Science.gov

Sample records for adenoviral expression system

  1. Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation

    PubMed Central

    Deinzer, Andrea

    2016-01-01

    To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B′ core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8+ T cells. Second, we generated the two-vector-based “modular promoter” system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B′ core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. PMID:27446966

  2. Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation.

    PubMed

    Knippertz, Ilka; Deinzer, Andrea; Dörrie, Jan; Schaft, Niels; Nettelbeck, Dirk M; Steinkasserer, Alexander

    2016-01-01

    To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B' core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8(+) T cells. Second, we generated the two-vector-based "modular promoter" system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B' core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. PMID:27446966

  3. Regulated Expression of Adenoviral Vectors-Based Gene Therapies

    PubMed Central

    Curtin, James F.; Candolfi, Marianela; Puntel, Mariana; Xiong, Weidong; Muhammad, A. K. M.; Kroeger, Kurt; Mondkar, Sonali; Liu, Chunyan; Bondale, Niyati; Lowenstein, Pedro R.; Castro, Maria G.

    2008-01-01

    Summary Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the “OFF” state, and expression should quickly reach therapeutic levels in the “ON” state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA2S-M2 inducer and tTSKid silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo. PMID:18470649

  4. Adenoviral vector which delivers FasL-GFP fusion protein regulated by the tet-inducible expression system.

    PubMed

    Rubinchik, S; Ding, R; Qiu, A J; Zhang, F; Dong, J

    2000-05-01

    Fas ligand (FasL) is a member of the tumor necrosis family and when bound to its receptor, Fas, induces apoptosis. It plays important roles in immune response, degenerative and lymphoproliferative diseases, development and tumorigenesis. It is also involved in generation of immune privilege sites in the eye and testis. Harnessing the power of this molecule is expected to lead to a powerful chemotherapeutic. We describe the construction and characterization of replication-deficient adenoviral vectors that express a fusion of murine FasL and green fluorescent protein (GFP). FasL-GFP retains full activity of wild-type FasL, at the same time allowing for easy visualization and quantification in both living and fixed cells. The fusion protein is under the control of a tetracycline-regulated gene expression system. Tight control of expression is achieved by creating a novel 'double recombinant' Ad vector, in which the tet-responsive element and the transactivator element are built into the opposite ends of the same vector to avoid enhancer interference. Expression can be conveniently regulated by tetracycline or its derivatives in a dose-dependent manner. The vector was able to deliver FasL-GFP gene to cells in vitro efficiently, and the expression level and function of the fusion protein was modulated by the concentration of doxycycline. This regulation allows us to produce high titers of the vector by inhibiting FasL expression in an apoptosis-resistant cell line. Induction of apoptosis was demonstrated in all cell lines tested. These results indicate that our vector is a potentially valuable tool for FasL-based gene therapy of cancer and for the study of FasL/Fas-mediated apoptosis and immune privilege. PMID:10845726

  5. Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging

    PubMed Central

    Sato, M; Figueiredo, ML; Burton, JB; Johnson, M; Chen, M; Powell, R; Gambhir, SS; Carey, M; Wu, L

    2009-01-01

    Effective treatment for recurrent, disseminated prostate cancer is notably limited. We have developed adenoviral vectors with a prostate-specific two-step transcriptional amplification (TSTA) system that would express therapeutic genes at a robust level to target metastatic disease. The TSTA system employs the prostate-specific antigen (PSA) promoter/enhancer to drive a potent synthetic activator, which in turn activates the expression of the therapeutic gene. In this study, we explored different configurations of this bipartite system and discovered that physical separation of the two TSTA components into E1 and E3 regions of adenovirus was able to enhance androgen regulation and cell-discriminatory expression. The TSTA vectors that express imaging reporter genes were assessed by noninvasive imaging technologies in animal models. The improved selectivity of the E1E3 configured vector was reflected in silenced ectopic expression in the lung. Significantly, the enhanced specificity of the E1E3 vector enabled the detection of lung metastasis of prostate cancer. An E1E3 TSTA vector that expresses the herpes simplex virus thymidine kinase gene can effectively direct positron emission tomography (PET) imaging of the tumor. The prostate-targeted gene delivery vectors with robust and cell-specific expression capability will advance the development of safe and effective imaging guided therapy for recurrent metastatic stages of prostate cancer. PMID:18305574

  6. Production of human epidermal growth factor using adenoviral based system

    PubMed Central

    Negahdari, Babak; Shahosseini, Zahra; Baniasadi, Vahid

    2016-01-01

    Epidermal growth factor (EGF), a growth factor involved in cell growth and differentiation, is a small polypeptide with molecular weight of approximately 6 kDa known to be present in a number of different mammalian species. Experimental studies in animals and humans have demonstrated that the topical application of EGF accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns. Due to its commercial applications, Human EGF (hEGF) has been cloned in several forms. In the present study, adenoviral based expression system was used to produce biologically active recombinant hEGF. The presence of secreted recombinant hEGF was confirmed by a dot blot and its expression level was determined by enzyme-linked immuno-sorbent assay. Moreover, biological activity of secreted hEGF was evaluated by a proliferation assay performed on A549 cells. For production of hEGF in a secretory form, a chimeric gene coding for the hEGF fused to the signal peptide was expressed using adenoviral based method. This method enables the production of hEGF at the site of interest and moreover it could be used for cell proliferation and differentiation assays in tissue engineering research experiments instead of using commercially available EGF. PMID:27051431

  7. A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression

    PubMed Central

    Ma, Jing; Wang, Wenbin; Zhang, Lu; Tikoo, Suresh K.; Yang, Zengqi

    2015-01-01

    Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes. PMID:26011074

  8. A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration.

    PubMed

    Boehme, Philip; Zhang, Wenli; Solanki, Manish; Ehrke-Schulz, Eric; Ehrhardt, Anja

    2016-01-01

    For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose. PMID:27434682

  9. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    SciTech Connect

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

    2012-05-01

    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.

  10. Development of an adenoviral vector with robust expression driven by p53

    SciTech Connect

    Bajgelman, Marcio C.; Strauss, Bryan E.

    2008-02-05

    Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG served as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.

  11. Development of Adenoviral Delivery Systems to Target Hepatic Stellate Cells In Vivo

    PubMed Central

    Meier, Claudia; Kowtharapu, Bhavani S.; Timm, Franziska; Vollmar, Brigitte; Herchenröder, Ottmar; Abshagen, Kerstin; Pützer, Brigitte M.

    2013-01-01

    Hepatic stellate cells (HSCs) are known as initiator cells that induce liver fibrosis upon intoxication or other noxes. Deactivation of this ongoing remodeling process of liver parenchyma into fibrotic tissue induced by HSCs is an interesting goal to be achieved by targeted genetic modification of HSCs. The most widely applied approach in gene therapy is the utilization of specifically targeted vectors based on Adenovirus (Ad) serotype 5. To narrow down the otherwise ubiquitous tropism of parental Ad, two modifications are required: a) ablating the native tropism and b) redirecting the vector particles towards a specific entity solely present on the cells of interest. Therefore, we designed a peptide of the nerve growth factor (NGFp) with specific affinity for the p75 neurotrophin receptor (p75NTR) present on HSCs. Coupling of this NGFp to vector particles was done either via chemical conjugation using bifunctional polyethylene glycol (PEG) or, alternatively, by molecular bridging with a fusion protein specific for viral fiber knob and p75NTR. Both Ad vectors transmit the gene for the green fluorescent protein (GFP). GFP expression was monitored in vitro on primary murine HSCs as well as after systemic administration in mice with healthy and fibrotic livers using intravital fluorescence microscopy. Coupling of NGFp to Ad via S11 and/or PEGylation resulted in markedly reduced liver tropism and an enhanced adenoviral-mediated gene transfer to HSCs. Transduction efficiency of both specific Ads was uniformly higher in fibrotic livers, whereas Ad.GFP-S11-NGFp transduce activated HSCs better than Ad.GFP-PEG-NGFp. These experiments contribute to the development of a targeted gene transfer system to specifically deliver antifibrotic compounds into activated HSCs by systemically applied adenoviral vector modified with NGFp. PMID:23825626

  12. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    NASA Astrophysics Data System (ADS)

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-06-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  13. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    PubMed Central

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  14. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness.

    PubMed

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  15. Adenoviral vector expressing murine β-defensin 2 enhances immunogenicity of an adenoviral vector based H5N1 influenza vaccine in aged mice.

    PubMed

    Vemula, Sai V; Pandey, Aseem; Singh, Neetu; Katz, Jacqueline M; Donis, Ruben; Sambhara, Suryaprakash; Mittal, Suresh K

    2013-10-01

    The ability to resist infections and respond to vaccinations is greatly reduced in the older adult population owing to a general decline in innate and adaptive immune functions with aging. Over the years several strategies such as increasing the vaccine dose, number of immunizations and using adjuvants have been evaluated to improve the immunogenicity and efficacy of vaccines in the older adult population. Murine β-defensin 2 (Mbd2) has been shown to function as a molecular adjuvant by recruiting and activating immature dendritic cells (DCs), professional antigen-presenting cells (APC), to the site of the immunization. In this study, we evaluated the potential utility of Mbd2 to enhance the efficacy of an adenoviral vector-based H5N1 influenza vaccine expressing hemagglutinin (HA) and nucleoprotein (NP) (HAd-HA-NP) in an aged mouse model. Our results indicated that immunostimulation with an adenoviral vector expressing Mbd2 (HAd-Mbd2) activated DCs and significantly enhanced the humoral and cellular immune responses induced by HAd-HA-NP. Furthermore, immunostimulation with HAd-Mbd2 followed by immunization with HAd-HA-NP resulted in significantly lower virus titers in the lungs following challenge with a H5N1 influenza virus compared to the group immunized with HAd-HA-NP without immunostimulation. Overall, our results highlight the potential utility of Mbd2 as a molecular adjuvant to enhance the immunogenicity and protective efficacy of vaccines for the elderly. PMID:23892144

  16. Adenoviral Vector Expressing Murine β-Defensin 2 Enhances Immunogenicity of an Adenoviral Vector based H5N1 Influenza Vaccine in Aged Mice

    PubMed Central

    Vemula, Sai V.; Pandey, Aseem; Singh, Neetu; Katz, Jacqueline M; Donis, Ruben; Sambhara, Suryaprakash; Mittal, Suresh K.

    2013-01-01

    The ability to resist infections and respond to vaccinations is greatly reduced in the older adult population owing to a general decline in innate and adaptive immune functions with aging. Over the years several strategies such as increasing the vaccine dose, number of immunizations and using adjuvants have been evaluated to improve the immunogenicity and efficacy of vaccines in the older adult population. Murine ß-defensin 2 (Mbd2) has been shown to function as a molecular adjuvant by recruiting and activating immature dendritic cells (DCs), professional antigen-presenting cells (APC), to the site of the immunization. In this study, we evaluated the potential utility of Mbd2 to enhance the efficacy of an adenoviral vector-based H5N1 influenza vaccine expressing hemagglutinin (HA) and nucleoprotein (NP) (HAd-HA-NP) in an aged mouse model. Our results indicated that immunostimulation with an adenoviral vector expressing Mbd2 (HAd-Mbd2) activated DCs and significantly enhanced the humoral and cellular immune responses induced by HAd-HA-NP. Furthermore, immunostimulation with HAd-Mbd2 followed by immunization with HAd-HA-NP resulted in significantly lower virus titers in the lungs following challenge with a H5N1 influenza virus compared to the group immunized with HAd-HA-NP without immunostimulation. Overall, our results highlight the potential utility of Mbd2 as a molecular adjuvant to enhance the immunogenicity and protective efficacy of vaccines for the elderly. PMID:23892144

  17. Adenoviral expression of murine serum amyloid A proteins to study amyloid fibrillogenesis.

    PubMed

    Kindy, M S; King, A R; Yu, J; Gerardot, C; Whitley, J; de Beer, F C

    1998-06-15

    Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro

  18. Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells.

    PubMed

    Lowe, S L; Rubinchik, S; Honda, T; McDonnell, T J; Dong, J Y; Norris, J S

    2001-09-01

    In prostate carcinoma, overexpression of the anti-apoptotic gene Bcl-2 has been found to be associated with resistance to therapies including radiation and androgen ablation. Restoring the balance of Bcl-2 family members may result in the induction of apoptosis in prostate cancer cells previously resistant to treatment. To accomplish this, a strategy involving overexpression of the pro-apoptotic gene Bax was executed. The use of cytotoxic genes such as Bax require selective expression of the gene. In this study, we examined the ability of selective expression of Bax protein directed by a prostate-specific promoter to induce apoptosis in human prostate carcinoma. A second-generation adenoviral vector was constructed with the modified prostate-specific probasin promoter, ARR2PB, directing expression of an HA-tagged Bax gene and a green fluorescent protein reporter translated from an internal ribosome entry site (ARR2PB.Bax.GFP). ARR2PB promoter activity is tightly regulated and highly prostate specific and is responsive to androgens and glucocorticoids. The prostate-specific promoter-Bax-GFP transgene cassette was inserted into a cloning site near the right inverted terminal repeat of the adenoviral vector to retain specificity of the promoter. LNCaP cells infected with Ad/ARR(2)PB.Bax.GFP showed high levels of Bax expression 48 h after infection resulting in an 85% reduction in cell viability. Importantly, LNCaP cells stably transfected to overexpress Bcl-2 showed similar patterns of cell death when infected with Ad/ARR(2)PB.Bax.GFP, an 82% reduction in cell viability seen 48 h after infection. Apoptosis was confirmed by measuring caspase activation and using the TUNEL assay. Tissue specificity was evaluated using A549 cells (lung adenocarcinoma), SK-Hep-1 (liver cancer) cells, and Hela (cervical cancer) cells which did not show detectable expression of virally delivered Bax protein or any increase in cell death. Systemic administration of Ad/ARR2PB. Bax.GFP in nude

  19. Helper virus-mediated downregulation of transgene expression permits production of recalcitrant helper-dependent adenoviral vector

    PubMed Central

    Palmer, Donna J; Grove, Nathan C; Ng, Philip

    2016-01-01

    Helper-dependent adenoviral vectors (HDAd) that express certain transgene products are impossible to produce because the transgene product is toxic to the producer cells, especially when made in large amounts during vector production. Downregulating transgene expression from the HDAd during vector production is a way to solve this problem. In this report, we show that this can be accomplished by inserting the target sequence for the adenoviral VA RNAI into the 3’ untranslated region of the expression cassette in the HDAd. Thus during vector production, when the producer cells are coinfected with both the helper virus (HV) and the HDAd, the VA RNAI produced by the HV will target the transgene mRNA from the HDAd via the endogenous cellular RNAi pathway. Once the HDAd is produced and purified, transduction of the target cells results in unimpeded transgene expression because of the absence of HV. This simple and universal strategy permits for the robust production of otherwise recalcitrant HDAds. PMID:27331077

  20. Intra-testicular injection of adenoviral constructs results in Sertoli cell-specific gene expression and disruption of the seminiferous epithelium

    PubMed Central

    Hooley, R P; Paterson, M; Brown, P; Kerr, K; Saunders, P T K

    2009-01-01

    Spermatogenesis is a complex process that cannot be modelled in vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-down in vivo. This paper describes a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC function in vivo and future work will therefore focus on the use of lentiviral delivery systems. PMID:18955374

  1. AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED CU,ZN-SOD AND MN-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED Cu,Zn-SOD AND Mn-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO. JB Smith1, PC Hartig3, MR Blanton3, KK Sulik1,2, and ES Hunter3. 1Department of Cell and Developmental Biology and 2Bowles Cente...

  2. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation

    PubMed Central

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo. PMID:26808122

  3. HIV-1 Adenoviral Vector Vaccines Expressing Multi-Trimeric BAFF and 4-1BBL Enhance T Cell Mediated Anti-Viral Immunity

    PubMed Central

    Gupta, Sachin; Raffa, Francesca N.; Fuller, Katherine A.; Rivas, Yaelis; Philip, Sakhi; Kornbluth, Richard S.; Stone, Geoffrey W.

    2014-01-01

    Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia

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

    PubMed Central

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

    2010-01-01

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

  5. Balloon Catheter Delivery of Helper-dependent Adenoviral Vector Results in Sustained, Therapeutic hFIX Expression in Rhesus Macaques

    PubMed Central

    Brunetti-Pierri, Nicola; Liou, Aimee; Patel, Priti; Palmer, Donna; Grove, Nathan; Finegold, Milton; Piccolo, Pasquale; Donnachie, Elizabeth; Rice, Karen; Beaudet, Arthur; Mullins, Charles; Ng, Philip

    2012-01-01

    Hemophilia B is an excellent candidate for gene therapy because low levels of factor IX (FIX) (≥1%) result in clinically significant improvement of the bleeding diathesis. Helper-dependent adenoviral (HDAd) vectors can mediate long-term transgene expression without chronic toxicity. To determine the potential for HDAd-mediated liver-directed hemophilia B gene therapy, we administered an HDAd expressing hFIX into rhesus macaques through a novel and minimally invasive balloon occlusion catheter-based method that permits preferential, high-efficiency hepatocyte transduction with low, subtoxic vector doses. Animals given 1 × 1012 and 1 × 1011 virus particle (vp)/kg achieved therapeutic hFIX levels for the entire observation period (up to 1,029 days). At 3 × 1010 and 1 × 1010 vp/kg, only subtherapeutic hFIX levels were achieved which were not sustained long-term. Balloon occlusion administration of HDAd was well tolerated with negligible toxicity. Five of six animals developed inhibitors to hFIX. These results provide important information in assessing the clinical utility of HDAd for hemophilia B gene therapy. PMID:22828499

  6. Capsid-Modified Adenoviral Vectors for Improved Muscle-Directed Gene Therapy

    PubMed Central

    Guse, Kilian; Suzuki, Masataka; Sule, Gautam; Bertin, Terry K.; Tyynismaa, Henna; Ahola-Erkkilä, Sofia; Palmer, Donna; Suomalainen, Anu; Ng, Philip; Cerullo, Vincenzo; Hemminki, Akseli

    2012-01-01

    Abstract Skeletal muscle represents an attractive target tissue for adenoviral gene therapy to treat muscle disorders and as a production platform for systemic expression of therapeutic proteins. However, adenovirus serotype 5 vectors do not efficiently transduce adult muscle tissue. Here we evaluated whether capsid modifications on adenoviral vectors could improve transduction in mature murine muscle tissue. First-generation and helper-dependent serotype 5 adenoviral vectors featuring the serotype 3 knob (5/3) showed significantly increased transduction of skeletal muscle after intramuscular injection in adult mice. Furthermore, we showed that full-length dystrophin could be more efficiently transferred to muscles of mdx mice using a 5/3-modified helper-dependent adenoviral vector. In contrast to first-generation vectors, helper-dependent adenoviral vectors mediated stable marker gene expression for at least 1 year after intramuscular injection. In conclusion, 5/3 capsid-modified helper-dependent adenoviral vectors show enhanced transduction in adult murine muscle tissue and mediate long-term gene expression, suggesting the suitability of these vectors for muscle-directed gene therapy. PMID:22888960

  7. The HDAC Inhibitor FK228 Enhances Adenoviral Transgene Expression by a Transduction-Independent Mechanism but Does Not Increase Adenovirus Replication

    PubMed Central

    Danielsson, Angelika; Dzojic, Helena; Rashkova, Victoria; Cheng, Wing-Shing; Essand, Magnus

    2011-01-01

    The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected. PMID:21379379

  8. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis

    PubMed Central

    Dolzhikova, Inna V.; Shcherbinin, Dmitry N.; Zubkova, Olga V.; Ivanova, Tatiana I.; Tukhvatulin, Amir I.; Shmarov, Maxim M.; Logunov, Denis Y.; Naroditsky, Boris S.; Gintsburg, Aleksandr L.

    2016-01-01

    Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection. PMID:26962869

  9. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

    PubMed

    Burmistrova, Daria A; Tillib, Sergey V; Shcheblyakov, Dmitry V; Dolzhikova, Inna V; Shcherbinin, Dmitry N; Zubkova, Olga V; Ivanova, Tatiana I; Tukhvatulin, Amir I; Shmarov, Maxim M; Logunov, Denis Y; Naroditsky, Boris S; Gintsburg, Aleksandr L

    2016-01-01

    Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection. PMID:26962869

  10. Enhanced antitumor response mediated by the codelivery of paclitaxel and adenoviral vector expressing IL-12.

    PubMed

    Cao, Linjie; Zeng, Qin; Xu, Chaoqun; Shi, Sanjun; Zhang, Zhirong; Sun, Xun

    2013-05-01

    It has been well-established that chemo-immunotherapy using cytotoxic drugs and appropriate cytokines offers a promising approach for the treatment of neoplastic diseases. In view of this, to improve melanoma treatment effect, our study developed a new codelivery system (AL/Ad5/PTX) that paclitaxel (PTX) and adenovirus encoding for murine interleukin-12 (Ad5-mIL-12) were incorporated into anionic liposomes (AL). First, AL/Ad5/PTX complexes were prepared by incorporating Ad5 into anionic PTX liposomes using calcium-induced phase change. Second, the size distribution and zeta potential of AL/Ad5/PTX were investigated. Third, the results of in vitro transduction assays showed that PTX introduced into AL/Ad-luc or AL/Ad5-mIL-12 highly enhanced gene transduction efficiency in B16 cells than naked Ad5 or AL/Ad complexes while it had no comparability in A549 cells. Finally, a melanoma-bearing mouse model was established to assess the antitumor effect. Tumor growth inhibition and prolonged survival time, accompanied by increased mIL-12 or interferon-γ (IFN-γ) expression levels in serum or tumor sites, were observed in mice treated with AL/Ad5-mIL-12/PTX, as compared with those treated with either AL/Ad5-mIL-12 or AL/PTX. In conclusion, these results suggested that codelivery of Ad5-mIL-12 and PTX incorporated into AL could be a relatively efficient strategy for the treatment of melanoma. PMID:23534449

  11. Induction of a Protective Heterosubtypic Immune Response Against the Influenza Virus by using Recombinant Adenoviral Vectors Expressing Hemagglutinin of the Influenza H5 Virus.

