmodium berghei was created using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant

Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

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Richard W Daniels

Full Text Available Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A and Thosea asigna virus (T2A worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

Gene delivery systems by the combination of lipid bubbles and ultrasound.

Science.gov (United States)

Negishi, Yoichi; Endo-Takahashi, Yoko; Maruyama, Kazuo

2016-11-28

Gene therapy is promising for the treatment of many diseases including cancers and genetic diseases. From the viewpoint of safety, ultrasound (US)-mediated gene delivery with nano/ microbubbles was recently developed as a novel non-viral vector system. US-mediated gene delivery using nano/microbubbles are able to produce transient changes in the permeability of the cell membrane after US-induced cavitation while reducing cellular damage and enables the tissue-specific or the site-specific intracellular delivery of gene both in vitro and in vivo. We have recently developed novel lipid nanobubbles (Lipid Bubbles). These nanobubbles can also be used to enhance the efficacy of the US-mediated genes (plasmid DNA, siRNA, and miRNA etc.) delivery. In this review, we describe US-mediated delivery systems combined with nano/microbubbles and discuss their feasibility as non-viral vector systems.

Adeno-associated virus vector-mediated transduction in the cat brain.

Science.gov (United States)

Vite, Charles H; Passini, Marco A; Haskins, Mark E; Wolfe, John H

2003-10-01

Adeno-associated virus (AAV) vectors are capable of delivering a therapeutic gene to the mouse brain that can result in long-term and widespread protein production. However, the human infant brain is more than 1000 times larger than the mouse brain, which will make the treatment of global neurometabolic disorders in children more difficult. In this study, we evaluated the ability of three AAV serotypes (1,2, and 5) to transduce cells in the cat brain as a model of a large mammalian brain. The human lysosomal enzyme beta-glucuronidase (GUSB) was used as a reporter gene, because it can be distinguished from feline GUSB by heat stability. The vectors were injected into the cerebral cortex, caudate nucleus, thalamus, corona radiata, internal capsule, and centrum semiovale of 8-week-old cats. The brains were evaluated for gene expression using in situ hybridization and enzyme histochemistry 10 weeks after surgery. The AAV2 vector was capable of transducing cells in the gray matter, while the AAV1 vector resulted in greater transduction of the gray matter than AAV2 as well as transduction of the white matter. AAV5 did not result in detectable transduction in the cat brain.

Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.

Science.gov (United States)

Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G; Scholler, John; Levine, Bruce L; Albelda, Steven M; June, Carl H

2010-11-15

Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(-/-) mice. Dramatic tumor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes showing that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. Copyright © 2010 AACR.

Endoscopic retrograde cholangiopancreatography causes reduced myocardial blood flow

DEFF Research Database (Denmark)

Christensen, M; Hendel, H W; Rasmussen, V

2002-01-01

BACKGROUND AND STUDY AIMS: Previous studies have shown that up to 50% of healthy patients may develop ST-segment changes during upper gastrointestinal endoscopy. The aim of the study was to evaluate myocardial blood flow in patients during endoscopic retrograde cholangiopancreatography (ERCP...

Coxsackievirus B3 vaccines: use as an expression vector for prevention of myocarditis.

Science.gov (United States)

Henke, Andreas; Jarasch, Nadine; Wutzler, Peter

2008-12-01

Coxsackievirus B3 (CVB3), a member of the Picornaviridae family, is considered to be one of the most important infectious agents to cause virus-induced myocarditis. Despite improvements in studying virus pathology, structure and molecular biology, as well as the diagnosis of this disease, there is still no virus-specific drug or vaccine in clinical use. During the last 20 years many investigations have been performed to develop classic and modern immunization techniques against CVB3-induced heart disease. One promising approach among others includes the insertion of coding sequences of cytokines into the viral genome. The application of an IFN-gamma-expressing recombinant coxsackievirus vector is especially efficient against CVB3-induced myocarditis. Beside direct IFN-gamma-mediated antiviral effects, the local and simultaneous expression of IFN-gamma by the virus itself activates the immune system in a strong and long-lasting manner, which protects animals completely against subsequent lethal infections independently of the age of the immunized individual and the route of vaccine administration.