    PubMed

    Shmarov, M M; Sedova, E S; Verkhovskaya, L V; Rudneva, I A; Bogacheva, E A; Barykova, Yu A; Shcherbinin, D N; Lysenko, A A; Tutykhina, I L; Logunov, D Y; Smirnov, Yu A; Naroditsky, B S; Gintsburg, A L

    2010-04-01

    Influenza viruses are characterized by a high degree of antigenic variability, which causes the annual emergence of flu epidemics and irregularly timed pandemics caused by viruses with new antigenic and biological traits. Novel approaches to vaccination can help circumvent this problem. One of these new methods incorporates genetic vaccines based on adenoviral vectors. Recombinant adenoviral vectors which contain hemagglutinin-encoding genes from avian H5N1 and H5N2 (Ad-HA5-1 and Ad-HA5-2) influenza viruses were obtained using the AdEasy Adenoviral Vector System (Stratagene). Laboratory mice received a double intranasal vaccination with Ad-HA5-1 and Ad-HA5-2. This study demonstrates that immunization with recombinant adenoviruses bearing the Н 5 influenza virus hemagglutinin gene induces a immune response which protects immunized mice from a lethal dose of the H5 influenza virus. Moreover, it also protects the host from a lethal dose of the H1 virus, which belongs to the same clade as H5, but does not confer protection from the subtype H3 influenza virus, which belongs to a different clade. PMID:22649637

  12. Adenoviral-based foot-and-mouth disease virus vaccine: effect of duration of antigen expression on immunogenicity in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An adenoviral (Ad5)-based foot-and-mouth disease virus (FMDV) subunit vaccine, Ad5-A24, is an effective alternative to the current inactivated whole virus vaccine and has a number of advantages including no requirement for infectious FMDV and DIVA (Differentiation of Infected from Vaccinated Animals...

  13. [Construction of recombinant adenoviral vector expressing genes of the conservative influenza proteins M2 and nucleoprotein].

    PubMed

    Esmagambetov, I B; Sedova, E S; Shcherbinin, D N; Lysenko, A A; Garas, M N; Shmarov, M M; Logunov, D Iu

    2014-01-01

    Influenza is a highly contagious and one of the most massive infection diseases. General epidemiological significance has a strain, which belongs to subtype A. A high degree of genetic variety leads to the permanent changes in the antigenic structure of the influenza virus. Therefore, the current influenza vaccines require periodic updating of the composition of strains. Presently, it is important to develop a universal vaccine that can protect against different strains of influenza A virus at the same time and is based on the conserved antigens of the influenza virus. The recombinant adenovirus vectors expressing genes of conserved viral antigenes may be a promising candidate vaccine against influenza A. Using the method of the homologous recombination, we developed in this study recombinant adenovirus of fifth serotype that expresses genes of the ion channel M2 and nucleoprotein NP of the influenza virus A. Genes of the consensus protein M2 and NP of human influenza A virus were included into the structure of the viral genome. The expression of the antigens M2 and NP using recombinant adenovirus vector was detected by a Western blot assay. The immunogenicity of the developed recombinant adenovirus vector was demonstrated by the intranasal immunization of laboratory mice. PMID:25080815

  14. Preclinical Efficacy and Safety Profile of Allometrically Scaled Doses of Doxycycline Used to Turn "On" Therapeutic Transgene Expression from High-Capacity Adenoviral Vectors in a Glioma Model.

    PubMed

    VanderVeen, Nathan; Raja, Nicholas; Yi, Elizabeth; Appelman, Henry; Ng, Philip; Palmer, Donna; Zamler, Daniel; Dzaman, Marta; Lowenstein, Pedro R; Castro, Maria G

    2016-06-01

    Glioblastoma multiforme (GBM) is the most commonly occurring primary brain cancer in adults, in whom its highly infiltrative cells prevent total surgical resection, often leading to tumor recurrence and patient death. Our group has discovered a gene therapy approach for GBM that utilizes high-capacity "gutless" adenoviral vectors encoding regulatable therapeutic transgenes. The herpes simplex type 1-thymidine kinase (TK) actively kills dividing tumor cells in the brain when in the presence of the prodrug, ganciclovir (GCV), whereas the FMS-like tyrosine kinase 3 ligand (Flt3L) is an immune-stimulatory molecule under tight regulation by a tetracycline-inducible "Tet-On" activation system that induces anti-GBM immunity. As a prelude to a phase I clinical trial, we evaluated the safety and efficacy of Food and Drug Administration (FDA)-approved doses of the tetracycline doxycycline (DOX) allometrically scaled for rats. DOX initiates the expression of Flt3L, which has been shown to recruit dendritic cells to the brain tumor microenvironment-an integral first step in the development of antitumor immunity. The data revealed a highly safe profile surrounding these human-equivalent doses of DOX under an identical therapeutic window as proposed in the clinical trial. This was confirmed through a neuropathological analysis, liver and kidney histopathology, detection of neutralizing antibodies, and systemic toxicities in the blood. Interestingly, we observed a significant survival advantage in rats with GBM receiving the 300 mg/day equivalent dosage of DOX versus the 200 mg/day equivalent. Additionally, rats rejected "recurrent" brain tumor threats implanted 90 days after their primary brain tumors. We also show that DOX detection within the plasma can be an indicator of optimal dosing of DOX to attain therapeutic levels. This work has significant clinical relevance for an ongoing phase I clinical trial in humans with primary GBM and for other therapeutic approaches using

  15. Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia

    PubMed Central

    Koehler, David R.; Sajjan, Umadevi; Chow, Yu-Hua; Martin, Bernard; Kent, Geraldine; Tanswell, A. Keith; McKerlie, Colin; Forstner, Janet F.; Hu, Jim

    2003-01-01

    We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens. PMID:14673110

  16. Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia.

    PubMed

    Koehler, David R; Sajjan, Umadevi; Chow, Yu-Hua; Martin, Bernard; Kent, Geraldine; Tanswell, A Keith; McKerlie, Colin; Forstner, Janet F; Hu, Jim

    2003-12-23

    We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens. PMID:14673110

  17. Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

    PubMed

    Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle; Dmitriev, Igor P; Kashentseva, Elena A; Yeh, Anthony J; Cheung, Gordon Y C; Curiel, David T; Leppla, Stephen; Shoemaker, Charles B

    2016-03-01

    Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. PMID:26740390

  18. Immunogenicity and protective efficacy of a recombinant adenoviral based vaccine expressing heat-stable enterotoxin (STa) and K99 adhesion antigen of enterotoxigenic Escherichia coli in mice.

    PubMed

    Deng, Guangcun; Li, Wu; Wu, Xiaoling; Bao, Shaowen; Zeng, Jin; Zhao, Ning; Luo, Meihui; Liu, Xiaoming; Wang, Yujiong

    2015-12-01

    The diarrheal disease of domestic animals or in humans caused by enterotoxigenic Escherichia coli (ETEC) infections remains a major issue for public health in developing countries. Unfortunately, there is no effective vaccine available for preventing from an ETEC infection. Therefore, the development of a safe and effective vaccine against ETEC is urgently needed. In the present study, A recombinant adenoviral vector Ad5-STa-K99 that capable of expressing a fusion protein of heat-stable enterotoxin (STa) and K99 adhesion antigen of ETEC was generated and its immunogenicity was evaluated in a murine model. The intestinal mucosal secretory IgA(sIgA), serum anti-STa-K99 antibody responses, antigen-specific CD4(+) and CD8(+) T cells frequencies, as well as T-cell proliferation of mice immunized with the viral vector were determined as immunological indexes. The results demonstrated that Ad5-STa-K99 was able to enhance humoral responses with a dramatically augmented antigen-specific serum IgG antibody, and an elevated production of intestinal sIgA in immunized mice, suggesting the elicitation of both of humoral and mucosal immune responses. In addition, this adenoviral vector could significantly promote splenic T cell proliferation and increase the frequencies of CD4(+) and CD8(+) T cell populations in mice, indicative of a capacity to activate T cell responses. More importantly, vaccination of the Ad5-STa-K99 showed a potential to evoke a protective effect from ETEC challenge in mice. These data indicate that the Ad5-STa-K99 is a highly immunogenic vector able to induce a broad range of antigen-specific immune responses in vivo, and evoke a protective immune response against ETEC infections, implying that it may be a novel vaccine candidate warranted for further investigation. PMID:26589454

  19. Adenoviral virotherapy for malignant brain tumors

    PubMed Central

    Nandi, Suvobroto; Lesniak, Maciej S

    2009-01-01

    Glioblastoma multiforme (GBM) is the most common form of primary brain cancer. In the past decade, virotherapy of tumors has gained credence, particularly in glioma management, as these tumors are not completely resectable and tend to micro-metastasize. Adenoviral vectors have an advantage over other viral vectors in that they are relatively non-toxic and do not integrate in the genome. However, the lack of coxsackie and adenovirus receptors (CAR) on surface of gliomas provides for inefficient transduction of wild-type adenoviral vectors in these tumors. By targeting receptors that are over-expressed in gliomas, modified adenoviral constructs have been shown to efficiently infect glioma cells. In addition, by taking advantage of tumor specific promoter (TSP) elements, oncolytic adenoviral vectors offer the promise of selective tumor-specific replication. This dual targeting strategy has enabled specificity in both laboratory and pre-clinical settings. This review looks at current trends in adenoviral virotherapy of gliomas, with an emphasis on targeting modalities and future clinical applications. PMID:19456208

  20. Helper-Dependent Adenoviral Vectors

    PubMed Central

    Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

    2012-01-01

    Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology. PMID:24533227

  1. Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

    PubMed Central

    CAO, Huibi; WANG, Anan; MARTIN, Bernard; KOEHLER, David R.; ZEITLIN, Pamela L.; TANAWELL, A. Keith; HU, Jim

    2015-01-01

    Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr−/−; C38, Cftr-corrected) stimulated with TNF-α, IL-1β or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels of IκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases. PMID:15740640

  2. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors

    PubMed Central

    Suzuki, Keiichiro; Mitsui, Kaoru; Aizawa, Emi; Hasegawa, Kouichi; Kawase, Eihachiro; Yamagishi, Toshiyuki; Shimizu, Yoshihiko; Suemori, Hirofumi; Nakatsuji, Norio; Mitani, Kohnosuke

    2008-01-01

    Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey (Macaca fasicularis) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was ≈100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only ≈1% with other gene delivery methods. Furthermore, in combination with negative selection, ≈45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells. PMID:18768795

  3. Recombinant adenoviral expression of IL-10 protects beta cell from impairment induced by pro-inflammatory cytokine.

    PubMed

    Xu, Ai-Jing; Zhu, Wei; Tian, Fei; Yan, Li-Hua; Li, Tang

    2010-11-01

    Interleukin-10 (IL-10) is a pleiotropic immunosuppressive and immunostimulatory cytokine. In autoimmune diabetes of the nonobese diabetic (NOD) mouse, IL-10 has exhibited paradoxical effects. Systemic IL-10 expression prevented or delayed diabetes onset in NOD mice while local expression of IL-10 did not. As antigen-presenting cells (APCs) play a central role in the generation of primary T cell responses, the direct role of this gene in pancreatic beta (β) cell is not clear. The effects of IL-10 on the protection of β cells in vitro were examined. In the present study, we examined the effects of adenovirus vector-mediated murine IL-10 (mIL-10) gene transfer to islet cell line RINm5F cells in vitro and to explore if IL-10 overexpression may prevent cytokine-mediated cytotoxicity. We had established the recombinant adenovirus vector containing mIL-10 genes (Ad-mIL-10) successfully. After infection of Ad-mIL-10, both mRNA and protein were expressed in RINm5F cells. Moreover, RINm5F cells secreted IL-10 protein into culture medium. Ad-mIL-10 prevented IL-1β-mediated nitric oxide production from β cells in vitro as well as the suppression of β cells function as determined by glucose-stimulated insulin production. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing β cells and caspase-3 activity which were induced by IL-1β and the apoptotic rates of Ad-mIL-10 group were decreased. These findings show that IL-10 gene transfer to β cells may be beneficial in maintaining cells function, protecting islet cells from apoptosis-mediated by factors, which showed the potential therapy for type 1 diabetes mellitus. PMID:20658311

  4. Targeting different types of human meningioma and glioma cells using a novel adenoviral vector expressing GFP-TRAIL fusion protein from hTERT promoter

    PubMed Central

    2011-01-01

    Objective The objective of this study was to evaluate the anti-tumor effects of Ad/gTRAIL (an adenoviral vector in which expression of GFP and TRAIL is driven by a human telomerase reverse transcriptase promoter, hTERT) on malignant meningiomas and gliomas. Background Gliomas and meningiomas are the two most common types of human brain tumors. Currently there is no effective cure for recurrent malignant meningiomas or for gliomas. Ad/gTRAIL has been shown to be effective in killing selected lung, colon and breast cancer cells, but there have been no studies reporting its antitumor effects on malignant meningiomas. Therefore, we tested the antitumor effect of Ad/gTRAIL for the first time in human malignant meningioma and glioma cell lines, and in intracranial M6 and U87 xenografts. Methods Materials and Methods: Human malignant meningioma and glioma cells were infected with adenoviruses, Ad/gTRAIL and Ad/CMV-GFP. Cell viability was determined by proliferation assay. FACS analysis and quantification of TRAIL were used to measure apoptosis in these cells. We injected Ad/gTRAIL viruses in intracranial M6 and U87 xenografts, and measured the brain tumor volume, quantified apoptosis by TUNEL assay in the brain tumor tissue. Results Our studies demonstrate that in vitro/in vivo treatment with Ad/gTRAIL virus resulted in significant increase of TRAIL activity, and elicited a greater tumor cell apoptosis in malignant brain tumor cells as compared to treatment with the control, Ad/CMV-GFP virus without TRAIL activity. Conclusions We showed for the first time that adenovirus Ad/gTRAIL had significant antitumor effects against high grade malignant meningiomas as well as gliomas. Although more work needs to be done, our data suggests that Ad/gTRAIL has the potential to be useful as a tool against malignant brain tumors. PMID:22035360

  5. Adenoviral Delivery of the EMX2 Gene Suppresses Growth in Human Gastric Cancer

    PubMed Central

    Li, Jie; Mo, Minli; Chen, Zhao; Chen, Zhe; Sheng, Qing; Mu, Hang; Zhang, Fang; Zhang, Yi; Zhi, Xiu-Yi; Li, Hui; He, Biao; Zhou, Hai-Meng

    2012-01-01

    Background EMX2 is a human orthologue of the Drosophila empty spiracles homeobox gene that has been implicated in embryogenesis. Recent studies suggest possible involvement of EMX2 in human cancers; however, the role of EMX2 in carcinogenesis needs further exploration. Results In this study, we reported that down-regulation of EMX2 expression was significantly correlated with EMX2 promoter hypermethylation in gastric cancer. Restoring EMX2 expression using an adenovirus delivery system in gastric cancer cell lines lacking endogenous EMX2 expression led to inhibition of cell proliferation and Wnt signaling pathway both in vitro and in a gastric cancer xenograft model in vivo. In addition, we observed that animals treated with the adenoviral EMX2 expression vector had significantly better survival than those treated with empty adenoviral vector. Conclusion Our study suggests that EMX2 is a putative tumor suppressor in human gastric cancer. The adenoviral-EMX2 may have potential as a novel gene therapy for the treatment of patients with gastric cancer. PMID:23029345

  6. Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.

    PubMed

    Souza, Sandra C; Muliro, Kizito V; Liscum, Laura; Lien, Ping; Yamamoto, Mia T; Schaffer, Jean E; Dallal, Gerard E; Wang, Xinzhong; Kraemer, Fredric B; Obin, Martin; Greenberg, Andrew S

    2002-03-01

    Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin treatment induced Peri A hyperphosphorylation and abrogated the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri A Delta 3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduced Peri A hyperphosphorylation and blocked constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells. PMID:11751901

  7. Evaluation of the immune response to recombinant DNA vaccine and adenoviral vaccine co-expressing the M1 and HA genes of H5N1 influenza virus in mice.

    PubMed

    Guo, Jianqiang; Yao, Lihong; Chen, Aijun; Liu, Xiaoyu; Fu, Jinqi; Xu, Pengwei; Zhang, Zhiqing

    2011-06-01

    In order to evaluate the response to vector-expressed M1 and HA genes of influenza virus in mice, we prepared recombinant plasmid pStar-M1/HA and recombinant adenovirus Ad-M1/HA containing both the full-length matrix protein 1(M1) and hemagglutinin (HA) genes of human H5N1 influenza virus strain A/Anhui/1/2005. We then combined the DNA vaccine and adenoviral vaccine in immunization of BALB/c mice with a prime-boost regime. We immunized the mice with DNA vaccine at day 0 and 28 and with recombinant adenoviral vaccines at day 14 and 42. We took blood samples before each injection and 14 days after the final injection for detection of humoral immune responses. At day 56, we sacrificed the mice and collected splenocytes for detection of cellular immune responses. ELISA and hemagglutination inhibition (HI) assay showed that specific IgG Abs against H5N1 influenza virus was induced in serum of the immunized mice. ELISPOT results confirmed that the specific cellular immune responses were successfully induced against the M1 and HA proteins of H5N1 influenza virus. This study provides new strategy for development of novel influenza vaccines. PMID:22034816

  8. Surmounting limited gene delivery into primary immune cell populations: Efficient cell type-specific adenoviral transduction by CAR.

    PubMed

    Clausen, Björn E; Brand, Anna; Karram, Khalad

    2015-06-01

    Ectopic gene expression studies in primary immune cells have been notoriously difficult to perform due to the limitations in conventional transfection and viral transduction methods. Although replication-defective adenoviruses provide an attractive alternative for gene delivery, their use has been hampered by the limited susceptibility of murine leukocytes to adenoviral infection, due to insufficient expression of the human coxsackie/adenovirus receptor (CAR). In this issue of the European Journal of Immunology, Heger et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] report the generation of transgenic mice that enable conditional Cre/loxP-mediated expression of human CAR. The authors demonstrate that this R26/CAG-CAR∆1(StopF) mouse strain facilitates the faithful monitoring of Cre activity in situ as well as the specific and efficient adenoviral transduction of primary immune cell populations in vitro. Further tweaking of the system towards more efficient gene transfer in vivo remains a future challenge. PMID:25903647

  9. Radiolabeled Adenoviral Sub-unit Proteins for Molecular Imaging and Therapeutic Applications in Oncology

    SciTech Connect

    Srivastava, S.; Meinken, G.; Springer, K. Awasthi, V.; Freimuth, P.

    2004-10-06

    The objective of this project was to develop and optimize new ligand systems, based on adenoviral vectors (intact adenovirus, adeno-viral fiber protein, and the knob protein), for delivering suitable radionuclides into tumor cells for molecular imaging and combined gene/radionuclide therapy of cancer.

  10. Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

    SciTech Connect

    Fu, Yuanhui; He, Jinsheng; Zheng, Xianxian; Wu, Qiang; Zhang, Mei; Wang, Xiaobo; Wang, Yan; Xie, Can; Tang, Qian; Wei, Wei; Wang, Min; Song, Jingdong; Qu, Jianguo; Zhang, Ying; Wang, Xin; Hong, Tao

    2009-04-17

    Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.

  11. Optimization of adenoviral vector-mediated transgene expression in the canine brain in vivo, and in canine glioma cells in vitro.

    PubMed

    Candolfi, Marianela; Pluhar, G Elizabeth; Kroeger, Kurt; Puntel, Mariana; Curtin, James; Barcia, Carlos; Muhammad, A K M Ghulam; Xiong, Weidong; Liu, Chunyan; Mondkar, Sonali; Kuoy, William; Kang, Terry; McNeil, Elizabeth A; Freese, Andrew B; Ohlfest, John R; Moore, Peter; Palmer, Donna; Ng, Phillip; Young, John D; Lowenstein, Pedro R; Castro, Maria G

    2007-07-01

    Expression of the immune-stimulatory molecule Fms-like tyrosine kinase 3 ligand (Flt3L) and the conditional cytotoxic enzyme herpes simplex virus type 1 thymidine kinase (HSV1-TK) provides long-term immune-mediated survival of large glioblastoma multiforme (GBM) models in rodents. A limitation for predictive testing of novel antiglioma therapies has been the lack of a glioma model in a large animal. Dogs bearing spontaneous GBM may constitute an attractive large-animal model for GBM, which so far has remained underappreciated. In preparation for a clinical trial in dogs bearing spontaneous GBMs, we tested and optimized adenovirus-mediated transgene expression with negligible toxicity in the dog brain in vivo and in canine J3T glioma cells. Expression of the marker gene beta-galactosidase (beta-Gal) was higher when driven by the murine (m) than the human (h) cytomegalovirus (CMV) promoter in the dog brain in vivo, without enhanced inflammation. In the canine brain, beta-Gal was expressed mostly in astrocytes. beta-Gal activity in J3T cells was also higher with the mCMV than the hCMV promoter driving tetracycline-dependent (TetON) transgene expression within high-capacity adenovirus vectors (HC-Ads). Dog glioma cells were efficiently transduced by HC-Ads expressing mCMV-driven HSV1-TK, which induced 90% reduction in cell viability in the presence of ganciclovir. J3T cells were also effectively transduced with HC-Ads expressing Flt3L under the control of the regulatable TetON promoter system, and as predicted, Flt3L release was stringently inducer dependent. HC-Ads encoding therapeutic transgenes under the control of regulatory sequences driven by the mCMV promoter are excellent vectors for the treatment of spontaneous GBM in dogs, which constitute an ideal preclinical animal model. PMID:17522335

  12. Peptide-Based Technologies to Alter Adenoviral Vector Tropism: Ways and Means for Systemic Treatment of Cancer

    PubMed Central

    Reetz, Julia; Herchenröder, Ottmar; Pützer, Brigitte M.

    2014-01-01

    Due to the fundamental progress in elucidating the molecular mechanisms of human diseases and the arrival of the post-genomic era, increasing numbers of therapeutic genes and cellular targets are available for gene therapy. Meanwhile, the most important challenge is to develop gene delivery vectors with high efficiency through target cell selectivity, in particular under in situ conditions. The most widely used vector system to transduce cells is based on adenovirus (Ad). Recent endeavors in the development of selective Ad vectors that target cells or tissues of interest and spare the alteration of all others have focused on the modification of the virus broad natural tropism. A popular way of Ad targeting is achieved by directing the vector towards distinct cellular receptors. Redirecting can be accomplished by linking custom-made peptides with specific affinity to cellular surface proteins via genetic integration, chemical coupling or bridging with dual-specific adapter molecules. Ideally, targeted vectors are incapable of entering cells via their native receptors. Such altered vectors offer new opportunities to delineate functional genomics in a natural environment and may enable efficient systemic therapeutic approaches. This review provides a summary of current state-of-the-art techniques to specifically target adenovirus-based gene delivery vectors. PMID:24699364

  13. An adenoviral vector for probing promoter activity in primary immune cells

    PubMed Central

    Tripathi, Pulak; Madan, Rajat; Chougnet, Claire; Divanovic, Senad; Ma, Xiaojing; Wahl, Larry M.; Gajewski, Thomas; Karp, Christopher L.; Hildeman, David A.

    2010-01-01

    Functional analysis of the DNA regulatory regions that control gene expression has largely been performed through transient transfection of promoter–reporter constructs into transformed cells. However, transformed cells are often poor models of primary cells. To directly analyze DNA regulatory regions in primary cells, we generated a novel adenoviral luciferase reporter vector, pShuttle-luciferase-GFP (pSLUG) that contains a promoterless luciferase cassette (with an upstream cloning site) for probing promoter activity, and a GFP expression cassette that allows for the identification of transduced cells. Recombinant adenoviruses generated from this vector can transduce a wide range of primary immune cells with high efficiency, including human macrophages, dendritic cells and T cells; and mouse T cells transgenic for the coxsackie and adenoviral receptor (CAR). In primary T cells, we show inducible nuclear factor of activated T cells (NF-AT) activity using a recombinant pSLUG adenovirus containing a consensus NF-AT promoter. We further show inducible IL-12/23 p40 promoter activity in primary macrophages and dendritic cells using a recombinant pSLUG adenovirus containing the proximal human IL-12/23 p40 promoter. The pSLUG system promises to be a powerful tool for the analysis of DNA regulatory regions in diverse types of primary immune cells. PMID:16563424

  14. Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products.

    PubMed

    Gilbert, R; Nalbantoglu, J; Howell, J M; Davies, L; Fletcher, S; Amalfitano, A; Petrof, B J; Kamen, A; Massie, B; Karpati, G

    2001-09-20

    Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer. PMID:11560768

  15. [Prokaryotic expression systems].

    PubMed

    Porowińska, Dorota; Wujak, Magdalena; Roszek, Katarzyna; Komoszyński, Michał

    2013-01-01

    For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities.  PMID:23475488

  16. Adenoviral virotherapy for malignant brain tumors.

    PubMed

    Nandi, Suvobroto; Lesniak, Maciej S

    2009-06-01

    Glioblastoma multiforme is the most common form of primary brain cancer. In the past decade, virotherapy of tumors has gained credence, particularly in glioma management, as these tumors are not completely resectable and tend to micro-metastasize. Adenoviral vectors have an advantage over other viral vectors in that they are relatively non-toxic and do not integrate in the genome. However, the lack of coxsackie and adenovirus receptors on surface of gliomas provides for inefficient transduction of wild-type adenoviral vectors in these tumors. By targeting receptors that are overexpressed in gliomas, modified adenoviral constructs have been shown to efficiently infect glioma cells. In addition, by taking advantage of tumor-specific promoter elements, oncolytic adenoviral vectors offer the promise of selective tumor-specific replication. This dual targeting strategy has enabled specificity in both laboratory and pre-clinical settings. This review examines current trends in adenoviral virotherapy of gliomas, with an emphasis on targeting modalities and future clinical applications. PMID:19456208

  17. Capsid modification strategies for detargeting adenoviral vectors.

    PubMed

    Parker, Alan L; Bradshaw, Angela C; Alba, Raul; Nicklin, Stuart A; Baker, Andrew H

    2014-01-01

    Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by "off target" interactions that preclude cell specific targeting to sites of disease. A number of "off target" interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Factor, p-selectin as well as neutralizing antibodies. Thus, to improve efficacious targeting to sites of disease and limit nonspecific uptake of virus to non-target tissues, specifically the liver and the spleen, it is necessary to develop suitable strategies for genetically modifying the capsid proteins to preclude these interactions. To this end we have developed versatile systems based on homologous recombination for modification of each of the major capsid proteins, which are described herein. PMID:24132476

  18. Magnetically Responsive Biodegradable Nanoparticles Enhance Adenoviral Gene Transfer in Cultured Smooth Muscle and Endothelial Cells

    PubMed Central

    Chorny, Michael; Fishbein, Ilia; Alferiev, Ivan; Levy, Robert J.

    2012-01-01

    Replication-defective adenoviral (Ad) vectors have shown promise as a tool for gene delivery-based therapeutic applications. Their clinical use is however limited by therapeutically suboptimal transduction levels in cell types expressing low levels of Coxsackie-Ad receptor (CAR), the primary receptor responsible for the cell entry of the virus, and by systemic adverse reactions. Targeted delivery achievable with Ad complexed with biodegradable magnetically responsive nanoparticles (MNP) may therefore be instrumental for improving both the safety and efficiency of these vectors. Our hypothesis was that magnetically driven delivery of Ad affinity-bound to biodegradable MNP can substantially increase transgene expression in CAR deficient vascular cells in culture. Fluorescently labeled MNP were formulated from polylactide with inclusion of iron oxide and surface-modified with the D1 domain of CAR as an affinity linker. MNP cellular uptake and GFP reporter transgene expression were assayed fluorimetrically in cultured endothelial and smooth muscle cells using λex/λem of 540 nm/575 nm and 485 nm/535 nm, respectively. Stable vector-specific association of Ad with MNP resulted in formation of MNP–Ad complexes displaying rapid cell binding kinetics following a brief exposure to a high gradient magnetic field with resultant gene transfer levels significantly increased compared to free vector or nonmagnetic control treatment. Multiple regression analysis suggested a mechanism of MNP–Ad mediated transduction distinct from that of free Ad, and confirmed the major contribution of the complexes to the gene transfer under magnetic conditions. The magnetically enhanced transduction was achieved without compromising the cell viability or growth kinetics. The enhancement of adenoviral gene delivery by affinity complexation with biodegradable MNP represents a promising approach with a potential to extend the applicability of the viral gene therapeutic strategies. PMID:19496618

  19. Adenoviral-mediated gene transfer to rabbit synovium in vivo.

    PubMed Central

    Roessler, B J; Allen, E D; Wilson, J M; Hartman, J W; Davidson, B L

    1993-01-01

    Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection. Images PMID:8349791

  20. Gene Transfer into Rat Brain Using Adenoviral Vectors

    PubMed Central

    Puntel, Mariana; Kroeger, Kurt M.; Sanderson, Nicholas S.R.; Thomas, Clare E.; Castro, Maria G.; Lowenstein, Pedro R.

    2010-01-01

    Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc. PMID:20066657

  1. Amplification of inflammation in emphysema and its association with latent adenoviral infection.

    PubMed

    Retamales, I; Elliott, W M; Meshi, B; Coxson, H O; Pare, P D; Sciurba, F C; Rogers, R M; Hayashi, S; Hogg, J C

    2001-08-01

    This study examines the hypothesis that the cigarette smoke-induced inflammatory process is amplified in severe emphysema and explores the association of this response with latent adenoviral infection. Lung tissue from patients with similar smoking histories and either no (n = 7), mild (n = 7), or severe emphysema (n = 7) was obtained by lung resection. Numbers of polymorphonuclear cells (PMN), macrophages, B cells, CD4, CD8 lymphocytes, and eosinophils present in tissue and airspaces and of epithelial cells expressing adenoviral E1A protein were determined using quantitative techniques. Severe emphysema was associated with an absolute increase in the total number of inflammatory cells in the lung tissue and airspaces. The computed tomography (CT) determined extent of lung destruction was related to the number of cells/m(2) surface area by R(2) values that ranged from 0.858 (CD8 cells) to 0.483 (B cells) in the tissue and 0.630 (CD4 cells) to 0.198 (B cells) in the airspaces. These changes were associated with a 5- to 40-fold increase in the number of alveolar epithelial cells expressing adenoviral E1A protein in mild and severe disease, respectively. We conclude that cigarette smoke-induced lung inflammation is amplified in severe emphysema and that latent expression of the adenoviral E1A protein expressed by alveolar epithelial cells influenced this amplification process. PMID:11500352

  2. Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

    PubMed Central

    2011-01-01

    Background Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. Methods In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. Results The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. Conclusion The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin. PMID:21255430

  3. Retrograde adenoviral vector targeting of nociresponsive pontospinal noradrenergic neurons in the rat in vivo

    PubMed Central

    Howorth, Patrick W; Teschemacher, Anja G; Pickering, Anthony E

    2009-01-01

    The spinal dorsal horn receives a dense innervation of noradrenaline-containing fibers that originate from pontine neurons in the A5, locus coeruleus (LC), and A7 cell groups. These pontospinal neurons are believed to constitute a component of the endogenous analgesic system. We used an adenoviral vector with a catecholaminergic-selective promoter (AVV-PRS) to retrogradely label the noradrenergic neurons projecting to the lumbar (L4–L5) dorsal horn with enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein (mRFP). Retrogradely labeled neurons (145 ± 12, n = 14) were found in A5-12%, LC-80% and A7-8% after injection of AVV-PRS-EGFP to the dorsal horn of L4–L5. These neurons were immunopositive for dopamine β-hydroxylase, indicating that they were catecholaminergic. Retrograde labeling was optimal 7 days after injection, persisted for over 4 weeks, and was dependent on viral vector titer. The spinal topography of the noradrenergic projection was examined using EGFP- and mRFP-expressing adenoviral vectors. Pontospinal neurons provide bilateral innervation of the cord and there was little overlap in the distribution of neurons projecting to the cervical and lumbar regions. The axonal arbor of the pontospinal neurons was visualized with GFP immunocytochemistry to show projections to the inferior olive, cerebellum, thalamus, and cortex but not to the hippocampus or caudate putamen. Formalin testing evoked c-fos expression in these pontospinal neurons, suggesting that they were nociresponsive (A5-21%, LC-16%, and A7-26%, n = 8). Thus, we have developed a viral vector-based strategy to selectively, retrogradely target the pontospinal noradrenergic neurons that are likely to be involved in the descending control of nociception. PMID:19003793

  4. Immunocompromised Children with Severe Adenoviral Respiratory Infection

    PubMed Central

    Tylka, Joanna C.; McCrory, Michael C.; Gertz, Shira J.; Custer, Jason W.; Spaeder, Michael C.

    2016-01-01

    Purpose. To investigate the impact of severe respiratory adenoviral infection on morbidity and case fatality in immunocompromised children. Methods. Combined retrospective-prospective cohort study of patients admitted to the intensive care unit (ICU) in four children's hospitals with severe adenoviral respiratory infection and an immunocompromised state between August 2009 and October 2013. We performed a secondary case control analysis, matching our cohort 1 : 1 by age and severity of illness score with immunocompetent patients also with severe respiratory adenoviral infection. Results. Nineteen immunocompromised patients were included in our analysis. Eleven patients (58%) did not survive to hospital discharge. Case fatality was associated with cause of immunocompromised state (p = 0.015), multiple organ dysfunction syndrome (p = 0.001), requirement of renal replacement therapy (p = 0.01), ICU admission severity of illness score (p = 0.011), and treatment with cidofovir (p = 0.005). Immunocompromised patients were more likely than matched controls to have multiple organ dysfunction syndrome (p = 0.01), require renal replacement therapy (p = 0.02), and not survive to hospital discharge (p = 0.004). One year after infection, 43% of immunocompromised survivors required chronic mechanical ventilator support. Conclusions. There is substantial case fatality as well as short- and long-term morbidity associated with severe adenoviral respiratory infection in immunocompromised children. PMID:27242924

  5. Specific NFκB subunit activation and kinetics of cytokine induction in adenoviral keratitis

    PubMed Central

    Rajaiya, Jaya; Sadeghi, Neda

    2009-01-01

    Purpose Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFκB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFκB in adenoviral infection of the cornea. Methods Primary keratocytes derived from human donor corneas were treated with signaling inhibitors and small interfering RNA specific to MAPKs, and infected with adenovirus for different time periods before analysis. Activation of specific NFκB subunits was analyzed by western blot, confocal microscopy, electromobility shift assay, and chromatin immunoprecipitation, and chemokine expression was quantified by enzyme-linked immunosorbent assay. Results Upon adenoviral infection, NFκB p65, p50, and cREL subunits translocate to the nucleus. This translocation is blocked by inhibitors of specific MAPK signaling pathways. Confocal microscopy showed that inhibitors of the p38, JNK, and ERK pathways differentially inhibited NFκB nuclear translocation, while PP2, an inhibitor of Src family kinases, completely inhibited NFκB nuclear translocation. Western blot analysis revealed that activation of specific NFκB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFκB p65 and p50 subunits to the IL-8 promoter upon viral infection was differentially reduced by chemical inhibitors of MAPKs. Electromobility shift assay and luciferase assay analysis

  6. Genetically engineering adenoviral vectors for gene therapy.

    PubMed

    Coughlan, Lynda

    2014-01-01

    Adenoviral (Ad) vectors are commonly used for various gene therapy applications. Significant advances in the genetic engineering of Ad vectors in recent years has highlighted their potential for the treatment of metastatic disease. There are several methods to genetically modify the Ad genome to incorporate retargeting peptides which will redirect the natural tropism of the viruses, including homologous recombination in bacteria or yeast. However, homologous recombination in yeast is highly efficient and can be achieved without the need for extensive cloning strategies. In addition, the method does not rely on the presence of unique restriction sites within the Ad genome and the reagents required for this method are widely available and inexpensive. Large plasmids containing the entire adenoviral genome (~36 kbp) can be modified within Saccharomyces cerevisiae yeast and genomes easily rescued in Escherichia coli hosts for analysis or amplification. A method for two-step homologous recombination in yeast is described in this chapter. PMID:24243238

  7. Adenoviral Vectors for Hemophilia Gene Therapy

    PubMed Central

    Brunetti-Pierri, N; Ng, Philip

    2013-01-01

    Hemophilia is an inherited blood clotting disorder resulting from deficiency of blood coagulation factors. Current standard of care for hemophilia patients is frequent intravenous infusions of the missing coagulation factor. Gene therapy for hemophilia involves the introduction of a normal copy of the deficient coagulation factor gene thereby potentially offering a definitive cure for the bleeding disorder. A variety of approaches have been pursued for hemophilia gene therapy and this review article focuses on those that use adenoviral vectors. PMID:24883229

  8. Transfection of Primary Hepatocytes with Liver-Enriched Transcription Factors Using Adenoviral Vectors.

    PubMed

    Benet, Marta; Jover, Ramiro; Bort, Roque

    2015-01-01

    Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infecting primary cultured hepatocytes. This recombinant adenovirus induces robust expression of the protein of interest in hepatocytes within a wide range of doses. PMID:26272145

  9. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    PubMed

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B; Iozzo, Renato V; Seth, Prem

    2015-12-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases. PMID:26467629

  10. Replication-deficient adenoviral vector for gene transfer potentiates airway neurogenic inflammation.

    PubMed

    Piedimonte, G; Pickles, R J; Lehmann, J R; McCarty, D; Costa, D L; Boucher, R C

    1997-03-01

    Human trials for the treatment of cystic fibrosis lung disease with adenoviral vectors have been complicated by acute inflammatory reactions of unknown etiology. Because replicating respiratory viruses can potentiate tachykinin-mediated neurogenic inflammatory responses in airways, we studied whether the endotracheal administration of a replication-deficient adenoviral vector potentiated this response. The vector Ad5CMVLacZ was administered endotracheally to rats and the leakage of Evans blue dye was used to measure the capsaicin-induced neurogenic albumin extravasation. These studies show that neurogenic albumin extravasation is significantly potentiated in the airways of rats after administration of Ad5CMVLacZ. This inflammatory response can be blocked by selective antagonists of the substance P receptor or by glucocorticoids. Therefore, (1) the acute airway inflammation observed in patients after exposure to adenoviral vectors may exhibit a neurogenic component, which can be blocked pharmacologically, and (2) preclinical adenoviral vector safety studies of other organs innervated by the tachykinin system, e.g., coronary arteries and gastrointestinal tract, should include assessment of neurogenic inflammation. PMID:9070609

  11. An adenoviral vector regulated by hypoxia for the treatment of ischaemic disease and cancer.

    PubMed

    Binley, K; Iqball, S; Kingsman, A; Kingsman, S; Naylor, S

    1999-10-01

    Recombinant adenoviral vectors have a number of advantages for gene therapy, including transduction of a range of dividing and non-dividing cell types. However, this broad range may be a disadvantage if non-target cells are transduced and are adversely affected by expression of the transferred gene. Here we describe a novel adenoviral vector in which transcription of the transgene is restricted to the patho-physiological condition of low oxygen tension (hypoxia). Hypoxia activates the expression of a number of genes, principally via the stabilisation of members of the bHLH/PAS family of transcription factors that bind to a con- sensus DNA sequence, the hypoxia response element (HRE). We have configured an optimised HRE expression cassette into an adenoviral vector, AdOBHRE. A range of cell types, including primary human skeletal muscle, when transduced with AdOBHRE display a low basal level of transgene expression that is highly induced in hypoxia to levels equivalent to that obtained from the CMV promoter. The AdOBHRE vector could be exploited for transcriptionally targeted gene therapy for the treatment of diseases such as cancer, peripheral arterial disease, arthritis and anaemia where tissue hypoxia is a cardinal feature. PMID:10516721

  12. Chemokine CXCL1/KC and its Receptor CXCR2 Are Responsible for Neutrophil Chemotaxis in Adenoviral Keratitis

    PubMed Central

    Chintakuntlawar, Ashish V.

    2009-01-01

    Epidemic keratoconjunctivitis (EKC), caused by human adenovirus (HAdV), is one of the most common ocular infections and results in corneal inflammation and subepithelial infiltrates. Adenoviral keratitis causes significant morbidity to the patients, and is characterized by infiltration of leukocytes in the corneal stroma, and expression of chemokines. The exact role of these chemokines in adenoviral infection has not been studied due to lack of animal models. Here, we have characterized the role of chemokine CXCL1/KC and receptor CXCR2 in adenoviral keratitis using a novel mouse model. Analysis of chemokine expression, leukocyte infiltration, and development of keratitis was performed by ELISA, flow cytometry, and histopathology, respectively. Deficiency of CXCL1 and CXCR2 resulted in delayed infiltration of neutrophils, but not inflammatory monocytes in HAdV-37 corneal infection. CXCL1−/− mice showed decreased expression of CXCL2/MIP-2, but not CCL2/MCP-1. CXCR2−/− mice showed increased expression of CXCL1 and CXCL2, but not CCL2. Both CXCL1−/− and CXCR2−/− mice demonstrated keratitis similar to wild-type mice. In conclusion, both CXCL1 and CXCR2 play an important role in chemokine expression and neutrophil infiltration following adenoviral corneal infection, but have a redundant role in the development of keratitis. PMID:19642907

  13. Adenoviral gene transfer of macrophage inflammatory protein-2 in rat lung.

    PubMed Central

    Foley, R.; Driscoll, K.; Wan, Y.; Braciak, T.; Howard, B.; Xing, Z.; Graham, F.; Gauldie, J.

    1996-01-01

    Replication-defective adenoviral vectors are capable of localized transfer and expression of incorporated gene product in lung tissue. We have constructed an adenoviral vector that expresses rat macrophage inflammatory protein (MIP)-2, a C-X-C chemokine specifically chemotactic for neutrophils, Supernatants from 293 cells, infected with the adenoviral MIP-2 (ADMIP-2) construct, showed potent chemotactic activity and the ability of the ADMIP-2 vector to transcribe and make functional protein was confirmed. In vivo analysis of bronchoalveolar lavage fluid from rats after intratracheal instillation of ADMIP-2 (10(9) plaque-forming units) showed a 10-fold increase in the absolute number of neutrophils in bronchoalveolar lavage fluid as opposed to rats treated with an equal titer of an E1-disabled control virus expressing firefly luciferase (ADCA-18). Neutrophils constituted 65% of total BAL cells with alveolar macrophages being the other major cell type recovered. Rat MIP-2 protein was increased (nanograms per milliliter) in bronchoalveolar lavage fluid over a period of 7 days in ADMIP-2-treated animals. MIP-2 mRNA was demonstrated by Northern blot analysis in lung tissue, and histological analysis confirmed the presence of massive localized tissue neutrophilia. Evidence of chronic tissue injury and repair (ie, fibrosis) was not detected up to 2 weeks after the neutrophil infiltrate had resolved, subsequent to decreased chemokine presence. Adenoviral gene transfer proved an effective tool for the assessment of lung tissue expression of this chemokine in vivo and is useful in developing rodent models of tissue neutrophilia. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 PMID:8863686

  14. Recombinant adenoviral microRNA-206 induces myogenesis in C2C12 cells

    PubMed Central

    Zhang, Weiwei; Wang, Tao; Su, Yongping; Li, Wang; Frame, Lynn T.; Ai, Guoping

    2011-01-01

    Summary Background The expression of microRNA-206 (miR-206) is high in skeletal muscle but low in most other tissues. The expression of miR-206 is increased in muscular dystrophy, suggesting its involvement in the pathogenesis of muscle diseases. To determine the role of miR-206 in muscle cell differentiation and explore a possible gene therapy vector, we constructed a miR-206 adenoviral expression vector (AdvmiR-206) and tested for transfection into C2C12 stem cells. Material/Methods A 355-bp PCR amplicon from C57B6 mouse skeletal muscle genomic DNA was inserted into the adenoviral shuttle vector pAdTrack-CMV, which was then co-transformed with the adenoviral backbone plasmid pAdEasy-1 into competent E. coli BJ5183 bacteria. The specificity and function of this recombinant adenoviral MiR-206 were studied in C2C12 cells by Northern blot, immunofluorescence, Western blot, and flow cytometry. Results Increased expression of miR-206 in AdvmiR-206 transfected C2C12 cells (P<0.001) and resulted in morphological and biochemical changes over time that were similar to serum deprivation, including elongated cells and increased myosin heavy chain proteins. Even in the absence of serum deprivation, miR-206 overexpression accounted for a 50% reduction of S-phase cells (P<0.01). Moreover, in untransfected C2C12 cells, the introduction of miR-206-specific antisense oligoribonucleotides inhibited the normal response to serum deprivation. Twenty-four hours after lipofection of antisense oligoribonucleotides, the number of elongated cells was reduced by half (P<0.01). Conclusions Collectively, these data support a role for miR-206 in myoblast differentiation. We foresee potential applications for the AdvmiR-206 vector in research and therapy. PMID:22129894

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

    PubMed Central

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

    2013-01-01

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

  16. Inhibition of apoptosis reduces immunogeneic potential of adenoviral-treated syngeneic liver grafts.

    PubMed

    Puellmann, Kerstin; Beham, Alexander; Kienle, Klaus; Vogel, Mandy; Schlitt, Hans Juergen; Jauch, Karl Walter; Rentsch, Markus

    2006-11-27

    Effects of adenoviral therapy and reduced apoptosis on immune response were investigated in a rat liver transplantation model after prolonged ischemia-reperfusion. Liver donors were treated i.v. either with an adenoviral construct, expressing bcl-2, green-fluorescent-protein, or doxycyclin. Intrahepatic apoptosis was assessed by terminal transferase dUTP nick end labeling assay. The intrahepatic presence of CD4, CD8a, CD163, immunoglobulin (Ig)beta, tumor necrosis factor (TNF)-alpha and myeloperoxidase (MPO) was quantified by realtime polymerase chain reaction at 24 hours and seven days after transplantation. Bcl-2 expression abrogated the TNF-alpha elevation and reduced apoptosis of hepatocytes and sinusoidal endothelial cells as compared to advCMV green fluorescent protein. No effects on CD4, CD8a, CD163 and MPO expression were noticed in bcl-2 pretreated livers, whereas Igbeta was slightly enhanced compared to controls. Adenoviral infected liver grafts trigger an immune response but reduced apoptosis resulted in down-regulation of TNF-alpha. Thus, bcl-2 transfer might simultaneously reduce graft ischemia reperfusion injury and immunogenicity. PMID:17130789

  17. Magnetofection Enhances Adenoviral Vector-based Gene Delivery in Skeletal Muscle Cells

    PubMed Central

    Pereyra, Andrea Soledad; Mykhaylyk, Olga; Lockhart, Eugenia Falomir; Taylor, Jackson Richard; Delbono, Osvaldo; Goya, Rodolfo Gustavo; Plank, Christian; Hereñu, Claudia Beatriz

    2016-01-01

    The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models. PMID:27274908

  18. Adenoviral-mediated Gene Transfer into the Canine Brain In Vivo

    PubMed Central

    Candolfi, Marianela; Kroeger, Kurt M.; Pluhar, G. Elizabeth; Bergeron, Josee; Puntel, Mariana; Curtin, James F.; McNiel, Elizabeth A.; Freese, Andrew B.; Ohlfest, John R.; Moore, Peter; Lowenstein, Pedro R.; Castro, Maria G.