Cardioplegia retrógrada seqüencial Sequencial retrograde cardioplegy

Directory of Open Access Journals (Sweden)

Otoni M Gomes

1996-09-01

extracorporeal circulation established. The following routes were employed for cardioplegic perfusion: 1 Antegrade - through ascending aortic canullation bellow the aortic occlusion clamp;2 Selective retrograde - through coronary synus (Co.S - 25 mmHg using a self-inflating ballooned cannula; 3 Total retrograde, (Co.S - 40 mmHg - through a cannula inserted in the right atrium (RA; 4 Sequencial retrograde, Co.S-RA - with the CS flowing first through the coronary synus lowering the interventricular septal temperature to 16 ºC and after through the RA cannula as in the total retrograde technique with the pulmonary artery occluded and;5 Sequencial retrograde, Co.S-RV - the RV chamber being directly cannulated through the tricuspid valve and perfused, instead of the RA in the latter technique. The temperature variation of the myocardium in the left ventricule (LV, RVt RA and sinus node region (SN was controlled employing an Omega needle termistor and thermometer. With the antegrade technique (70 mmHg pressure the most uniform myocardial cooling, the lowest CS volume and perfusion time duration was observed, followed In excelence by the Co.S-RA Sequencial retrograde technique and the Co.S-RV sequencial technique. The present data indicate that sequencial retrograde cardioplegic perfusion techinique is significantly better than the usual Co.S or RA total retrograde technique alone for myocardial protection when compared with the aortic root antegrade perfusion technique.

Wide Awake and Ready to Move: 20 Years of Non-Viral Therapeutic Genome Engineering with the Sleeping Beauty Transposon System.

Science.gov (United States)

Hodge, Russ; Narayanavari, Suneel A; Izsvák, Zsuzsanna; Ivics, Zoltán

2017-10-01

Gene therapies will only become a widespread tool in the clinical treatment of human diseases with the advent of gene transfer vectors that integrate genetic information stably, safely, effectively, and economically. Two decades after the discovery of the Sleeping Beauty (SB) transposon, it has been transformed into a vector system that is fulfilling these requirements. SB may well overcome some of the limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are being used in the majority of ongoing clinical trials. The SB system has achieved a high level of stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, representing crucial steps that may permit its clinical use in the near future. This article reviews the most important aspects of SB as a tool for gene therapy, including aspects of its vectorization and genomic integration. As an illustration, the clinical development of the SB system toward gene therapy of age-related macular degeneration and cancer immunotherapy is highlighted.

Lentiviral vectors in cancer immunotherapy.

Science.gov (United States)

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

2015-01-01

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

Mutagenesis-mediated virus extinction: virus-dependent effect of viral load on sensitivity to lethal defection.

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Héctor Moreno

Full Text Available BACKGROUND: Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI on progeny production of several RNA viruses under enhanced mutagenesis. RESULTS: The effect of the mutagenic base analogue 5-fluorouracil (FU on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI, or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV, but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV and encephalomyocarditis virus (EMCV. The increase in mutation frequency and Shannon entropy (mutant spectrum complexity as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results. CONCLUSIONS: (i Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.

Non-viral gene delivery strategies for cancer therapy, tissue engineering and regenerative medicine

Science.gov (United States)

Bhise, Nupura S.

Gene therapy involves the delivery of deoxyribonucleic acid (DNA) into cells to override or replace a malfunctioning gene for treating debilitating genetic diseases, including cancer and neurodegenerative diseases. In addition to its use as a therapeutic, it can also serve as a technology to enable regenerative medicine strategies. The central challenge of the gene therapy research arena is developing a safe and effective delivery agent. Since viral vectors have critical immunogenic and tumorogenic safety issues that limit their clinical use, recent efforts have focused on developing non-viral biomaterial based delivery vectors. Cationic polymers are an attractive class of gene delivery vectors due to their structural versatility, ease of synthesis, biodegradability, ability to self-complex into nanoparticles with negatively charged DNA, capacity to carry large cargo, cellular uptake and endosomal escape capacity. In this thesis, we hypothesized that developing a biomaterial library of poly(betaamino esters) (PBAE), a newer class of cationic polymers consisting of biodegradable ester groups, would allow investigating vector design parameters and formulating effective non-viral gene delivery strategies for cancer drug delivery, tissue engineering and stem cell engineering. Consequently, a high-throughput transfection assay was developed to screen the PBAE-based nanoparticles in hard to transfect fibroblast cell lines. To gain mechanistic insights into the nanoparticle formulation process, biophysical properties of the vectors were characterized in terms of molecular weight (MW), nanoparticle size, zeta potential and plasmid per particle count. We report a novel assay developed for quantifying the plasmid per nanoparticle count and studying its implications for co-delivery of multiple genes. The MW of the polymers ranged from 10 kDa to 100 kDa, nanoparticle size was about 150 run, zeta potential was about 30 mV in sodium acetate buffer (25 mM, pH 5) and 30 to 100

Multiple-indicator dilution technique for characterization of normal and retrograde flow in once-through rat liver perfusions

International Nuclear Information System (INIS)

St-Pierre, M.V.; Schwab, A.J.; Goresky, C.A.; Lee, W.F.; Pang, K.S.