    2007-01-01

    OBJECTIVE: Glioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding β-galactosidase (βGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo. METHODS: J3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding βGal (Ad-βGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, βGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration. RESULTS: Adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and βGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm2 area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed. CONCLUSION: We demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials. PMID:17228266

  19. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

    PubMed Central

    Ruan, Merry ZC; Cerullo, Vincenzo; Cela, Racel; Clarke, Chris; Lundgren-Akerlund, Evy; Barry, Michael A; Lee, Brendan HL

    2016-01-01

    Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting. PMID:27626040

  20. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes.

    PubMed

    Ruan, Merry Zc; Cerullo, Vincenzo; Cela, Racel; Clarke, Chris; Lundgren-Akerlund, Evy; Barry, Michael A; Lee, Brendan Hl

    2016-01-01

    Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting. PMID:27626040

  1. GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells.

    PubMed

    Honda, Tsuyoshi; Coppola, Simona; Ghibelli, Lina; Cho, Song H; Kagawa, Shunsuke; Spurgers, Kevin B; Brisbay, Shawn M; Roth, Jack A; Meyn, Raymond E; Fang, Bingliang; McDonnell, Timothy J

    2004-04-01

    The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes. PMID:15002033

  2. Construction and evaluation of replication-defective recombinant optimized triosephosphate isomerase adenoviral vaccination in Schistosoma japonicum challenged mice.

    PubMed

    Dai, Yang; Wang, Xiaoting; Zhao, Song; Tang, Jianxia; Zhang, Lu; Dai, Jianrong; Zeng, Mingtao; Lu, Shan; Zhu, Yinchang; Su, Chuan

    2014-02-01

    Schistosomiasis is an endemic, zoonotic parasitic disease that remains a public health concern in China. Development of transmission blocking veterinary vaccines against Schistosoma japonicum infection is urgently needed. Replication-defective adenoviral vector is an efficient vaccine delivery system that has been widely used. Its use is associated with high levels of gene insertion and expression. It is easy to construct and prepare, and is safe. It is not known whether this delivery system can improve the protective effect of schistosome vaccination. Triosephosphate isomerase from S. japonicum (SjTPI) is a promising vaccine candidate. Thus far it has induced only partial protection in animal models and needs to be further enhanced to be effective. We constructed a replication-defective adenoviral vector-based vaccine with optimized SjTPI (rAdV-SjTPI.opt). The specific immune responses and protective efficiency in mice were evaluated. Results showed that intramuscular rAdV-SjTPI.opt induced Th1 biased immune responses in the host, while subcutaneous rAdV-SjTPI.opt induced Th2 predominant immune responses. Oral rAdV-SjTPI.opt induced low levels of immune responses and no significant protection. Intramuscular rAdV-SjTPI.opt provided a consistent and repeatable higher protective effect in mice (more than 50%). These findings may be due to the associated higher levels of specific Th1, antibody responses and partially lower level of IL-17A. This report provides a foundation for developing transmission-blocking veterinary vaccines in larger animals. PMID:24397904

  3. Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.

    PubMed

    Blum, Jeremy S; Parrott, M Brandon; Mikos, Antonios G; Barry, Michael A

    2004-03-01

    We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone. PMID:15013104

  4. Leishmania-based expression systems.

    PubMed

    Taheri, Tahereh; Seyed, Negar; Mizbani, Amir; Rafati, Sima

    2016-09-01

    Production of therapeutic or medical recombinant proteins, such as monoclonal antibodies, proteins, or active enzymes, requires a highly efficient system allowing natural folding and perfect post-translation modifications of the expressed protein. These requirements lead to the generation of a variety of gene expression systems from bacteria to eukaryotes. To achieve the best form of eukaryotic proteins, two factors need to be taken into consideration: choosing a suitable organism to express the protein of interest, and selecting an efficient delivery system. For this reason, the expression of recombinant proteins in eukaryotic nonpathogenic Leishmania parasites is an interesting approach which meets both criteria. Here, new Leishmania-based expression systems are compared with current systems that have long histories in research and industry. PMID:27435294

  5. Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

    PubMed Central

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-01-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

  6. A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells

    PubMed Central

    Buo, Atum M; Williams, Mark S; Kerr, Jaclyn P; Stains, Joseph P

    2016-01-01

    We report here a method for the use of poly-l-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein (GFP)-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research. PMID:27547486

  7. Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV-1-Specific T-Cell Responses in Macaques

    PubMed Central

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN (‘A’), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 (‘P’), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques

  8. A novel Cre recombinase reporter mouse strain facilitates selective and efficient infection of primary immune cells with adenoviral vectors.

    PubMed

    Heger, Klaus; Kober, Maike; Rieß, David; Drees, Christoph; de Vries, Ingrid; Bertossi, Arianna; Roers, Axel; Sixt, Michael; Schmidt-Supprian, Marc

    2015-06-01

    Replication-deficient recombinant adenoviruses are potent vectors for the efficient transient expression of exogenous genes in resting immune cells. However, most leukocytes are refractory to efficient adenoviral transduction as they lack expression of the coxsackie/adenovirus receptor (CAR). To circumvent this obstacle, we generated the R26/CAG-CARΔ1(StopF) (where R26 is ROSA26 and CAG is CMV early enhancer/chicken β actin promoter) knock-in mouse line. This strain allows monitoring of in situ Cre recombinase activity through expression of CARΔ1. Simultaneously, CARΔ1 expression permits selective and highly efficient adenoviral transduction of immune cell populations, such as mast cells or T cells, directly ex vivo in bulk cultures without prior cell purification or activation. Furthermore, we show that CARΔ1 expression dramatically improves adenoviral infection of in vitro differentiated conventional and plasmacytoid dendritic cells (DCs), basophils, mast cells, as well as Hoxb8-immortalized hematopoietic progenitor cells. This novel dual function mouse strain will hence be a valuable tool to rapidly dissect the function of specific genes in leukocyte physiology. PMID:25787118

  9. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    PubMed Central

    Coughlan, Lynda; Alba, Raul; Parker, Alan L.; Bradshaw, Angela C.; McNeish, Iain A.; Nicklin, Stuart A.; Baker, Andrew H.

    2010-01-01

    Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated

  10. Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

    PubMed Central

    Sakurai, Takashi; Lanahan, Anthony; Woolls, Melissa J.; Li, Na; Tirziu, Daniela; Murakami, Masahiro

    2014-01-01

    Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope’s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators. PMID:24998400

  11. Live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy.

    PubMed

    Sakurai, Takashi; Lanahan, Anthony; Woolls, Melissa J; Li, Na; Tirziu, Daniela; Murakami, Masahiro

    2014-01-01

    Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope's autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators. PMID:24998400

  12. Pluto Express power system architecture

    SciTech Connect

    Carr, G.A.

    1996-12-31

    The Pluto Express power system must answer the challenge of the next generation spacecraft by reducing its power, mass and volume envelopes. Technology developed by the New Millennium Program will enable the power system to meet the stringent requirements for the Pluto Express mission without exceeding the spacecraft mass and volume budgets. Traditionally, there has been an increasing trend of the percentage of mass of the power system electronics with respect to the total spacecraft mass. With all of the previous technology focus on high density digital packaging, the power system electronics have not been keeping pace forcing the spacecraft to absorb a relative increase in the power system mass. The increasing trend can be reversed by using mixed signal ASICs and high density multi-chip-module (MCM) packaging techniques validated by the New Millennium Program. As the size of the spacecraft shrinks, the power system electronics must become tightly integrated with the spacecraft loads. The power system architecture needs the flexibility to accommodate the specific load requirements without sacrificing the capability for growth or reduction as the spacecraft requirements change throughout the development. Modularity is a key requirement that will reduce the overall power system cost. Although the focus has been on shrinking the power system volume and mass, the efficiency and functionality cannot be ignored. Increased efficiency and functionality will only enhance the power systems capability to reduce spacecraft power requirements. The combination of the New Millennium packaging technologies with the Pluto Express power system architecture will produce a product with the capability to meet a wide range of mission profiles while reducing system development costs.

  13. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  14. SR-A and SREC-I Are Kupffer and Endothelial Cell Receptors for Helper-dependent Adenoviral Vectors

    PubMed Central

    Piccolo, Pasquale; Vetrini, Francesco; Mithbaokar, Pratibha; Grove, Nathan C; Bertin, Terry; Palmer, Donna; Ng, Philip; Brunetti-Pierri, Nicola

    2013-01-01

    Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes with no chronic toxicity. However, a toxic acute response with potentially lethal consequences has hindered their clinical applications. Liver sinusoidal endothelial cells (LSECs) and Kupffer cells are major barriers to efficient hepatocyte transduction. Understanding the mechanisms of adenoviral vector uptake by non-parenchymal cells may allow the development of strategies aimed at overcoming these important barriers and to achieve preferential hepatocyte gene transfer with reduced toxicity. Scavenger receptors on Kupffer cells bind adenoviral particles and remove them from the circulation, thus preventing hepatocyte transduction. In the present study, we show that HDAd particles interact in vitro and in vivo with scavenger receptor-A (SR-A) and with scavenger receptor expressed on endothelial cells-I (SREC-I) and we exploited this knowledge to increase the efficiency of hepatocyte transduction by HDAd vectors in vivo through blocking of SR-A and SREC-I with specific fragments antigen-binding (Fabs). PMID:23358188

  15. Retrograde optogenetic characterization of the pontospinal module of the locus coeruleus with a canine adenoviral vector.

    PubMed

    Li, Yong; Hickey, Louise; Perrins, Ray; Werlen, Emilie; Patel, Amisha A; Hirschberg, Stefan; Jones, Matt W; Salinas, Sara; Kremer, Eric J; Pickering, Anthony E

    2016-06-15

    Noradrenergic neurons of the brainstem extend projections throughout the neuraxis to modulate a wide range of processes including attention, arousal, autonomic control and sensory processing. A spinal projection from the locus coeruleus (LC) is thought to regulate nociceptive processing. To characterize and selectively manipulate the pontospinal noradrenergic neurons in rats, we implemented a retrograde targeting strategy using a canine adenoviral vector to express channelrhodopsin2 (CAV2-PRS-ChR2-mCherry). LC microinjection of CAV2-PRS-ChR2-mCherry produced selective, stable, transduction of noradrenergic neurons allowing reliable opto-activation in vitro. The ChR2-transduced LC neurons were opto-identifiable in vivo and functional control was demonstrated for >6 months by evoked sleep-wake transitions. Spinal injection of CAV2-PRS-ChR2-mCherry retrogradely transduced pontine noradrenergic neurons, predominantly in the LC but also in A5 and A7. A pontospinal LC (ps:LC) module was identifiable, with somata located more ventrally within the nucleus and with a discrete subset of projection targets. These ps:LC neurons had distinct electrophysiological properties with shorter action potentials and smaller afterhyperpolarizations compared to neurons located in the core of the LC. In vivo recordings of ps:LC neurons showed a lower spontaneous firing frequency than those in the core and they were all excited by noxious stimuli. Using this CAV2-based approach we have demonstrated the ability to retrogradely target, characterise and optogenetically manipulate a central noradrenergic circuit and show that the ps:LC module forms a discrete unit. This article is part of a Special Issue entitled SI: Noradrenergic System. PMID:26903420

  16. Helper-Dependent Adenoviral Vectors and Their Use for Neuroscience Applications.

    PubMed

    Montesinos, Mónica S; Satterfield, Rachel; Young, Samuel M

    2016-01-01

    Neuroscience research has been revolutionized by the use of recombinant viral vector technology from the basic, preclinical and clinical levels. Currently, multiple recombinant viral vector types are employed with each having its strengths and weaknesses depending on the proposed application. Helper-dependent adenoviral vectors (HdAd) are emerging as ideal viral vectors that solve a major need in the neuroscience field: (1) expression of transgenes that are too large to be packaged by other viral vectors and (2) rapid onset of transgene expression in the absence of cytotoxicity. Here, we describe the methods for large-scale production of HdAd viral vectors for in vivo use with neurospecific transgene expression. PMID:27515075

  17. Immunization with Recombinant Adenoviral Vectors Expressing HCV Core or F Proteins Leads to T Cells with Reduced Effector Molecules Granzyme B and IFN-γ: A Potential New Strategy for Immune Evasion in HCV Infection.

    PubMed

    Samrat, Subodh Kumar; Vedi, Satish; Singh, Shakti; Li, Wen; Kumar, Rakesh; Agrawal, Babita

    2015-01-01

    Multispecific, broad, and potent T cell responses have been correlated with viral clearance in hepatitis C virus (HCV) infection. However, the majority of infected patients develop chronic infection, suggesting that natural infection mostly leads to development of inefficient T cell immunity. Multiple mechanisms of immune modulation and evasion have been shown in HCV infection through various investigations. This study examined the generation and modulation of T cell responses against core and frameshift (F) proteins of HCV. A single immunization of mice with replication incompetent recombinant adenovirus vectors encoding for F or core antigens induces poor T cell responses and leads to generation of CD4+ and CD8+ T cells with low granzyme B (GrB) expression. These T cells have impaired GrB enzyme activity and are unable to kill peptide loaded target cells. The low intracellular expression of GrB is not due to degranulation of cytotoxic granules containing cytotoxic T cells. Addition of exogenous IL-2 in in vitro cultures leads to partial recovery of GrB production, whereas immunization with the Toll-like receptor (TLR) agonist poly I:C leads to complete restoration of GrB expression in both CD4+ and CD8+ T cells. Thus, a possible new strategy of T cell modulation is recognized wherein effector T cells are caused to be dysfunctional by HCV-derived antigens F or core, and strategies are also delineated to overcome this dysfunction. These studies are important in the investigation of prophylactic vaccine and immunotherapy strategies for HCV infection. PMID:26133045

  18. Systems Biophysics of Gene Expression

    PubMed Central

    Vilar, Jose M.G.; Saiz, Leonor

    2013-01-01

    Gene expression is a process central to any form of life. It involves multiple temporal and functional scales that extend from specific protein-DNA interactions to the coordinated regulation of multiple genes in response to intracellular and extracellular changes. This diversity in scales poses fundamental challenges to the use of traditional approaches to fully understand even the simplest gene expression systems. Recent advances in computational systems biophysics have provided promising avenues to reliably integrate the molecular detail of biophysical process into the system behavior. Here, we review recent advances in the description of gene regulation as a system of biophysical processes that extend from specific protein-DNA interactions to the combinatorial assembly of nucleoprotein complexes. There is now basic mechanistic understanding on how promoters controlled by multiple, local and distal, DNA binding sites for transcription factors can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including precision and flexibility of the transcriptional responses. PMID:23790365

  19. Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5.

    PubMed

    Ehrke-Schulz, Eric; Zhang, Wenli; Schiwon, Maren; Bergmann, Thorsten; Solanki, Manish; Liu, Jing; Boehme, Philip; Leitner, Theo; Ehrhardt, Anja

    2016-01-01

    High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks and shortcuts developed over the past years working with this HCAdV vector system are presented. PMID:26863087

  20. Large-Scale Production of High-Quality Helper-Dependent Adenoviral Vectors Using Adherent Cells in Cell Factories

    PubMed Central

    Suzuki, Masataka; Cela, Racel; Clarke, Christian; Bertin, Terry K.; Mouriño, Susana

    2010-01-01

    Abstract The most efficient and widely used system for generating helper-dependent adenoviral vectors (HDAds) is the Cre/loxP system developed by Graham and co-workers (Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M.A., and Graham, F.L. [1996]. Proc. Natl. Acad. Sci. U. S. A. 93, 13565–13570). Alternative systems have been developed for HDAd production, but all are limited by the technical complexity of a three-component vector production system for reproducibly generating large quantities of adenovirus with high infectivity and low helper virus (HV) contamination. Recently, these problems were addressed by Ng and co-workers (Palmer, D., and Ng, P. [2003]. Mol Ther. 8, 846–852), who developed an improved system that combines the use of a suspension-adapted producer cell line expressing high levels of Cre recombinase, a HV resistant to mutation, and a refined purification protocol. With this system, >1 × 1013 highly infectious vector particles are easily produced without vector genome rearrangements and having very low HV contamination levels. However, the Ng system incorporates a spinner flask culture system that involves considerable time, effort, and tissue culture medium to produce HDAds. We have an alternative system to obtain comparable quantities with equivalent quality to the spinner flask approach but requiring reduced labor and lower volumes of medium. This method utilizes a 10-chamber cell factory with adherent cells to produce high infectivity of HDAds with minimal HV contamination while improving yield and reducing technical complexity, effort, and medium requirements. This system is easily translatable to the production of clinical-grade HDAds for human trials. PMID:19719388

  1. Molecular epidemiology of adenoviral keratoconjunctivitis in Saudi Arabia

    PubMed Central

    Omar, Nazri; Hammouda, Ehab; Akanuma, Masataka; Ohguchi, Takeshi; Ariga, Toshihide; Tagawa, Yoshitsugu; Kitaichi, Nobuyoshi; Ishida, Susumu; Aoki, Koki; Ishiko, Hiroaki; Ohno, Shigeaki

    2010-01-01

    Purpose Adenoviral keratoconjunctivitis is a major cause of ocular morbidity and may lead to visual loss. Adenovirus types 8, 19, and 37 may cause epidemic keratoconjunctivitis. The main objective of this study was to determine the types of adenoviruses causing keratoconjunctivitis in Saudi Arabia. Methods We conducted a non-interventional observational clinical study. Seventy three eyes from 65 patients who presented to The Eye Center in Riyadh, Saudi Arabia with clinical features of acute adenoviral keratoconjunctivitis were included. Each patient underwent complete clinical examination and features such as membranous reaction, conjunctival hemorrhage, subepithelial corneal infiltrates, and preauricular lymph node enlargement were recorded. Conjunctival swabs were obtained from patients with presumed acute viral conjunctivitis. Immunochromatography (IC) and restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) were performed on the conjunctival swabs obtained from each eye. Serotype identification was performed using direct sequencing technique. Results Forty-nine (67.1%) were adenovirus type 8, 8 (11.0%) were adenovirus type 3, 6 (8.2%) type 37, 5 (6.8%) were adenovirus type 4, and 2 (2.3%) type 19. The remaining 5 were types 14, 19, and 22. The prevalence of membranous conjunctivitis was highest (83%) among eyes with adenovirus type 37 while subepithelial corneal opacities were most commonly seen among eyes with adenovirus type 8 (47%). Immunochromatography tests were positive for adenovirus in 48 (65.7%) out of 73 eyes. Conclusions This study determined the types of adenoviruses causing keratoconjunctivitis at one center in Saudi Arabia. Direct sequencing techniques is an efficient, accurate, and rapid means of diagnosing adenoviral keratoconjunctivitis. The most common causes of adenoviral keratoconjunctivitis in Saudi Arabia were adenovirus types 8, 3, and 37. Membranous conjunctivitis and subepithelial opacities had the highest

  2. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

    PubMed

    Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

    2003-10-01

    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle. PMID:12960972

  3. Specific interaction between adenoviral 55-kDa E1B protein and in vivo produced p53 fusion proteins.

    PubMed

    Chumakov, A; Koeffler, H P

    1993-09-15

    Several protein fusion systems have been used in recent years to study protein-protein and DNA-protein interactions. Most of them use bacterially produced proteins which have several inherent disadvantages, notably, the absence of correct post-translational modifications and the frequent insolubility of recombinant proteins. We sought to develop a system to study proteins interacting with the nuclear phosphoprotein p53, which is believed to be a tumor suppressor. To prepare fusions of p53, we developed a convenient system that permits both in vivo and in vitro production and easy affinity purification of peptides and protein fragments as glutathione-transferase fusions. We placed the coding sequence of the Schistosoma japonica glutathione S-transferase (GST) under the control of the strong CMV/T7 promoter and SV40 splice and polyadenylation signals. An extensive polylinker (MCS) at the 3' end of the GST gene is preceded by the sequence encoding the cleavage site of the site-specific protease. We cloned the complete coding sequences of human wild-type p53, as well as p53 mutants representing all four mutational hotspots (codons 141, 175, 248, and 273), into our expression vector. In vitro transcription using the upstream T7 promoter and translation in reticulocyte lysates form an easy way to produce hybrid proteins; affinity purification on a glutathione-agarose column removes proteins that are present in reticulocyte lysates. We have also studied specific in vivo interactions of human p53 with the adenoviral 55-kDa E1B protein by transfecting expression constructs of GST-p53 fusions into human Ad5-transformed 293 cells. PMID:8406015

  4. Disseminated adenoviral infection masquerading as lower urinary tract voiding dysfunction in a kidney transplant recipient.

    PubMed

    Aboumohamed, Ahmed; Flechner, Stuart M; Chiesa-Vottero, Andres; Srinivas, Titte R; Mossad, Sherif B

    2014-11-01

    Viral infections continue to cause significant morbidity in immunosuppressed kidney transplant patients. Although cytomegalovirus, Epstein-Barr virus and polyoma "BK" virus are more frequently encountered, the Adenovirus can cause multi-organ system infections, and may be difficult to diagnose because it is not often considered in the initial work up in kidney transplant recipients. We present an unusual case of a kidney recipient 1 year post-transplant with disseminated adenoviral infection, who had an initial presentation of lower urinary tract voiding dysfunction with hematuria and sterile pyuria. This progressed to a severe tubulointerstitial nephritis and acute kidney injury that improved with reduction of immunosuppression. Serial blood viral loads are useful for monitoring the course of infection. Urinary adenoviral infection should be considered in the differential diagnosis whenever a kidney transplant recipient presents with unexplained lower tract voiding dysfunction, hematuria, and sterile pyuria. The allograft kidney and bladder can be targets of viral proliferation. Early diagnosis with reduction of immunosuppressive therapy is essential to clear the virus and maintain allograft function. PMID:23816478

  5. Improved Gene Delivery to Intestinal Mucosa by Adenoviral Vectors Bearing Subgroup B and D Fibers

    PubMed Central

    Lecollinet, S.; Gavard, F.; Havenga, M. J. E.; Spiller, O. B.; Lemckert, A.; Goudsmit, J.; Eloit, M.; Richardson, J.

    2006-01-01

    A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses. PMID:16501084

  6. Enhancement of Aerosol Cisplatin Chemotherapy with Gene Therapy Expressing ABC10 protein in Respiratory System

    PubMed Central

    Hohenforst-Schmidt, Wolfgang; Zarogoulidis, Paul; Linsmeier, Bernd; Kioumis, Ioannis; Li, Qiang; Huang, Haidong; Sachpatzidou, Despoina; Lampaki, Sofia; Organtzis, John; Domvri, Kalliopi; Sakkas, Leonidas; Zachariadis, George A.; Archontas, Konstantinos N.; Kallianos, Anastasios; Rapti, Aggeliki; Yarmus, Lonny; Zarogoulidis, Konstantinos; Brachmann, Johannes

    2014-01-01

    Inhaled therapy for lung cancer is a local form of treatment. Currently inhaled non-specific cytotoxic agents have been evaluated as a future treatment for local disease control and distant metastasis control. There are few information regarding the influence of local transporters and gene expression of the respiratory epithelium to the absorption of administered drugs. In the current work we used adenoviral-type 5(dE1/E3) (Cytomegalovirus promoter) with human ABCA10 transgene (Ad-h-ABCA10) purchased from Vector Labs® in order to investigate whether gene therapy can be used as a pre-treatment to enhance the efficiency of inhaled cisplatin. We included the following groups to our work: a) control, b) aerosol vector, c) aerosol vector plus cisplatin, d) aerosol cisplatin, e) intratumoral cisplatin administration, f) intratumoral vector plus cisplatin administration. The results indicate that the aerosol cisplatin group had a long term survival with the intratumoral cisplatin group following. The enhancement of the ABCA family locally to the respiratory system prior to the aerosol cisplatin administration can be used safely and efficiently. Future treatment design of local therapies should include the investigation of local transporters and genes. PMID:24723977

  7. Hepatic Delivery of Artificial Micro RNAs Using Helper-Dependent Adenoviral Vectors.

    PubMed

    Crowther, Carol; Mowa, Betty; Arbuthnot, Patrick

    2016-01-01

    The potential of RNA interference (RNAi)-based gene therapy has been demonstrated in many studies. However, clinical application of this technology has been hampered by a paucity of efficient and safe methods of delivering the RNAi activators. Prolonged transgene expression and improved safety of helper-dependent adenoviral vectors (HD AdVs) makes them well suited to delivery of engineered artificial intermediates of the RNAi pathway. Also, AdVs' natural hepatotropism makes them potentially useful for liver-targeted gene delivery. HD AdVs may be used for efficient delivery of cassettes encoding short hairpin RNAs and artificial primary microRNAs to the mouse liver. Methods for the characterization of HD AdV-mediated delivery of hepatitis B virus-targeting RNAi activators are described here. PMID:26472456

  8. Factors involved in the maturation of murine dendritic cells transduced with adenoviral vector variants

    SciTech Connect

    Kanagawa, Naoko; Koretomo, Ryosuke; Murakami, Sayaka |; Sakurai, Fuminori; Mizuguchi, Hiroyuki |; Nakagawa, Shinsaku; Fujita, Takuya |; Yamamoto, Akira; Okada, Naoki |

    2008-05-10

    Adenoviral vector (Ad)-mediated gene transfer is an attractive method for manipulating the immunostimulatory properties of dendritic cells (DCs) for cancer immunotherapy. DCs treated with Ad have phenotype alterations (maturation) that facilitate T cell sensitization. We investigated the mechanisms of DC maturation with Ad transduction. Expression levels of a maturation marker (CD40) on DCs treated with conventional Ad, fiber-modified Ads (AdRGD, AdF35, AdF35{delta}RGD), or a different serotype Ad (Ad35) were correlated with their transduction efficacy. The {alpha}{sub v}-integrin directional Ad, AdRGD, exhibited the most potent ability to enhance both foreign gene expression and CD40 expression, and induced secretion of interleukin-12, tumor necrosis factor-{alpha}, and interferon-{alpha} in DCs. The presence of a foreign gene expression cassette in AdRGD was not necessary for DC maturation. Maturation of DCs treated with AdRGD was suppressed by destruction of the Ad genome, inhibition of endocytosis, or endosome acidification, whereas proteasome inhibition increased CD40 expression levels on DCs. Moreover, inhibition of {alpha}{sub v}-integrin signal transduction and blockade of cytokine secretion affected the maturation of DCs treated with AdRGD only slightly or not at all, respectively. Thus, our data provide evidence that Ad-induced DC maturation is due to Ad invasion of the DCs, followed by nuclear transport of the Ad genome, and not to the expression of foreign genes.

  9. Comparison of different Bacillus subtilis expression systems.

    PubMed

    Vavrová, Ludmila; Muchová, Katarína; Barák, Imrich

    2010-11-01

    Bacillus subtilis is considered to have great potential as a host for the production and secretion of recombinant proteins. Many different expression systems have been developed for B. subtilis. Here we compare two widely used expression systems, the IPTG-inducible derivative of spac system (hyper-spank) and the xylose-inducible (xyl) to the SURE (subtilin-regulated gene expression) system. Western blot analysis of the membrane protein SpoIISA together with its protein partner SpoIISB showed that the highest expression level of this complex is obtained using the SURE system. Measurement of β-galactosidase activities of the promoter-lacZ fusions in individual expression systems confirmed that the P(spaS) promoter of the SURE system is the strongest of those compared, although the induction/repression ratio reached only 1.84. Based on these results, we conclude that the SURE system is the most efficient of these three B. subtilis expression systems in terms of the amount of expressed product. Remarkably, the yield of the SpoIISA-SpoIISB complex obtained from B. subtilis was comparable to that normally obtained from the Escherichia coli arabinose-inducible expression system. PMID:20863884

  10. Efficacy of Topical Immunoglobulins against Experimental Adenoviral Ocular Infection

    PubMed Central

    Nwanegbo, Edward C.; Romanowski, Eric G.; Gordon, Y. Jerold; Gambotto, Andrea

    2007-01-01

    Purpose Presently, there is no U.S. Federal Drug Administration (FDA)–approved antiviral therapy for the treatment of adenoviral (Ad) ocular infections. The goal of the present study was to determine the antiviral efficacy of human immunoglobulin (Ig), a preparation of highly purified and concentrated immunoglobulin (IgG) antibodies isolated from a large pool of human plasma donors, in vitro and on acute Ad replication in the Ad5 New Zealand White (NZW) rabbit ocular model. Methods The antiviral activity of human Ig against multiple wild-type and human ocular isolates of adenovirus serotypes was investigated in vitro by using neutralizing assays in different human epithelial cell lines. In vivo bilateral topical ocular toxicity and antiviral efficacy were evaluated with established Ad5/NZW rabbit ocular models. In vivo Ig antiviral results were compared with those obtained with topical 0.5% cidofovir and saline. Results In three different epithelial cell lines, ≤6.25 mg/mL of the Ig neutralized several wild-type adenoviral serotypes that cause ocular infections. A dose of ≤10 mg/mL neutralized 88% of ocular isolates of the adenovirus serotypes. After treatment of infected animals, adenovirus-positive cultures per total cultures (days 1–14; P = 0.021), the duration of Ad5 shedding, (P = 0.008), and the mean combined ocular viral titer during the early (days 1–5; P = 0.0001) and the late (days 7–14; P = 0.013) phases of infection were significantly lower in Ig-treated animals than in saline-treated animals and were similar to those in cidofovir-treated animals. Conclusions Ig demonstrated antiviral properties against multiple adenoviral serotypes in vitro and in the Ad5/NZW rabbit ocular model. Further studies are needed to advance topical immunoglobulin for treatment and prophylaxis of ocular infections. PMID:17724203

  11. Adenoviral vector-based strategies against infectious disease and cancer

    PubMed Central

    Zhang, Chao; Zhou, Dongming

    2016-01-01

    ABSTRACT Adenoviral vectors are widely employed against infectious diseases or cancers, as they can elicit specific antibody responses and T cell responses when they are armed with foreign genes as vaccine carriers, and induce apoptosis of the cancer cells when they are genetically modified for cancer therapy. In this review, we summarize the biological characteristics of adenovirus (Ad) and the latest development of Ad vector-based strategies for the prevention and control of emerging infectious diseases or cancers. Strategies to circumvent the pre-existing neutralizing antibodies which dampen the immunogenicity of Ad-based vaccines are also discussed. PMID:27105067

  12. Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

    PubMed Central

    Campos, Samuel K.; Barry, Michael A.

    2008-01-01

    Gene delivery vectors based on Adenoviral (Ad) vectors have enormous potential for the treatment of both hereditary and acquired disease. Detailed structural analysis of the Ad virion, combined with functional studies has broadened our knowledge of the structure/function relationships between Ad vectors and host cells/tissues and substantial achievement has been made towards a thorough understanding of the biology of Ad vectors. The widespread use of Ad vectors for clinical gene therapy is compromised by their inherent immunogenicity. The generation of safer and more effective Ad vectors, targeted to the site of disease, has therefore become a great ambition in the field of Ad vector development. This review provides a synopsis of the structure/function relationships between Ad vectors and host systems and summarizes the many innovative approaches towards achieving Ad vector targeting. PMID:17584037

  13. Evaluation of CD46 re-targeted adenoviral vectors for clinical ovarian cancer intraperitoneal therapy.

    PubMed

    Hulin-Curtis, S L; Uusi-Kerttula, H; Jones, R; Hanna, L; Chester, J D; Parker, A L

    2016-07-01

    Ovarian cancer accounts for >140 000 deaths globally each year. Typically, disease is asymptomatic until an advanced, incurable stage. Although response to cytotoxic chemotherapy is frequently observed, resistance to conventional platinum-based therapies develop rapidly. Improved treatments are therefore urgently required. Virotherapy offers great potential for ovarian cancer, where the application of local, intraperitoneal delivery circumvents some of the limitations of intravenous strategies. To develop effective, adenovirus (Ad)-based platforms for ovarian cancer, we profiled the fluid and cellular components of patient ascites for factors known to influence adenoviral transduction. Levels of factor X (FX) and neutralizing antibodies (nAbs) in ascitic fluid were quantified and tumor cells were assessed for the expression of coxsackie virus and adenovirus receptor (CAR) and CD46. We show that clinical ascites contains significant levels of FX but consistently high CD46 expression. We therefore evaluated in vitro the relative transduction of epithelial ovarian cancers (EOCs) by Ad5 (via CAR) and Ad5 pseudotyped with the fiber of Ad35 (Ad5T*F35++) via CD46. Ad5T*F35++ achieved significantly increased transduction in comparison to Ad5 (P<0.001), independent of FX and nAb levels. We therefore propose selective transduction of CD46 over-expressing EOCs using re-targeted, Ad35-pseudotyped Ad vectors may represent a promising virotherapy for ovarian cancer. PMID:27229159

  14. A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma.

    PubMed

    Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Morshed, Ramin A; Miska, Jason; Ahmed, Atique U; Balyasnikova, Irina V; Han, Yu; Zhang, Lingjiao; Curiel, David T; Lesniak, Maciej S

    2015-09-01

    The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy. PMID:26058317

  15. An Adenoviral Vector Based Vaccine for Rhodococcus equi

    PubMed Central

    Giles, Carla; Ndi, Olasumbo; Barton, Mary D.; Vanniasinkam, Thiru

    2016-01-01

    Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals. PMID:27008624

  16. An Adenoviral Vector Based Vaccine for Rhodococcus equi.

    PubMed

    Giles, Carla; Ndi, Olasumbo; Barton, Mary D; Vanniasinkam, Thiru

    2016-01-01

    Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals. PMID:27008624

  17. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors. PMID:17525704

  18. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    SciTech Connect

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  19. INSM1 promoter-driven adenoviral herpes simplex virus thymidine kinase cancer gene therapy for the treatment of primitive neuroectodermal tumors.

    PubMed

    Wang, Hong-Wei; Breslin, Mary B; Chen, Chiachen; Akerstrom, Victoria; Zhong, Qiu; Lan, Michael S

    2009-11-01

    The INSM1 gene encodes a developmentally regulated zinc finger transcription factor. INSM1 expression is normally absent in adult tissues, but is reactivated in neuroendocrine tumor cells. In the present study, we analyzed the therapeutic potential of an adenoviral INSM1 promoter-driven herpes simplex virus thymidine kinase (HSV-tk) construct in primitive neuroectodermal tumors (PNETs). We constructed an adenoviral INSM1 promoter-driven HSV-tk gene for therapy in PNETs. The PNET-specific adeno-INSM1 promoter HSV-tk construct was tested both in vitro and in vivo in a nude mouse tumor model. Northern blot analysis and transient transfection of an INSM1 promoter-driven luciferase reporter gene indicated that the INSM1 promoter was active in neuroblastoma (IMR-32), retinoblastoma (Y79), and medulloblastoma (D283 Med) cells, but not in glioblastoma (U-87 MG) cells. After Ad-INSM1p-HSV-tk infection, the levels of HSV-tk protein expression were consistent with INSM1 promoter activities. Furthermore, in vitro multiplicity of infection and ganciclovir (GCV) sensitivity studies indicated that the INSM1 promoter could mediate specific expression of the HSV-tk gene and selective killing of INSM1-positive PNETs. In vivo intratumoral adenoviral delivery demonstrated that the INSM1 promoter could direct HSV-tk gene expression in a nude mouse tumor model and effectively repressed tumor growth in response to GCV treatment. Taken together, our data show that the INSM1 promoter is specific and effective for targeted cancer gene therapy in PNETs. PMID:19604042

  20. Intranasal vaccination with a helper-dependent adenoviral vector enhances transgene-specific immune responses in BALB/c mice.

    PubMed

    Fu, Yuan-hui; He, Jin-sheng; Zheng, Xian-xian; Wang, Xiao-bo; Xie, Can; Shi, Chang-xin; Zhang, Mei; Tang, Qian; Wei, Wei; Qu, Jian-guo; Hong, Tao

    2010-01-01

    Helper-dependent adenoviral (HDAd) vectors were developed primarily for genetic disease therapy by deleting all coding regions for attenuating the host cellular immune response to adenovirus (Ad) and long-lasting gene expression. Recently Harui et al. reported that HDAd vaccine could stimulate superior transgene-specific cytotoxic T lymphocyte (CTL) and antibody responses via the intraperitoneal route, compared to first-generation adenoviral (FGAd) vaccine. This prompted us to explore the potential of HDAd as a vaccine vector administrated intranasally. In this study, we prepared HDAd and FGAd vectors expressing enhanced green fluorescent protein (EGFP), respectively, and compared their efficacy in mice. Mice were immunized intranasally with 5x10(9) vp HDAd or FGAd vector particles. Despite stimulating similar anti-Ad antibody responses with FGAd vaccine in the prime/boost strategy, HDAd vector expressing EGFP displayed superior transgene-specific serum IgG, mucosal IgA and cellular immune response, with the characterization of balanced or mixed Th1/Th2 CD4+ T-cell responses. Meanwhile, a single dose of intranasal (i.n.) vaccine of HDAd-EGFP induced a serum IgG response with more efficacy than FGAd-EGFP. In addition, i.n. boost immunization enhanced transgene-specific humoral and cellular responses, compared to single i.n. HDAd-EGFP immunization. Our results suggest that HDAd has potential for a mucosal vaccine vector via i.n. route, which will be useful for the development of vaccines against respiratory viruses, such as respiratory syncytial virus and influenza virus. PMID:19945423

  1. Highly efficient adenoviral transduction of pancreatic islets using a microfluidic device.

    PubMed

    Silva, Pamuditha N; Atto, Zaid; Regeenes, Romario; Tufa, Uilki; Chen, Yih Yang; Chan, Warren C W; Volchuk, Allen; Kilkenny, Dawn M; Rocheleau, Jonathan V

    2016-08-01

    Tissues are challenging to genetically manipulate due to limited penetration of viral particles resulting in low transduction efficiency. We are particularly interested in expressing genetically-encoded sensors in ex vivo pancreatic islets to measure glucose-stimulated metabolism, however poor viral penetration biases these measurements to only a subset of cells at the periphery. To increase mass transfer of viral particles, we designed a microfluidic device that holds islets in parallel hydrodynamic traps connected by an expanding by-pass channel. We modeled viral particle flow into the tissue using fluorescently-labelled gold nanoparticles of varying sizes and showed a penetration threshold of only ∼5 nm. To increase this threshold, we used EDTA to transiently reduce cell-cell adhesion and expand intercellular space. Ultimately, a combination of media flow and ETDA treatment significantly increased adenoviral transduction to the core of the islet. As proof-of-principle, we used this protocol to transduce an ER-targeted redox sensitive sensor (eroGFP), and revealed significantly greater ER redox capacity at core islet cells. Overall, these data demonstrate a robust method to enhance transduction efficiency of islets, and potentially other tissues, by using a combination of microfluidic flow and transient tissue expansion. PMID:27378588

  2. Evaluation of recombinant adenovirus-mediated gene delivery for expression of tracer genes in catecholaminergic neurons

    PubMed Central

    Kim, Mi-La; Han, Shengjun; Lee, Sat-Byol; Kim, Jung Hye; Ahn, Hee Kyung

    2010-01-01

    Selective labeling of small populations of neurons of a given phenotype for conventional neuronal tracing is difficult because tracers can be taken up by all neurons at the injection site, resulting in nonspecific labeling of unrelated pathways. To overcome these problems, genetic approaches have been developed that introduce tracer proteins as transgenes under the control of cell-type-specific promoter elements for visualization of specific neuronal pathways. The aim of this study was to explore the use of tracer gene expression for neuroanatomical tracing to chart the complex interconnections of the central nervous system. Genetic tracing methods allow for expression of tracer molecules using cell-type-specific promoters to facilitate neuronal tracing. In this study, the rat tyrosine hydroxylase (TH) promoter and an adenoviral delivery system were used to express tracers specifically in dopaminergic and noradrenergic neurons. Region-specific expression of the transgenes was then analyzed. Initially, we characterized cell-type-specific expression of GFP or RFP in cultured cell lines. We then injected an adenovirus carrying the tracer transgene into several brain regions using a stereotaxic apparatus. Three days after injection, strong GFP expression was observed in the injected site of the brain. RFP and WGA were expressed in a cell-type-specific manner in the cerebellum, locus coeruleus, and ventral tegmental regions. Our results demonstrate that selective tracing of catecholaminergic neuronal circuits is possible in the rat brain using the TH promoter and adenoviral expression. PMID:21189997

  3. The systemic control of circadian gene expression.

    PubMed

    Gerber, A; Saini, C; Curie, T; Emmenegger, Y; Rando, G; Gosselin, P; Gotic, I; Gos, P; Franken, P; Schibler, U

    2015-09-01

    The mammalian circadian timing system consists of a central pacemaker in the brain's suprachiasmatic nucleus (SCN) and subsidiary oscillators in nearly all body cells. The SCN clock, which is adjusted to geophysical time by the photoperiod, synchronizes peripheral clocks through a wide variety of systemic cues. The latter include signals depending on feeding cycles, glucocorticoid hormones, rhythmic blood-borne signals eliciting daily changes in actin dynamics and serum response factor (SRF) activity, and sensors of body temperature rhythms, such as heat shock transcription factors and the cold-inducible RNA-binding protein CIRP. To study these systemic signalling pathways, we designed and engineered a novel, highly photosensitive apparatus, dubbed RT-Biolumicorder. This device enables us to record circadian luciferase reporter gene expression in the liver and other organs of freely moving mice over months in real time. Owing to the multitude of systemic signalling pathway involved in the phase resetting of peripheral clocks the disruption of any particular one has only minor effects on the steady state phase of circadian gene expression in organs such as the liver. Nonetheless, the implication of specific pathways in the synchronization of clock gene expression can readily be assessed by monitoring the phase-shifting kinetics using the RT-Biolumicorder. PMID:26332965

  4. Isolation and Characterization of Anti-Adenoviral Secondary Metabolites from Marine Actinobacteria

    PubMed Central

    Strand, Mårten; Carlsson, Marcus; Uvell, Hanna; Islam, Koushikul; Edlund, Karin; Cullman, Inger; Altermark, Björn; Mei, Ya-Fang; Elofsson, Mikael; Willassen, Nils-Peder; Wadell, Göran; Almqvist, Fredrik

    2014-01-01

    Adenovirus infections in immunocompromised patients are associated with high mortality rates. Currently, there are no effective anti-adenoviral therapies available. It is well known that actinobacteria can produce secondary metabolites that are attractive in drug discovery due to their structural diversity and their evolved interaction with biomolecules. Here, we have established an extract library derived from actinobacteria isolated from Vestfjorden, Norway, and performed a screening campaign to discover anti-adenoviral compounds. One extract with anti-adenoviral activity was found to contain a diastereomeric 1:1 mixture of the butenolide secondary alcohols 1a and 1b. By further cultivation and analysis, we could isolate 1a and 1b in different diastereomeric ratio. In addition, three more anti-adenoviral butenolides 2, 3 and 4 with differences in their side-chains were isolated. In this study, the anti-adenoviral activity of these compounds was characterized and substantial differences in the cytotoxic potential between the butenolide analogs were observed. The most potent butenolide analog 3 displayed an EC50 value of 91 μM and no prominent cytotoxicity at 2 mM. Furthermore, we propose a biosynthetic pathway for these compounds based on their relative time of appearance and structure. PMID:24477283

  5. Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

    SciTech Connect

    Ugai, Hideyo; Dobbins, George C.; Wang, Minghui; Le, Long P.; Matthews, David A.; Curiel, David T.

    2012-10-25

    Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.

  6. Oncolytic Adenoviral Mutants with E1B19K Gene Deletions Enhance Gemcitabine-induced Apoptosis in Pancreatic Carcinoma Cells and Anti-Tumor Efficacy In vivo

    PubMed Central

    Leitner, Stephan; Sweeney, Katrina; Öberg, Daniel; Davies, Derek; Miranda, Enrique; Lemoine, Nick R.; Halldén, Gunnel

    2010-01-01

    Purpose Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal.We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. Experimental Design Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdΔE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. Results The ΔE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdΔE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. Conclusions Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways.These findings imply that less toxic doses than currently practicedin the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants. PMID:19223497

  7. The Evolution of Adenoviral Vectors through Genetic and Chemical Surface Modifications

    PubMed Central

    Capasso, Cristian; Garofalo, Mariangela; Hirvinen, Mari; Cerullo, Vincenzo

    2014-01-01

    A long time has passed since the first clinical trial with adenoviral (Ad) vectors. Despite being very promising, Ad vectors soon revealed their limitations in human clinical trials. The pre-existing immunity, the marked liver tropism and the high toxicity of first generation Ad (FG-Ad) vectors have been the main challenges for the development of new approaches. Significant effort toward the development of genetically and chemically modified adenoviral vectors has enabled researchers to create more sophisticated vectors for gene therapy, with an improved safety profile and a higher transduction ability of different tissues. In this review, we will describe the latest findings in the high-speed, evolving field of genetic and chemical modifications of adenoviral vectors, a field in which different disciplines, such as biomaterial research, virology and immunology, co-operate synergistically to create better gene therapy tools for modern challenges. PMID:24549268

  8. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

    PubMed

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. PMID:24995995

  9. Baculovirus expression system and method for high throughput expression of genetic material

    DOEpatents

    Clark, Robin; Davies, Anthony

    2001-01-01

    The present invention provides novel recombinant baculovirus expression systems for expressing foreign genetic material in a host cell. Such expression systems are readily adapted to an automated method for expression foreign genetic material in a high throughput manner. In other aspects, the present invention features a novel automated method for determining the function of foreign genetic material by transfecting the same into a host by way of the recombinant baculovirus expression systems according to the present invention.