1989-01-01

The technique of normal and retrograde rat liver perfusion has been widely used to probe zonal differences in drug-metabolizing activities. The validity of this approach mandates the same tissue spaces being accessed by substrates during both normal and retrograde perfusions. Using the multiple-indicator dilution technique, we presently examine the extent to which retrograde perfusion alters the spaces accessible to noneliminated references. A bolus dose of 51Cr-labeled red blood cells, 125I-albumin, 14C-sucrose and 3H2O was injected into the portal (normal) or hepatic (retrograde) vein of rat livers perfused at 10 ml per min per liver. The outflow perfusate was serially collected over 220 sec to characterize the transit times and the distribution spaces of the labels. During retrograde perfusion, red blood cells, albumin and sucrose profiles peaked later and lower than during normal perfusion, whereas the water curves were similar. The transit times of red blood cells, albumin and sucrose were longer (p less than 0.005), whereas those for water did not change. Consequently, retrograde flow resulted in significantly larger sinusoidal blood volumes (45%), albumin Disse space (42%) and sucrose Disse space (25%) than during normal flow, whereas the distribution spaces for total and intracellular water remained unaltered. The distension of the vascular tree was confirmed by electron microscopy, by which occasional isolated foci of widened intercellular recesses and spaces of Disse were observed. Cellular ultrastructure was otherwise unchanged, and there was no difference found between normal and retrograde perfusion for bile flow rates, AST release, perfusion pressure, oxygen consumption and metabolic removal of ethanol, a substrate with flow-limited distribution, which equilibrates rapidly with cell water (hepatic extraction ratios were virtually identical: normal vs. retrograde, 0.50 vs. 0.48 at 6 to 7.4 mM input concentration)

Twelve months follow-up after retrograde recanalization of superficial femoral artery chronic total occlusion

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Joanna Wojtasik-Bakalarz

2017-03-01

Full Text Available Introduction : Fifty percent of cases of peripheral artery disease are caused by chronic total occlusion (CTO of the superficial femoral artery (SFA. Ten–fifteen percent of percutaneous SFA recanalization procedures are unsuccessful. In those cases the retrograde technique can increase the success rate of the procedure, but the long-term follow-up of such procedures is still unknown. Aim : To assess the efficacy and clinical outcomes during long-term follow-up after retrograde recanalization of the SFA. Material and methods: We included patients after at least one unsuccessful percutaneous antegrade recanalization of the SFA. Patients were evaluated for the procedural and clinical follow-up of mean time 13.9 months. Results: The study included 17 patients (7 females, 10 males who underwent percutaneous retrograde recanalization of the SFA from June 2011 to June 2015. The mean age of patients was 63 ±7 years. Retrograde puncture of the distal SFA was successful in all cases. A retrograde procedure was performed immediately after antegrade failure in 4 (23.5% patients and after a previously failed attempt in 13 (76.5% patients. The procedure was successful in 15 (88.2% patients, and unsuccessful in 2 (11.8% patients. Periprocedural complications included 1 peripheral distal embolization (successfully treated with aspiration thrombectomy, 1 bleeding event from the puncture site and 7 puncture site hematomas. During follow-up the all-cause mortality rate was 5.8% (1 patient, non-cardiac death. The primary patency rate at 12 months was 88.2% and secondary patency 100%. Conclusions : The retrograde SFA puncture seems to be a safe and successful technique for CTO recanalization and is associated with a low rate of perioperative and long-term follow-up complications.

An antivector vaccine protects against a lethal vector-borne pathogen.

Directory of Open Access Journals (Sweden)

Milan Labuda

2006-04-01

Full Text Available Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV transmitted by infected Ixodes ricinus ticks. The vaccine has a "dual action" in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: "transmission," number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; "support," number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and "survival," number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD that targets only the tick's midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding site.

Preserved memory in retrograde amnesia: sparing of a recently acquired skill.

Science.gov (United States)

Squire, L R; Cohen, N J; Zouzounis, J A

1984-01-01

Recent work with amnesic patients has revealed a preserved capacity for acquiring and retaining new skills despite otherwise profound anterograde impairment. In addition to their anterograde impairment, amnesic patients also have retrograde memory loss for some information acquired prior to the amnesic event. The present experiment addresses for the first time the question of whether preservation of memory for skills is also a feature memory impairment. To determine the susceptibility of a recently learned skill to retrograde amnesia, we taught patients to read mirror-reversed words before and during the early part of a prescribed course of electroconvulsive therapy (ECT) and then tested retention of the skill after the course of treatment had been completed. Patients prescribed bilateral or right unilateral ECT and depressed patients not receiving ECT acquired the mirror-reading skill at the same rate and then retained it at the same level. For the patients prescribed ECT, intact learning and retention of the skill occurred despite retrograde amnesia for the previous testing sessions and for the words that they had read previously.