  10. Combination recombinant simian or chimpanzee adenoviral vectors for vaccine development.

    PubMed

    Cheng, Cheng; Wang, Lingshu; Ko, Sung-Youl; Kong, Wing-Pui; Schmidt, Stephen D; Gall, Jason G D; Colloca, Stefano; Seder, Robert A; Mascola, John R; Nabel, Gary J

    2015-12-16

    Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development. PMID:26514419

  11. Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII

    PubMed Central

    Cela, Racel G.; Suzuki, Masataka; Lee, Brendan; Lipshutz, Gerald S.

    2011-01-01

    Neonatal gene therapy is a promising strategy for treating a number of congenital diseases diagnosed shortly after birth as expression of therapeutic proteins during postnatal life may limit the pathologic consequences and result in a potential “cure.” Hemophilia A is often complicated by the development of antibodies to recombinant protein resulting in treatment failure. Neonatal administration of vectors may avoid inhibitory antibody formation to factor VIII (FVIII) by taking advantage of immune immaturity. A helper-dependent adenoviral vector expressing human factor VIII was administered i.v. to neonatal hemophilia A knockout mice. Three days later, mice produced high levels of FVIII. Levels declined rapidly with animal growth to 5 wk of age with stable factor VIII expression thereafter to >1 y of age. Decline in factor VIII expression was not related to cell-mediated or humoral responses with lack of development of antibodies to capsid or human factor VIII proteins. Subsequent readministration and augmentation of expression was possible as operational tolerance was established to factor VIII without development of inhibitors; however, protective immunity to adenovirus remained. PMID:21245323

  12. Orbital Express fluid transfer demonstration system

    NASA Astrophysics Data System (ADS)

    Rotenberger, Scott; SooHoo, David; Abraham, Gabriel

    2008-04-01

    Propellant resupply of orbiting spacecraft is no longer in the realm of high risk development. The recently concluded Orbital Express (OE) mission included a fluid transfer demonstration that operated the hardware and control logic in space, bringing the Technology Readiness Level to a solid TRL 7 (demonstration of a system prototype in an operational environment). Orbital Express (funded by the Defense Advanced Research Projects Agency, DARPA) was launched aboard an Atlas-V rocket on March 9th, 2007. The mission had the objective of demonstrating technologies needed for routine servicing of spacecraft, namely autonomous rendezvous and docking, propellant resupply, and orbital replacement unit transfer. The demonstration system used two spacecraft. A servicing vehicle (ASTRO) performed multiple dockings with the client (NextSat) spacecraft, and performed a variety of propellant transfers in addition to exchanges of a battery and computer. The fluid transfer and propulsion system onboard ASTRO, in addition to providing the six degree-of-freedom (6 DOF) thruster system for rendezvous and docking, demonstrated autonomous transfer of monopropellant hydrazine to or from the NextSat spacecraft 15 times while on orbit. The fluid transfer system aboard the NextSat vehicle was designed to simulate a variety of client systems, including both blowdown pressurization and pressure regulated propulsion systems. The fluid transfer demonstrations started with a low level of autonomy, where ground controllers were allowed to review the status of the demonstration at numerous points before authorizing the next steps to be performed. The final transfers were performed at a full autonomy level where the ground authorized the start of a transfer sequence and then monitored data as the transfer proceeded. The major steps of a fluid transfer included the following: mate of the coupling, leak check of the coupling, venting of the coupling, priming of the coupling, fluid transfer, gauging

  13. Enhancing the expressiveness of structured reporting systems.

    PubMed

    Langlotz, C P

    2000-05-01

    The overall goal of this research is to build a structured reporting system that reduces the cost, delays, and inconvenience associated with conventional dictation and speech recognition systems. We have implemented such a structured reporting system for radiology that replaces current dictation and transcription processes by allowing radiologists and other imaging professionals to select imaging findings from a medical lexicon. The system uses an imaging-specific information model, called a "description set,' to organize selected terms in a relational database. Unique features of the knowledge representation that enhance its expressiveness include its ability to codify uncertainty about an imaging observation and to represent explicitly the logical relationships among imaging findings. In addition, the system does not require the user to fill in "blanks' in a static text template. Instead, it allows entry of terms in arbitrary order and uses automated text-generation techniques to create a text report that referring physicians are accustomed to receiving. In parallel, the system also produces a multimedia report that the referring physician can use as a quick reference. Unlike the results of conventional dictation or speech recognition, each finding is coded in a relational database for later information processing. Thus, the structured report database can be used to index images by content, to provide real-time decision support, to enhance radiologists' performance, to conduct exploratory clinical research, and to transmit imaging report data to computer-based patient record systems. PMID:10847362

  14. [Eukaryonization of T7 RNA polymerase prokaryotic expression system and development of its couple expression system].

    PubMed

    Zheng, Hai-Xue; Jin, Ye; Yin, Shuang-Hui; Guo, Hui-Chen; Shang, You-Jun; Bai, Xing-Wen; Liu, Xiang-Tao; Xie, Qing-Ge

    2007-09-01

    To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c (+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5' end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, resulted in plasmid pIERS-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIERS-EGFP-ET. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results show that the IRES element from FMDV has the role of initiating CAP-independent translation, and lay foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system. PMID:18051880

  15. Production of serpins using yeast expression systems.

    PubMed

    Pemberton, Philip A; Bird, Phillip I

    2004-02-01

    Serpins occupy a unique niche in the field of biology. As more of them are discovered, the need to produce sufficient quantities of each to aid experimental and therapeutic research increases. Yeast expression systems are well suited for the production of recombinant serpins. The genetics of many yeast species is well understood and readily manipulated to induce the targeted over-production of many different serpins. In addition, protease-deficient strains of certain species are available and a few species carry out post-translational modifications resembling those of humans. Yeasts are easy to grow and multiply readily in simple culture media hence the cost of production is low, while the scale of production can be small or large. The disadvantages are the inability of most yeast(s) to perform complex post-translational modifications and a lower product yield of secreted protein compared to intracellular protein production. However, for the intracellular production of serpins, in particular the clade B serpins that do not have complex post-translational modifications, yeast expression systems should be among the first systems considered. PMID:14698631

  16. Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy

    PubMed Central

    Weymann, Alexander; Arif, Rawa; Weber, Antje; Zaradzki, Marcin; Richter, Karsten; Ensminger, Stephan; Robinson, Peter Nicholas; Wagner, Andreas H.; Karck, Matthias; Kallenbach, Klaus

    2016-01-01

    Objectives Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis. Methods We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM). Results IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1

  17. Adenoviral E4orf3 and E4orf6 Proteins, But Not E1B55K, Increase Killing of Cancer Cells by Radiotherapy in vivo

    SciTech Connect

    Liikanen, Ilkka; Dias, Joao D.; Nokisalmi, Petri; Sloniecka, Marta; Kangasniemi, Lotta; Rajecki, Mari; Dobner, Thomas; Tenhunen, Mikko; Kanerva, Anna; Pesonen, Sari; Ahtiainen, Laura Ph.D.; Hemminki, Akseli

    2010-11-15

    Purpose: Radiotherapy is widely used for treatment of many tumor types, but it can damage normal tissues. It has been proposed that cancer cells can be selectively sensitized to radiation by adenovirus replication or by using radiosensitizing transgenes. Adenoviral proteins E1B55K, E4orf3, and E4orf6 play a role in radiosensitization, by targeting the Mre11, Rad50, and NBS1 complex (MRN) and inhibiting DNA double-strand break (DSB) repair. We hypothesize that combined with irradiation, these adenoviral proteins increase cell killing through the impairment of DSB repair. Methods and Materials: We assessed the radiosensitizing/additive potential of replication-deficient adenoviruses expressing E1B55K, E4orf3, and E4orf6 proteins. Combination treatments with low-dose external photon beam radiotherapy were studied in prostate cancer (PC-3MM2 and DU-145), breast cancer (M4A4-LM3), and head and neck cancer (UT-SCC8) cell lines. We further demonstrated radiosensitizing or additive effects in mice with PC-3MM2 tumors. Results: We show enhanced cell killing with adenovirus and radiation combination treatment. Co-infection with several of the viruses did not further increase cell killing, suggesting that both E4orf6 and E4orf3 are potent in MRN inhibition. Our results show that adenoviral proteins E4orf3 and E4orf6, but not E1B55K, are effective also in vivo. Enhanced cell killing was due to inhibition of DSB repair resulting in persistent double-strand DNA damage, indicated by elevated phospho-H2AX levels at 24 h after irradiation. Conclusions: This knowledge can be applied for improving the treatment of malignant tumors, such as prostate cancer, for development of more effective combination therapies and minimizing radiation doses and reducing side effects.

  18. Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

    PubMed Central

    2013-01-01

    Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the

  19. AN UPDATE OF ADENOVIRAL HEMORRHAGIC DISEASE IN MULE DEER IN CALIFORNIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the summer and fall of 1993, a newly recognized disease, adenoviral hemorrhagic disease, caused widespread mortality in black-tailed (Odocoileus hemionus columbianus) and California mule deer (Odocoileus hemionus californicus) in northern California. Greater than a thousand deer were estimated t...

  20. Effect of 6-azacytidine on the course of experimental adenoviral infection in newborn Syrian hamsters.

    PubMed

    Zarubalev, V V; Slita, A V; Sukhinin, V P; Nosach, L N; Dyachenko, N S; Povnitsa, O Y; Zhovnovataya, V L; Alexeeva, I V; Palchikovskaya, L I

    2007-02-01

    Adenoviral infection is a serious human pathology leading to respiratory, gastrointestinal and ocular disorders and epidemic outbreaks, especially in children's groups. Here we present the results from an investigation of anti- adenoviral effect of 6-azacytidine (6-AC) both in vitro and in vivo. The selectivity index of 6-AC for adenovirus type 5 in HEp-2 cells was 374, the 50% effective concentration was 0.5 mg/ml. For in vivo investigations we developed a model of disseminated adenoviral infection in newborn Syrian hamsters. The infectious virus was recovered from the liver, kidney, lungs and heart. Application of 6-AC led to a reduced period of the virus presence (7 days in the liver and 4 days in the kidney and heart) and lowered virus titers on day 3 post-inoculation (p.i.) (liver - 2.7 and 4.1, heart - 0 and 3.2, kidney - 0 and 2.4 log(10 )CPD(50)/mg tissue weight, in the presence and absence of 6-AC, respectively). Application of 6-AC to newborn Syrian hamsters led to partial destruction of their splenocytes. The results obtained suggest that 6-AC or 6-ACbased drugs with lower toxicity or applied topically may be suitable for therapy and prevention of adenoviral infection in humans. PMID:17309850

  1. The prevalence of adenoviral conjunctivitis at the Clinical Hospital of the State University of Campinas, Brazil

    PubMed Central

    Pinto, Roberto Damian Pacheco; Lira, Rodrigo Pessoa Cavalcanti; Arieta, Carlos Eduardo Leite; de Castro, Rosane Silvestre; Bonon, Sandra Helena Alves

    2015-01-01

    OBJECTIVES: Viral conjunctivitis is a common, highly contagious disease that is often caused by an adenovirus. The aim of this study was to evaluate the prevalence of adenoviral conjunctivitis by analyzing data from a prospective clinical study of 122 consecutively enrolled patients who were treated at the Clinical Hospital of the State University of Campinas (UNICAMP) after a clinical diagnosis of infectious conjunctivitis between November 2011 and June 2012. METHODS: Polymerase chain reaction was used to evaluate all cases of clinically diagnosed infectious conjunctivitis and based on the laboratory findings, the prevalence of adenoviral infections was determined. The incidence of subepithelial corneal infiltrates was also investigated. RESULTS: Of the 122 patients with acute infectious conjunctivitis included, 72 had positive polymerase chain reaction results for adenoviruses and 17 patients developed subepithelial corneal infiltrates (13.93%). CONCLUSIONS: The polymerase chain reaction revealed that the prevalence of adenoviral conjunctivitis was 59% in all patients who presented with a clinical diagnosis of infectious conjunctivitis from November 2011 to June 2012. The prevalence of adenoviral conjunctivitis in the study population was similar to its prevalence in other regions of the world. PMID:26602522

  2. Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors.

    PubMed

    Hanayama, Hiroyuki; Ohashi, Kazuo; Utoh, Rie; Shimizu, Hirofumi; Ise, Kazuya; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Tsuchiya, Hiroyuki; Okano, Teruo; Gotoh, Mitsukazu

    2015-12-17

    To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells. PMID:26858906

  3. Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors

    PubMed Central

    Hanayama, Hiroyuki; Ohashi, Kazuo; Utoh, Rie; Shimizu, Hirofumi; Ise, Kazuya; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Tsuchiya, Hiroyuki; Okano, Teruo; Gotoh, Mitsukazu

    2015-01-01

    To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells. PMID:26858906

  4. Prime/boost immunization with DNA and adenoviral vectors protects from hepatitis D virus (HDV) infection after simultaneous infection with HDV and woodchuck hepatitis virus.

    PubMed

    Fiedler, Melanie; Kosinska, Anna; Schumann, Alexandra; Brovko, Olena; Walker, Andreas; Lu, Mengji; Johrden, Lena; Mayer, Anja; Wildner, Oliver; Roggendorf, Michael

    2013-07-01

    Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8(+) and CD4(+) T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven. PMID:23637419

  5. Bone formation in vivo induced by Cbfa1-carrying adenoviral vectors released from a biodegradable porous β-tricalcium phosphate (β-TCP) material

    NASA Astrophysics Data System (ADS)

    Uemura, Toshimasa; Kojima, Hiroko

    2011-06-01

    Overexpression of Cbfa1 (a transcription factor indispensable for osteoblastic differentiation) is expected to induce the formation of bone directly and indirectly in vivo by accelerating osteoblastic differentiation. Adenoviral vectors carrying the cDNA of Cbfa1/til-1(Adv-Cbf1) were allowed to be adsorbed onto porous blocks of β-tricalcium phosphate (β-TCP), a biodegradable ceramic, which were then implanted subcutaneously and orthotopically into bone defects. The adenoviral vectors were released sustainingly by biodegradation, providing long-term expression of the genes. Results of the subcutaneous implantation of Adv-Cbfa1-adsorbed β-TCP/osteoprogenitor cells suggest that a larger amount of bone formed in the pores of the implant than in the control material. Regarding orthotopic implantation into bone defects, the released Adv-Cbfa1 accelerated regeneration in the cortical bone, whereas it induced bone resorption in the marrow cavity. A safer gene transfer using a smaller amount of the vector was achieved using biodegradable porous β-TCP as a carrier.

  6. INGN 201: Ad-p53, Ad5CMV-p53, Adenoviral p53, INGN 101, p53 gene therapy--Introgen, RPR/INGN 201.

    PubMed

    2003-01-01

    undergoing phase I trials for the potential treatment of lung, breast, ovarian, bladder, liver and brain cancers. Introgen and Aventis Pharma had signed a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute (NCI). NCI will sponsor clinical trials to evaluate and develop RPR/INGN 201 as a potential anticancer agent for these cancer indications. The trials conducted under a NCI-sponsored IND will evaluate RPR/INGN 201 alone and in combination with other anticancer agents. This agreement was originally signed by Rhône-Poulenc Rorer's Gencell. Introgen has completed three phase I clinical trials with INGN 201 in patients with bronchioalveolar cell lung carcinoma, ovarian cancer and recurrent glioblastomas, respectively. Intratumoural injection of RPR/INGN 201 in patients with recurrent glioblastomas was well tolerated and resulted in expression of the p53 protein. Direct administration of RPR/INGN 201 to the lower airways of patients with bronchioalveolar cell lung carcinoma resulted in symptomatic improvement and improved lung function in some patients. In February 2003, Introgen announced that the US Patent and Trademark Office has issued to The Board of Regents of The University of Texas System, patent No. 6,511,847 entitled "Recombinant p53 Adenovirus Methods and Compositions". Introgen Therapeutics is the exclusive licensee of this patent. The patent covers any adenoviral DNA molecules that encode the p53 gene positioned under the control of a promoter. Such a DNA molecule forms the genetic core of Introgen's ADVEXIN cancer therapy. Introgen's ADVEXIN therapy is now covered by up to ten separate US patents relevant to the product including compositions, therapeutic methods of administering the product in virtually any form, alone and in conjunction with the most widely used chemotherapeutic and radiation treatments, as well as its production. Introgen has a number of US patents that relate to the clinical use of ADVEXIN in cancer as

  7. Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.

    PubMed

    Armendáriz-Borunda, Juan; Bastidas-Ramírez, Blanca Estela; Sandoval-Rodríguez, Ana; González-Cuevas, Jaime; Gómez-Meda, Belinda; García-Bañuelos, Jesús

    2011-11-01

    Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture. PMID:21856222

  8. HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins.

    PubMed

    Yadavalli, Raghavendra; Sam-Yellowe, Tobili

    2015-01-01

    Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed "in vitro human cell free expression systems". The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 µl of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer's Cleft - 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 µl of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford's assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be

  9. Cloning and Expression of Leishmania infantum LPG3 Gene by the Lizard Leishmania Expression System

    PubMed Central

    Pirdel, Leila; Hosseini, Ahmad Zavaran; Kazemi, Bahram; Rasouli, Manoochehr; Bandehpour, Mojgan; Soudi, Sara

    2012-01-01

    Background Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum (L.infantum). Methods The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Results Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. Conclusion These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 (rLPG3) to further research in vaccine designing against leishmaniasis. PMID:23407850

  10. The Effects of Adenoviral Transfection of the Keratinocyte Growth Factor Gene on Epidermal Stem Cells: an In Vitro Study

    PubMed Central

    Li, Xinping; Liang, Ling; Zhao, Pin; Uchida, Kenzo; Baba, Hisatoshi; Huang, Hong; Bai, Wenfang; Bai, Liming; Zhang, Mingsheng

    2013-01-01

    Epidermal stem cells (ESCs) are characterized as slow-cycling, multi-potent, and self-renewing cells that not only maintain somatic homeostasis but also participate in tissue regeneration and repair. To examine the feasibility of adenoviral vector-mediated keratinocyte growth factor (KGF) gene transfer into in vitro-expanded ESCs, ESCs were isolated from samples of human skin, cultured in vitro, and then transfected with recombinant adenovirus (Ad) carrying the human KGF gene (AdKGF) or green fluorescent protein gene (AdGFP). The effects of KGF gene transfer on cell proliferation, cell cycle arrest, cell surface antigen phenotype, and β-catenin expression were investigated. Compared to ESCs transfected with AdGFP, AdKGF-transfected ESCs grew well, maintained a high proliferative capacity in keratinocyte serum-free medium, and expressed high levels of β-catenin. AdKGF infection increased the number of ESCs in the G0/G1 phase and promoted ESCs entry into the G2/M phase, but had no effect on cell surface antigen phenotype (CD49f+/CD71−). The results suggest that KGF gene transfer can stimulate ESCs to grow and undergo cell division, which can be applied to enhance cutaneous wound healing. PMID:24170090

  11. Alanine-glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer.

    PubMed

    Salido, Eduardo C; Li, Xiao M; Lu, Yang; Wang, Xia; Santana, Alfredo; Roy-Chowdhury, Namita; Torres, Armando; Shapiro, Larry J; Roy-Chowdhury, Jayanta

    2006-11-28

    Mutations in the alanine-glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt(-/-) mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria. PMID:17110443

  12. Inhibition of rabies virus multiplication by siRNA delivered through adenoviral vector in vitro in BHK-21 cells and in vivo in mice.

    PubMed

    Sonwane, Arvind A; Dahiya, Shyam S; Saini, Mohini; Chaturvedi, V K; Singh, R P; Gupta, Praveen K

    2012-08-01

    To evaluate antiviral potential of adenoviral vector-delivered small interfering RNA (siRNA) against rabies, recombinant, replication-defective adenoviral vectors (rAdV) encoding siRNAs targeting rabies virus (RV) polymerase (L) and nucleoprotein (N) genes were developed. The siRNAs were delivered as small hairpin RNAs (shRNAs) through these vectors. Treatment of BHK-21 cells with rAdV expressing siRNA targeting L gene (rAdV-L) and N gene (rAdV-N) (100 MOI) and their subsequent infection with RV (0.001 MOI, RV PV-11), reduced RV fluorescent foci by 48.2% (mean±SEM; 48.17±0.6540, N=6) and 41.8% (mean±SEM; 41.83±0.3073, N=6), respectively, with respect to that of BHK-21 cells treated with rAdV expressing negative control siRNA (rAdV-Neg) indicating inhibition of multiplication of RV in BHK-21 cells in response to adenoviral vector mediated siRNA delivery. Also, the similar treatment of BHK-21 cells with rAdV-L and rAdV-N and similar subsequent infection of them with RV resulted in reduction in RV mRNA transcript levels for their respective targets (RV L gene for rAdV-L and N gene for rAdV-N). mRNA transcript level for RV L gene was reduced by 17.88-fold (mean±SEM; 17.88±0.06638, N=6) in cells treated with rAdV-L and that for RV N gene was reduced by 5.7-fold (mean±SEM; 5.7±0.04472, N=6), in cells treated with rAdV-N, in comparison with that in cells treated with rAdV-Neg, as analyzed by using real-time PCR. These in vitro studies showed that between these two, adenoviral vector mediated delivery of siRNA targeting RV L gene was comparatively more effective in inhibiting RV multiplication in BHK-21 cells than that of siRNA targeting RV N gene (p<0.0001). Localized treatment (intramuscular injection in masseter muscle) of mice with 10(7) plaque forming units of either rAdV-L or rAdV-N and subsequent lethal RV infection (15-20LD(50) of CVS-11) at the same site, through the same route, although resulted in 50% protection (3 out of 6 mice survived) against lethal

  13. Synthesis and Comparative Study of Anti-Adenoviral Activity of 6-Azacytidine and Its Analogues.

    PubMed

    Alexeeva, Inna; Nosach, Lydia; Palchykovska, Larisa; Usenko, Lyubov; Povnitsa, Olga

    2015-01-01

    This paper presents the results of synthesis and study of cytotoxicity and the anti-adenoviral activity of new N4-derivatives of 6-azacytidine and its α-L-glycopyranosyl analogues obtained by the simplified one-pot version of the silyl condensation method. The resulting acylated 4-methylmercapto-1,2,4-triazin-3(2Н)-one glycosides then underwent the amination and/or ammonolysis to provide 6-azacytidine glycoside analogues (2-6, 12, 15, 17) and compounds with modifications at both base and sugar fragments (11, 15). The evaluation of cytotoxicity and antiviral activity of new compounds against AdV5 showed high selectivity indexes for N4-methyl-6-azacytidine (2) and N,O-tetraacetyl-6-azacytidine (8). High anti-adenoviral activity of N4-methyl-6-azacytidine as well as very low cytotoxicity may suggest its further investigation as potential compound for the therapy of AdV infection. PMID:26167665

  14. Treatment for retinopathy of prematurity in an infant with adenoviral conjunctivitis.

    PubMed

    Gunay, Murat; Celik, Gokhan; Con, Rahim

    2015-01-01

    Retinopathy of prematurity (ROP) has been a major problematic disorder during childhood. Laser photocoagulation (LPC) has been proven to be effective in most of the ROP cases. Adenoviral conjunctivitis (AVC) is responsible for epidemics among adult and pediatric population. It has also been reported to be a cause of outbreaks in neonatal intensive care units (NICU) several times. We herein demonstrate a case with AVC who underwent LPC for ROP. And we discuss the treatment methodology in such cases. PMID:25874149

  15. Expression of UNC-5 in the developing Xenopus visual system.

    PubMed

    Anderson, Richard B; Holt, Christine E

    2002-10-01

    Netrin-1 and its receptors deleted in colorectal cancer (DCC) and neogenin are expressed within the developing visual system and have been implicated in mediating axon guidance and cell migration. However, the expression of the netrin-1 receptor UNC-5 has not yet been addressed. Here we clone Xenopus UNC-5 (XUNC-5) and describe its expression within the developing visual system. Xunc-5 transcripts are expressed within the developing optic vesicles and later become restricted to the dorsal ciliary marginal zone, a site of retinoblast proliferation and differentiation. This highly restricted expression pattern could make XUNC-5 an excellent marker of dorsal retinal precursor cells. PMID:12351179

  16. Advances and future challenges in recombinant adenoviral vectored H5N1 influenza vaccines.

    PubMed

    Zhang, Jianfeng

    2012-11-01

    The emergence of a highly pathogenic avian influenza virus H5N1 has increased the potential for a new pandemic to occur. This event highlights the necessity for developing a new generation of influenza vaccines to counteract influenza disease. These vaccines must be manufactured for mass immunization of humans in a timely manner. Poultry should be included in this policy, since persistent infected flocks are the major source of avian influenza for human infections. Recombinant adenoviral vectored H5N1 vaccines are an attractive alternative to the currently licensed influenza vaccines. This class of vaccines induces a broadly protective immunity against antigenically distinct H5N1, can be manufactured rapidly, and may allow mass immunization of human and poultry. Recombinant adenoviral vectors derived from both human and non-human adenoviruses are currently being investigated and appear promising both in nonclinical and clinical studies. This review will highlight the current status of various adenoviral vectored H5N1 vaccines and will outline novel approaches for the future. PMID:23202501

  17. Adenoviral vectors for prodrug activation-based gene therapy for cancer

    PubMed Central

    Doloff, Joshua C.; Waxman, David J.

    2013-01-01

    Cancer cell heterogeneity is a common feature - both between patients diagnosed with the same cancer and within an individual patient’s tumor - and leads to widely different response rates to cancer therapies and the potential for the emergence of drug resistance. Diverse therapeutic approaches have been developed to combat the complexity of cancer, including individual treatment modalities designed to target tumor heterogeneity. This review discusses adenoviral vectors and how they can be modified to replicate in a cancer-specific manner and deliver therapeutic genes under multi-tiered regulation to target tumor heterogeneity, including heterogeneity associated with cancer stem cell-like subpopulations. Strategies that allow for combination of prodrug-activation gene therapy with a novel replication-conditional, heterogeneous tumor-targeting adenovirus are discussed, as are the benefits of using adenoviral vectors as tumor-targeting oncolytic vectors. While the anticancer activity of many adenoviral vectors has been well established in preclinical studies, only limited successes have been achieved in the clinic, indicating a need for further improvements in activity, specificity, tumor cell delivery and avoidance of immunogenicity. PMID:23869779

  18. Advances and Future Challenges in Recombinant Adenoviral Vectored H5N1 Influenza Vaccines

    PubMed Central

    Zhang, Jianfeng

    2012-01-01

    The emergence of a highly pathogenic avian influenza virus H5N1 has increased the potential for a new pandemic to occur. This event highlights the necessity for developing a new generation of influenza vaccines to counteract influenza disease. These vaccines must be manufactured for mass immunization of humans in a timely manner. Poultry should be included in this policy, since persistent infected flocks are the major source of avian influenza for human infections. Recombinant adenoviral vectored H5N1 vaccines are an attractive alternative to the currently licensed influenza vaccines. This class of vaccines induces a broadly protective immunity against antigenically distinct H5N1, can be manufactured rapidly, and may allow mass immunization of human and poultry. Recombinant adenoviral vectors derived from both human and non-human adenoviruses are currently being investigated and appear promising both in nonclinical and clinical studies. This review will highlight the current status of various adenoviral vectored H5N1 vaccines and will outline novel approaches for the future. PMID:23202501

  19. Antitumor activity of adenoviral vector containing T42 and 4xT42 peptide gene through inducing apoptosis of tumor cells and suppressing angiogenesis.

    PubMed

    Zhang, Xiong; Qi, Dong-Dong; Zhang, Ting-Ting; Chen, Qing-Xin; Wang, Guang-Zhi; Sui, Guang-Yu; Hao, Xue-Wei; Sun, Shouli; Song, Xue; Chen, Ying-Li

    2015-03-01

    The T42 peptide, generated from two active fragments of tumstatin, has been shown to have anti‑tumor activity. The adenoviral vector is the most frequently used vector in research and clinical trials for gene therapy. In the present study, the anti‑tumor activity of the T42 peptide and quadruple T42 (4xT42) peptide adenoviral vectors were elucidated for the first time, to the best of our knowledge. Human embryonic kidney 293 cells were infected with plasmid adenovirus (pAd)‑enhanced green fluorescent protein (EGFP)‑T42 or pAd‑EGFP‑4xT42 and the expression of the T42 and 4xT42 genes was confirmed by the identification of GFP expression and reverse transcription polymerase chain reaction experiments. The anti‑cancer effects of pAd‑EGFP‑T42 and pAd‑EGFP‑4xT42 on breast cancer cells in vivo and in vitro were subsequently investigated. The results indicated that the packaging of the recombinant adenoviruses with the viral titer was successful, following purification at 5x109 plaque forming units/ml. The results also revealed that the recombinant adenoviruses promoted apoptosis in MCF‑7 breast cancer cells and inhibited cancer growth. Through the analysis of caspase‑3, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein expression, it was demonstrated that the T42/4xT42 peptide may induce apoptosis via the mitochondrial pathway. In addition, mouse xenograft experiments confirmed that the T42 peptide inhibited tumor growth and reduced angiogenesis in vivo. In conclusion, the results of the present study indicated that the T42 and 4xT42 peptide genes, transfected by a recombinant adenovirus, may provide a potential novel strategy for the treatment of breast cancer. PMID:25384346

  20. Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

    SciTech Connect

    Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

    2011-11-25

    We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: > We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. > Cre/loxP recombination was used to modify the adenovirus genome. > A targeting ligand present on capsid protein IX was removed or replaced using recombination. > Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

  1. Oncolytic adenoviral vectors which employ the survivin promoter induce glioma oncolysis via a process of beclin-dependent autophagy

    PubMed Central

    ULASOV, ILYA V.; TYLER, MATHEW A.; ZHU, ZENG B.; HAN, YU; HE, TONG-CHUAN; LESNIAK, MACIEJ S.

    2009-01-01

    Survivin has gained attention as a tumor-specific marker which is upregulated in a variety of neoplasms. Although the survivin protein is implicated in anti-apoptotic tumor pathways, little is known about the function of the survivin promoter. In this study, we constructed a conditionally replicative adenoviral vector (CRAd) that utilizes the survivin promoter and examined the mechanism of CRAd induced cell death in malignant glioma. Our results indicate that CRAd vectors which utilize the survivin promoter effectively replicate in glioma cells and exhibit a high oncolytic effect. The survivin-mediated CRAd appeared to induce apoptosis as measured by Annexin/7-AAD. Caspase-3 and BAX mRNAs were upregulated based on microarray data, however, Western blot analysis of infected cells showed no evidence of elevated caspase-3, BAX, or p53 protein expression. Of note, at each time point infected glioma cells showed no evidence of activated BAD or AKT. The inhibition of AKT signaling led us to examine autophagy in infected cells. Electron micrographs of virally infected glioma cells suggested autophagosomal-mediated cell death and selective blocking of beclin with siRNA prevented autophagy. These results indicate that the survivin promoter enhances viral replication and induces autophagy of infected glioma cells via a beclin-dependent mechanism. PMID:19212678

  2. Neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer.

    PubMed

    Li, Rongying; Oka, Kazuhiro; Yechoor, Vijay

    2012-01-01

    Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes. Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression(1). They lack chronic toxicity because they lack viral coding sequences(2-5) and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes. In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice. PMID:23093064

  3. Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71.

    PubMed

    Zhang, Chao; Yang, Yong; Chi, Yudan; Yin, Jieyun; Yan, Lijun; Ku, Zhiqiang; Liu, Qingwei; Huang, Zhong; Zhou, Dongming

    2015-09-22

    Hand, foot and mouth disease (HFMD) is a major public health concern in Asia; more efficient vaccines against HFMD are urgently required. Adenoviral (Ad) capsids have been used widely for the presentation of foreign antigens to induce specific immune responses in the host. Here, we describe a novel bivalent vaccine for HFMD based on the hexon-modified, E1-deleted chimpanzee adenovirus serotype 68 (AdC68). The novel vaccine candidate was generated by incorporating the neutralising epitope of Coxsackievirus A16 (CA16), PEP71, into hypervariable region 1 (HVR1), and a shortened neutralising epitope of Enterovirus 71 (EV71), sSP70, into HVR2 of the AdC68 hexon. In order to enhance the immunogenicity of EV71, VP1 of EV71 was cloned into the E1-region of the AdC68 vectors. The results demonstrated that these two epitopes were well presented on the virion surface and had high affinity towards specific antibodies, and VP1 of EV71 was also significantly expressed. In pre-clinical mouse models, the hexon-modified AdC68 elicited neutralising antibodies against both CA16 and EV71, which conferred protection to suckling mice against a lethal challenge of CA16 and EV71. In summary, this study demonstrates that the hexon-modified AdC68 may represent a promising bivalent vaccine carrier against EV71 and CA16 and an epitope-display platform for other pathogens. PMID:26296491

  4. Adenoviral delivery of the beta2-adrenoceptor gene in sepsis: a subcutaneous approach in rat for kidney protection.

    PubMed

    Nakamura, Akio; Imaizumi, Akira; Niimi, Ryo; Yanagawa, Yukishige; Kohsaka, Takao; Johns, Edward J

    2005-12-01

    Successful gene therapy requires gene delivery that is efficient, has an optimal route of administration and has biosafety. The aims of the present study were to evaluate the safety and applicability of the subcutaneous delivery route for adenoviral transgenes containing the human beta(2)-adrenoceptor (adeno-beta(2)-AR) and to investigate whether this approach prevented renal dysfunction in a rat model of endotoxaemic shock induced by LPS (lipopolysaccharide). Subcutaneous administration of adeno-beta(2)-AR (a total of 10(10) viral particles) significantly increased beta-AR density in the kidney, lung and liver, but was without effect on physiological and plasma biochemical parameters. Moreover, this dose of virus did not cause any of the potential toxic responses of viral administration, such as inflammation and tissue TNF (tumour necrosis factor)-alpha expression. Although the LPS challenge caused a decrease in glomerular filtration rate, fractional excretion of sodium and renal beta-AR density in all groups, the reduction in renal function was significantly less in the rats given adeno-beta(2)-AR compared with non-treated rats. Thus, although further evaluation will be required, this initial study demonstrated that the subcutaneous injection of adeno-beta(2)-AR was efficient, comparatively non-pathogenic and potentially therapeutic to deal with acute renal failure associated with sepsis. PMID:16076286

  5. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    PubMed

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury. PMID:15474356

  6. Thrombomodulin Expression in Tissues From Dogs With Systemic Inflammatory Disease.

    PubMed

    Kim, S D; Baker, P; DeLay, J; Wood, R D

    2016-07-01

    Thrombomodulin (TM) is a membrane glycoprotein expressed on endothelial cells, which plays a major role in the protein C anticoagulation pathway. In people with inflammation, TM expression can be down-regulated on endothelial cells and a soluble form released into circulation, resulting in increased risk of thrombosis and disseminated intravascular coagulation. TM is present in dogs; however, there has been minimal investigation of its expression in canine tissues, and the effects of inflammation on TM expression in canine tissues have not been investigated. The objective of this study was to evaluate endothelial TM expression in tissues from dogs with systemic inflammatory diseases. A retrospective evaluation of tissue samples of lung, spleen, and liver from dogs with and without systemic inflammatory diseases was performed using immunohistochemistry (IHC) and a modified manual IHC scoring system. TM expression was significantly reduced in all examined tissues in dogs diagnosed with septic peritonitis or acute pancreatitis. PMID:26926084

  7. An IPTG Inducible Conditional Expression System for Mycobacteria

    PubMed Central

    Ravishankar, Sudha; Ambady, Anisha; Ramu, Haripriya; Mudugal, Naina Vinay; Tunduguru, Ragadeepthi; Anbarasu, Anand; Sharma, Umender K.; Sambandamurthy, Vasan K.; Ramaiah, Sudha

    2015-01-01

    Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of β-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression

  8. An IPTG Inducible Conditional Expression System for Mycobacteria.

    PubMed

    Ravishankar, Sudha; Ambady, Anisha; Ramu, Haripriya; Mudugal, Naina Vinay; Tunduguru, Ragadeepthi; Anbarasu, Anand; Sharma, Umender K; Sambandamurthy, Vasan K; Ramaiah, Sudha

    2015-01-01

    Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of β-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression

  9. Adenoviral-E2F-1 radiosensitizes p53{sup wild-type} and p53{sup null} human prostate cancer cells

    SciTech Connect

    Nguyen, Khanh H.; Hachem, Paul; Khor, L.-Y.; Salem, Naji; Hunt, Kelly K.; Calkins, Peter R.; Pollack, Alan . E-mail: Alan.Pollack@fccc.edu

    2005-09-01

    Purpose: E2F-1 is a transcription factor that enhances the radiosensitivity of various cell lines by inducing apoptosis. However, there are conflicting data concerning whether this enhancement is mediated via p53 dependent pathways. Additionally, the role of E2F-1 in the response of human prostate cancer to radiation has not been well characterized. In this study, we investigated the effect of Adenoviral-E2F-1 (Ad-E2F-1) on the radiosensitivity of p53{sup wild-type} (LNCaP) and p53{sup null} (PC3) prostate cancer cell lines. Methods and Materials: LNCaP and PC3 cells were transduced with Ad-E2F-1, Adenoviral-Luciferase (Ad-Luc) control vector, or Adenoviral-p53 (Ad-p53). Expression of E2F-1 and p53 was examined by Western blot analysis. Annexin V and caspase 3 + 7 assays were performed to estimate the levels of apoptosis. Clonogenic survival assays were used to determine overall cell death. Statistical significance was determined by analysis of variance, using the Bonferroni method to correct for multiple comparisons. Results: Western blot analysis confirmed the efficacy of transductions with Ad-E2F-1 and Ad-p53. Ad-E2F-1 transduction significantly enhanced apoptosis and decreased clonogenic survival in both cell lines. These effects were compounded by the addition of RT. Although E2F-1-mediated radiosensitization was independent of p53 status, this effect was more pronounced in p53{sup wild-type} LNCaP cells. When PC3 cells were treated with Ad-p53 in combination with RT and Ad-E2F-1, there was at least an additive reduction in clonogenic survival. Conclusions: Our results suggest that Ad-E2F-1 significantly enhances the response of p53{sup wild-type} and p53{sup null} prostate cancer cells to radiation therapy, although radiosensitization is more pronounced in the presence of p53. Ad-E2F-1 may be a useful adjunct to radiation therapy in the treatment of prostate cancer.

  10. Relationships between Written Expression Achievement and the Cognitive Assessment System.

    ERIC Educational Resources Information Center

    Johnson, Judy A.; Bardos, Achilles N.; Tayebi, Kandi A.

    The purpose of this study was to explore the relationships between the Cognitive Assessment System (CAS) and writing achievement in junior high students (aged 11-15 years) with and without written expression disabilities. Ninety-six students with (n=48) and without (n=48) written expression disabilities were administered the Das-Naglieri:…

  11. The Facial Expression Coding System (FACES): Development, Validation, and Utility

    ERIC Educational Resources Information Center

    Kring, Ann M.; Sloan, Denise M.

    2007-01-01

    This article presents information on the development and validation of the Facial Expression Coding System (FACES; A. M. Kring & D. Sloan, 1991). Grounded in a dimensional model of emotion, FACES provides information on the valence (positive, negative) of facial expressive behavior. In 5 studies, reliability and validity data from 13 diverse…

  12. Local and Systemic Cytokine Expression in Patients with Postherpetic Neuralgia

    PubMed Central

    Üçeyler, Nurcan; Valet, Michael; Kafke, Waldemar; Tölle, Thomas R.; Sommer, Claudia

    2014-01-01

    Background Postherpetic neuralgia (PHN) is the painful complication of a varicella zoster virus reactivation. We investigated the systemic and local gene expression of pro- and anti-inflammatory cytokine expression in patients with PHN. Methods Thirteen patients with PHN at the torso (Th4-S1) were recruited. Skin punch biopsies were obtained from the painful and the contralateral painless body area for intraepidermal nerve fiber density (IENFD) and cytokine profiling. Additionally, blood was withdrawn for systemic cytokine expression and compared to blood values of healthy controls. We analyzed the gene expression of selected pro- and anti-inflammatory cytokines (tumor necrosis factor-alpha [TNF] and interleukins [IL]-1β, IL-2, and IL-8). Results IENFD was lower in affected skin compared to unaffected skin (p<0.05), while local gene expression of pro- and anti-inflammatory cytokines did not differ except for two patients who had 7fold higher IL-6 and 10fold higher IL-10 gene expression in the affected skin compared to the contralateral unaffected skin sample. Also, the systemic expression of cytokines in patients with PHN and in healthy controls was similar. Conclusion While the systemic and local expression of the investigated pro- and anti-inflammatory cytokines was not different from controls, this may have been influenced by study limitations like the low number of patients and different disease durations. Furthermore, other cytokines or pain mediators need to be considered. PMID:25127283

  13. Controlled expression of functional miR-122 with a ligand inducible expression system

    PubMed Central

    2010-01-01

    Background To study the biological function of miRNAs, and to achieve sustained or conditional gene silencing with siRNAs, systems that allow controlled expression of these small RNAs are desirable. Methods for cell delivery of siRNAs include transient transfection of synthetic siRNAs and expression of siRNAs in the form of short hairpins using constitutive RNA polymerase III promoters. Systems employing constitutive RNA polymerase II promoters have been used to express miRNAs. However, for many experimental systems these methods do not offer sufficient control over expression. Results We present an inducible mammalian expression system that allows for the conditional expression of short hairpin RNAs that are processed in vivo to generate miRNAs or siRNAs. Using modified nuclear receptors in a two hybrid format and a synthetic ligand, the Rheoswitch system allows rapid and reversible induction of mRNA expression. We evaluated the system's properties using miR-122 as a model miRNA. A short hairpin encoding miR-122 cloned into the expression vector was correctly processed to yield mature miRNA upon induction with ligand and the amount of miRNA produced was commensurate with the concentration of ligand. miR-122 produced in this way was capable of silencing both endogenous target genes and appropriately designed reporter genes. Stable cell lines were obtained, resulting in heritable, consistent and reversible expression of miR-122, a significant advantage over transient transfection. Based on these results, obtained with a microRNA we adapted the method to produce a desired siRNA by designing short hairpins that can be accurately and efficiently processed. Conclusion We established an Inducible expression system with a miR-122 backbone that can be used for functional studies of miRNAs and their targets, in heterologous cells that do not normally express the miRNA. Additionally we demonstrate the feasibility of using the miR-122 backbone to express shRNA with a desired

  14. Vertical transmission and clinical signs in broiler breeders and broilers experiencing adenoviral gizzard erosion.

    PubMed

    Grafl, Beatrice; Aigner, Franz; Liebhart, Dieter; Marek, Ana; Prokofieva, Irina; Bachmeier, Josef; Hess, Michael

    2012-12-01

    The present report documents an outbreak of adenoviral gizzard erosion in 22 broiler flocks in Germany. The clinical picture was characterized by uneven growth of affected broilers that resulted in considerably lower than average weight at slaughtering. Fowl adenovirus serotype 1 (FAdV-1) was isolated from gizzard lesions and histological examinations demonstrated FAdV-1-positive intranuclear inclusion bodies in gizzard epithelial cells of affected broilers by in-situ hybridization. Birds from all affected flocks originated from one broiler breeder farm. During production of affected birds, broiler breeders were between 27 and 32 weeks old. Enzyme-linked immunosorbent assay and specific virus neutralization assay of sera from parent birds demonstrated an acute FAdV-1 infection within the first 5 weeks of the production cycle. Clinically, broiler breeders exhibited a moderate fall in the hatchability of their chicks, while egg production remained normal. No further clinical signs could be observed. Genetically identical FAdV-1 strains were isolated from gizzards of embryos at the lowest point of hatchability and from affected broiler flocks raised on independent farms. For the first time, direct detection of viable FAdV-1 from gizzards of embryos and progenies of one FAdV-1-seropositive broiler breeder farm in the course of an outbreak of adenoviral gizzard erosion could be demonstrated, highlighting the importance of vertical transmission of this disease. Additionally, growth retardation and subsequent reduced average weight at the time of slaughter of broiler chickens underline the economic impact of adenoviral gizzard erosion for poultry production. PMID:23237373

  15. Sequence dependent interaction of hnRNP proteins with late adenoviral transcripts.

    PubMed Central

    van Eekelen, C; Ohlsson, R; Philipson, L; Mariman, E; van Beek, R; van Venrooij, W

    1982-01-01

    Irradiation with ultraviolet light was used to induce covalent linkage between hnRNA and its associated proteins in intact HeLa cells, late after infection with adenovirus type 2. Covalently linked hnRNA-protein complexes, containing polyadenylated adenoviral RNA, were isolated and their protein moiety characterized. Host 42,000 Mr hnRNP proteins proved to be the major proteins crosslinked to viral hnRNA. To investigate their possible involvement in RNA processing, the localization of these cross-linked polypeptides on adenoviral late transcripts was determined. Sequences of RNA around the attachment sites of the protein were isolated. After in vitro labeling they were hybridized to Southern blots of adeno DNA fragments. The hybridization patterns revealed that the 42,000 Mr polypeptides can be linked to adenoviral transcripts over the entire length of the RNA, corresponding to 16.2-91.5 m.u. of the viral genome. Fine mapping within the Hind III B region (16.8-31.5 m.u.) established, however, that the localization of the cross-linked polypeptides was not random in all parts of the transcript. Sequences around the third leader and the 3' part of the i-leader were overrepresented, whereas the regions encoding VA I and VA II RNA and the late region 1 mRNA bodies were underrepresented in the cross-linked RNA. Using genomic DNA fragments and a cDNA clone containing the tripartite leader it appeared that leader and intervening sequences were represented about equally in cross-linked RNA fragments. Although these results do not support the notion that introns or exons are specifically interacting with one RNP protein, they demonstrate that the 42,000 hnRNP proteins are non randomly positioned on the RNA sequence. Images PMID:6296766

  16. Increased Mucosal CD4+ T Cell Activation in Rhesus Macaques following Vaccination with an Adenoviral Vector

    PubMed Central

    Bukh, Irene; Calcedo, Roberto; Roy, Soumitra; Carnathan, Diane G.; Grant, Rebecca; Qin, Qiuyue; Boyd, Surina; Ratcliffe, Sarah J.; Veeder, Christin L.; Bellamy, Scarlett L.; Betts, Michael R.

    2014-01-01

    ABSTRACT The possibility that vaccination with adenovirus (AdV) vectors increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of HIV acquisition within the Step trial. Modeling this within rhesus macaques is complicated because human adenoviruses, including human adenovirus type 5 (HAdV-5), are not endogenous to macaques. Here, we tested whether vaccination with a rhesus macaque-derived adenoviral vector (simian adenovirus 7 [SAdV-7]) enhances mucosal T cell activation within rhesus macaques. Following intramuscular SAdV-7 vaccination, we observed a pronounced increase in SAdV-7-specific CD4+ T cell responses in peripheral blood and, more dramatically, in rectal mucosa tissue. Vaccination also induced a significant increase in the frequency of activated memory CD4+ T cells in SAdV-7- and HAdV-5-vaccinated animals in the rectal mucosa but not in peripheral blood. These fluctuations within the rectal mucosa were also associated with a pronounced decrease in the relative frequency of naive resting CD4+ T cells. Together, these results indicate that peripheral vaccination with an AdV vector can increase the activation of mucosal CD4+ T cells, potentially providing an experimental model to further evaluate the role of host-vector interactions in increased HIV acquisition after AdV vector vaccination. IMPORTANCE The possibility that vaccination with a human adenovirus 5 vector increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of human immunodeficiency virus (HIV) acquisition within the Step trial. In this study, we tested whether vaccination with a rhesus macaque-derived adenoviral vector in rhesus macaques enhances mucosal CD4+ T cell activation, the main cell target of simian immunodeficiency virus (SIV)/HIV. The results showed that vaccination with an adenoviral vector indeed increases activation of mucosal CD4+ T cells and potentially increases susceptibility to SIV

  17. Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

    PubMed Central

    Fishbein, Ilia; Forbes, Scott P.; Adamo, Richard F.; Chorny, Michael; Levy, Robert J.; Alferiev, Ivan S.

    2014-01-01

    In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of

  18. [Clinical syndrome of convulsive cough of adenoviral etiology in a children's collective].

    PubMed

    Grobnicu, M; Andreescu, V; Caffé, I; Măgureanu, E; Marion, M; Ivan, I; Botez, D; Cuteanu, I; Barbu, I

    1976-01-01

    Bacteriological, viral and serological investigators were carried out in a community with 100 prescholar children (Kindergarden), 34 of whom presented a clinical syndrome of whooping cough, in order to establish the bacteriologic or viral etiology of the syndrome. The etiologic role of organisms of the Bordetella, B. pertussis and B. parapertussis was invalidated by the bacteriologic and serological tests. Viral and serological tests, performed to demonstrate the participation of viral agents in the causation of this clinical syndrome, established an adenoviral diagnosis in 13 (43.3%) of the 30 children. Adenovirus type 6 was isolated and there was a significant increase in the titers of antibodies to adenoviruses. PMID:184515

  19. Establishment of a transient expression system for Dictyostelium discoideum.

    PubMed Central

    Howard, P K; Ahern, K G; Firtel, R A

    1988-01-01

    We have established a rapid and sensitive transient expression system for Dictyostelium discoideum. We constructed a gene fusion containing the promoter from the Dictyostelium Actin 15 gene fused to the firefly luciferase gene. The enzymatic activity of this gene fusion, expressed at very high levels in stable transformants, was measured to determine optimum conditions for transient expression using electroporation to introduce the DNA into cells. With these conditions, we show that a luciferase gene fusion driven by a prestalk, cell-type specific promoter from the pst-cathepsin gene expresses luciferase at the appropriate developmental stage. In addition, we present results suggesting that the system will be useful for expressing genes in non-axenic cell lines. Finally, we observe that electroporation is more efficient for obtaining stable transformations than the standard calcium phosphate procedure using extrachromosomally replicating shuttle vectors but less efficient for vectors that integrate into the Dictyostelium chromosomes. PMID:3362676

  20. Regulation of cadherin expression in nervous system development

    PubMed Central

    Paulson, Alicia F; Prasad, Maneeshi S; Thuringer, Amanda Henke; Manzerra, Pasquale

    2014-01-01

    This review addresses our current understanding of the regulatory mechanisms for classical cadherin expression during development of the vertebrate nervous system. The complexity of the spatial and temporal expression patterns is linked to morphogenic and functional roles in the developing nervous system. While the regulatory networks controlling cadherin expression are not well understood, it is likely that the multiple signaling pathways active in the development of particular domains also regulate the specific cadherins expressed at that time and location. With the growing understanding of the broader roles of cadherins in cell–cell adhesion and non-adhesion processes, it is important to understand both the upstream regulation of cadherin expression and the downstream effects of specific cadherins within their cellular context. PMID:24526207

  1. Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

    PubMed

    Iiyama, Kazuhiro; Lee, Jae Man; Tatsuke, Tuneyuki; Mon, Hiroaki; Kusakabe, Takahiro

    2016-06-01

    Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway. PMID:27059494

  2. Plasmid-free T7-based Escherichia coli expression systems.

    PubMed

    Striedner, Gerald; Pfaffenzeller, Irene; Markus, Luchner; Nemecek, Sabine; Grabherr, Reingard; Bayer, Karl

    2010-03-01

    In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing. PMID:19891007

  3. Construction and development of a novel expression system of Streptomyces.

    PubMed

    Guan, Chengran; Cui, Wenjing; He, Xiaotian; Hu, Xu; Xu, Jun; Du, Guocheng; Chen, Jian; Zhou, Zhemin

    2015-09-01

    Streptomyces is well known to be an attractive host for producing large amounts of proteins with potent biological activities into the culture supernatant. To expand its expression system, we constructed a novel expression plasmid for gene expression in Streptomyces by inserting the promoter (P(tg)) and the signal peptide (SP(tg)) of transglutaminase (TGase) from Streptomyces hygroscopicus WSH03-13 into vector pIJ86, followed by multiple cloning sites and a transcriptional terminator fd (fd-ter). The secretion capacity of the vector was further enhanced by optimizing the signal peptidase cleavage site and a rare codon of SP(tg), yielding expression vector pSG02. Using this vector, TGase was actively and greatly expressed in the supernatant in several Streptomyces strains. In addition, the heterologous proteins aminopeptidase from Bacillus subtilis Zj016 (BSAP) and phenylalanine ammonia-lyase from Rhodotorula glutinis (PAL) were also expressed in various Streptomyces strains by this vector. This expression system should be useful for the expression of other proteins. PMID:25956536

  4. Stem cells as delivery vehicles for oncolytic adenoviral virotherapy.

    PubMed

    Kranzler, Justin; Tyler, Matthew A; Sonabend, Adam M; Ulasov, Ilya V; Lesniak, Maciej S

    2009-10-01

    Glioblastoma multiforme is the most common primary intracranial tumor in humans. Despite continued advances in cancer therapy, the outcome for patients diagnosed with this disease remains bleak. Novel treatments involving the use of conditionally replicating adenoviruses (CRAds) to target malignant brain tumors have undergone extensive research and proven to be a promising mode of glioblastoma therapy. CRAds are genetically manipulated to replicate within tumor cells, exhibiting a high degree of infectivity, cytotoxicity, and transgene expression. While the use of various CRAds has been deemed safe for intracranial injection in preclinical trials, a significant therapeutic effect has yet to be seen in patients. This shortcoming stems from the distribution limitations involved with local delivery of virolytic agents. To enhance this modality of treatment, stem cells have been explored as cellular vehicles in virotherapy applications, given that they possess an intrinsic tropism for malignant brain tumors. Stem cell loaded CRAd delivery offers a more specific and effective method of targeting disseminated tumor cells and forms the basis for this review. PMID:19860653

  5. Stem Cells as Delivery Vehicles for Oncolytic Adenoviral Virotherapy

    PubMed Central

    Kranzler, Justin; Tyler, Matthew A.; Sonabend, Adam M.; Ulasov, Ilya V.; Lesniak, Maciej S.

    2009-01-01

    Glioblastoma multiforme is the most common primary intracranial tumor in humans. Despite continued advances in cancer therapy, the outcome for patients diagnosed with this disease remains bleak. Novel treatments involving the use of conditionally replicating adenoviruses (CRAds) to target malignant brain tumors have undergone extensive research and proven to be a promising mode of glioblastoma therapy. CRAds are genetically manipulated to replicate within tumor cells, exhibiting a high degree of infectivity, cytotoxicity, and transgene expression. While the use of various CRAds has been deemed safe for intracranial injection in preclinical trials, a significant therapeutic effect has yet to be seen in patients. This shortcoming stems from the distribution limitations involved with local delivery of virolytic agents. To enhance this modality of treatment, stem cells have been explored as cellular vehicles in virotherapy applications, given that they possess an intrinsic tropism for malignant brain tumors. Stem cell loaded CRAd delivery offers a more specific and effective method of targeting disseminated tumor cells and forms the basis for this review. PMID:19860653

  6. Linearized oncolytic adenoviral plasmid DNA delivered by bioreducible polymers

    PubMed Central

    Kim, Jaesung; Kim, Pyung-Hwan; Nam, Hye Yeong; Lee, Jung-Sun; Yun, Chae-Ok; Kim, Sung Wan

    2011-01-01

    As an effort to overcome limits of adenovirus (Ad) as a systemic delivery vector for cancer therapy, we developed a novel system using oncolytic Ad plasmid DNA with two bioreducible polymers: arginine-grafted bioreducible poly(disulfide amine)polymer (ABP) and PEG5k-conjugated ABP (ABP5k) in expectation of oncolytic effect caused by progeny viral production followed by replication. The linearized Ad DNAs for active viral replication polyplexed with each polymer were able to replicate only in humancancer cells and produce progeny viruses. The non-immunogenic polymers delivering the DNAs markedly elicited to evade the innate and adaptive immune response. The biodistribution ratio of the polyplexes administered systemically was approximately 99% decreased in liver when compared with naked Ad. Moreover, tumor-to-liver ratio of the Ad DNA delivered by ABP or ABP5k was significantly elevated at 229- or 419-fold greater than that of naked Ad, respectively. The ABP5k improved the chance of the DNA to localize within tumor versus liver with 1.8-fold increased ratio. In conclusion, the innovative and simple system for delivering oncolytic Ad plasmid DNA with the bioreducible polymers, skipping time-consuming steps such as generation and characterization of oncolytic Ad vectors, can be utilized as an alternative approach for cancer therapy. PMID:22207073

  7. Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

    PubMed

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  8. Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

    PubMed Central

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  9. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins. PMID:25851716

  10. Comparison of the effect of adenoviral delivery of three superoxide dismutase genes against hepatic ischemia-reperfusion injury.

    PubMed

    Wheeler, M D; Katuna, M; Smutney, O M; Froh, M; Dikalova, A; Mason, R P; Samulski, R J; Thurman, R G

    2001-12-10

    The purpose of this study was to investigate the effectiveness of superoxide dismutase (SOD) overexpression in an acute model of hepatic oxidative stress. Oxidative stress was established using a warm ischemia-reperfusion model, where nearly 70% of the liver was made hypoxic by clamping the hepatic artery and a branch of the portal vein for 1 hr followed by restoration of blood flow. Animals were infected i.v. with 1 x 10(9) plaque-forming units (PFU) of adenovirus containing the transgene for cytosolic Cu/Zn-SOD (Ad.SOD1), mitochondrial Mn-SOD (Ad.SOD2), extracellular Cu/Zn-SOD (Ad.SOD3), or the bacterial reporter gene for beta-galactosidase (Ad.lacZ) 3 days prior to experiments. Ad.SOD1 and Ad.SOD2 caused a three-fold increase in SOD expression and activity in liver compared to Ad.lacZ-treated control animals. Intravenous administration of Ad.SOD3 increased SOD activity slightly in serum but not in liver. Increases in serum transaminases and pathology due to ischemia-reperfusion were blunted by Ad.SOD1 and Ad.SOD2; however, extracellular SOD had no significant effect. Moreover, lipid-derived free radical adducts (a(N) = 15.65 G and a(H)(beta) = 2.78 G) were increased by ischemia-reperfusion. This effect was blunted by about 60% in Ad.SOD1- and Ad.SOD2-infected animals, but was unaffected by Ad.SOD3. However, when high doses of Ad.SOD3 (3 x 10(10) PFU) were administered. serum SOD activity was elevated three-fold and was protective against hepatic ischemia-reperfusion injury under these conditions. These data demonstrate that adenoviral delivery of superoxide dismutase can effectively reduce hepatic oxidative stress. PMID:11779401

  11. Adenoviral infection or deferoxamine? Two approaches to overexpress VEGF in beta-cell lines.

    PubMed

    Langlois, Allan; Bietiger, William; Sencier, Marie-Christine; Maillard, Elisa; Pinget, Michel; Kessler, Laurence; Sigrist, Severine

    2009-07-01

    Rapid and adequate revascularization of transplanted islets is important for their survival and function during transplantation. Vascular endothelial growth factor (VEGF) could play a critical role with respect to islet revascularization. The aim of this study was to compare two strategies that are used to overexpress VEGF in beta-cells: (1) gene therapy through adenoviral infection and (2) a pharmacological approach using deferoxamine (DFO). beta-Cell lines from rat insulinoma (RINm5F) were either infected using an adenovirus encoding the gene of human VEGF 165 or incubated with DFO. One day after treatment, the viability of RINm5F cells was preserved with 10 micromol/L of DFO (103.95 +/- 5.66% toward control; n = 4). In addition, adenoviral infection maintained the viability of cells for all the concentrations used. In both treatments, overexpression of VEGF was in a comparable level. Finally, the ratio of Bax/Bcl-2 indicated that the apoptosis increased in infected beta-cells whereas treatment with DFO seems to be antiapoptotic. Our results suggest that the use of DFO could be a realistic approach to improve the vascularization of islets during transplantation. PMID:19527112

  12. Thymidine kinase-mediated shut down of bone morphogenetic protein-4 expression allows regulated bone production.

    PubMed

    Lombardo, Barbara; Rocco, Teresa; Esposito, Maria T; Cantilena, Bruno; Gargiulo, Sara; Greco, Adelaide; Montanaro, Donatella; Brunetti, Arturo; Pastore, Lucio

    2013-06-01

    Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor. PMID:23317056

  13. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

  14. Pannexin-1 expression in developing mouse nervous system: new evidence for expression in sensory ganglia.

    PubMed

    Raslan, Abdulrahman; Hainz, Nadine; Beckmann, Anja; Tschernig, Thomas; Meier, Carola

    2016-04-01

    Pannexin1 (Panx1) is one of three members of the pannexin protein family. The expression of Panx1 mRNA has been extensively investigated from late embryonic to adult stages. In contrast, expression during early embryonic development is largely unknown. Our aim is to examine the temporal and spatial expression of Panx1 in mouse embryonic development by focusing on embryonic days (E) 9.5 to 12.5. Whole embryos are investigated in order to provide a comprehensive survey. Analyses were performed at the mRNA level by using reverse transcription plus the polymerase chain reaction and whole-mount in situ hybridization. Panx1 mRNA was detected in the heads and bodies of embryos at all developmental stages investigated (E9.5, E10.5, E11.5, E12.5). In particular, the nervous system expressed Panx1 at an early time point. Interestingly, Panx1 expression was found in afferent ganglia of the cranial nerves and spinal cord. This finding is of particular interest in the context of neuropathic pain and other Panx1-related neurological disorders. Our study shows, for the first time, that Panx1 is expressed in the central and peripheral nervous system during early developmental stages. The consequences of Panx1 deficiency or inhibition in a number of experimental paradigms might therefore be predicated on changes during early development. PMID:26453396

  15. A time- and dose-dependent STAT1 expression system

    PubMed Central

    Leitner, Nicole R; Strobl, Birgit; Bokor, Marion; Painz, Ronald; Kolbe, Thomas; Rülicke, Thomas; Müller, Mathias; Karaghiosoff, Marina

    2006-01-01

    Background The signal transducer and activator of transcription (STAT) family of transcription factors mediates a variety of cytokine dependent gene regulations. STAT1 has been mainly characterized by its role in interferon (IFN) type I and II signaling and STAT1 deficiency leads to high susceptibility to several pathogens. For fine-tuned analysis of STAT1 function we established a dimerizer-inducible system for STAT1 expression in vitro and in vivo. Results The functionality of the dimerizer-induced STAT1 system is demonstrated in vitro in mouse embryonic fibroblasts and embryonic stem cells. We show that this two-vector based system is highly inducible and does not show any STAT1 expression in the absence of the inducer. Reconstitution of STAT1 deficient cells with inducible STAT1 restores IFNγ-mediated gene induction, antiviral responses and STAT1 activation remains dependent on cytokine stimulation. STAT1 expression is induced rapidly upon addition of dimerizer and expression levels can be regulated in a dose-dependent manner. Furthermore we show that in transgenic mice STAT1 can be induced upon stimulation with the dimerizer, although only at low levels. Conclusion These results prove that the dimerizer-induced system is a powerful tool for STAT1 analysis in vitro and provide evidence that the system is suitable for the use in transgenic mice. To our knowledge this is the first report for inducible STAT1 expression in a time- and dose-dependent manner. PMID:17184522

  16. Transient expression systems for plant-derived biopharmaceuticals.

    PubMed

    Komarova, Tatiana V; Baschieri, Selene; Donini, Marcello; Marusic, Carla; Benvenuto, Eugenio; Dorokhov, Yuri L

    2010-08-01

    In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems. PMID:20673010

  17. An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications

    PubMed Central

    Sochor, Matthew A.; Vasireddy, Vidyullatha; Drivas, Theodore G.; Wojno, Adam; Doung, Thu; Shpylchak, Ivan; Bennicelli, Jeannette; Chung, Daniel; Bennett, Jean; Lewis, Mitchell

    2015-01-01

    The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl β-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy. PMID:26597678

  18. Regulation of gene expression in the nervous system

    SciTech Connect

    Giuffrida Stella, A.M.; Perez-Polo, J.R.; deVellis, J.

    1990-01-01

    This book offers an up-to-date account of the latest research findings concerned with the regulatory mechanisms of gene expression in neuronal and glial cells under different conditions. The book explores the cellular and neurobiological aspects of important phenomena of the nervous system and its role in health, disease and injury. Contributions form prominent scientists in the field address a variety of specific topics concerned with gene expression in the nervous system - from growth, hormonal and trophic factors to neural tissue reactions in injury or aging.

  19. Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels

    PubMed Central

    Dronadula, Nagadhara; Du, Liang; Flynn, Rowan; Buckler, Joshua; Kho, Jordan; Jiang, Zhilong; Tanaka, Shinji; Dichek, David A.

    2010-01-01

    The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity, and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin-10) in large vessel endothelial cells (EC) at levels as high as obtained with the CMV immediate-early promoter, while retaining EC-specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC-specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely 4 copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining—and possibly enhancing—EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large vessel EC, while minimizing systemic effects. PMID:21179172

  20. Interactive analysis of systems biology molecular expression data

    PubMed Central

    Zhang, Mingwu; Ouyang, Qi; Stephenson, Alan; Kane, Michael D; Salt, David E; Prabhakar, Sunil; Burgner, John; Buck, Charles; Zhang, Xiang

    2008-01-01

    Background Systems biology aims to understand biological systems on a comprehensive scale, such that the components that make up the whole are connected to one another and work through dependent interactions. Molecular correlations and comparative studies of molecular expression are crucial to establishing interdependent connections in systems biology. The existing software packages provide limited data mining capability. The user must first generate visualization data with a preferred data mining algorithm and then upload the resulting data into the visualization package for graphic visualization of molecular relations. Results Presented is a novel interactive visual data mining application, SysNet that provides an interactive environment for the analysis of high data volume molecular expression information of most any type from biological systems. It integrates interactive graphic visualization and statistical data mining into a single package. SysNet interactively presents intermolecular correlation information with circular and heatmap layouts. It is also applicable to comparative analysis of molecular expression data, such as time course data. Conclusion The SysNet program has been utilized to analyze elemental profile changes in response to an increasing concentration of iron (Fe) in growth media (an ionomics dataset). This study case demonstrates that the SysNet software is an effective platform for interactive analysis of molecular expression information in systems biology. PMID:18312669

  1. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins. PMID:20457256

  2. Induction of Specific Humoral and Cellular Immune Responses in a Mouse Model following Gene Fusion of HSP70C and Hantaan Virus Gn and S0.7 in an Adenoviral Vector

    PubMed Central

    Li, Kai; Wang, Fang; Zhang, Liang; Ye, Wei; Li, Puyuan; Zhang, Fanglin; Xu, Zhikai

    2014-01-01

    Heat shock proteins (HSPs) display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV) glycoprotein (GP) and nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359–610 aa, HSP70C) to the Gn and 0.7 kb fragment of the NP (aa1–274-S0.7). C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7) and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV. PMID:24505421

  3. Power system comparison for the Pluto Express mission

    SciTech Connect

    Harty, R.B.

    1995-12-31

    This paper presents a comparison of three advanced radioisotope power systems, along with a down sized RTG for the Pluto Express mission. These three advanced radioisotope power systems were the Radioisotope Alkali Metal Thermal--to-Electric Converter (RAMTEC), Radioisotope Stirling, and Radioisotope Thermophotovoltaic (RTPV). For the Pluto Express mission, the power requirement at the end of the 10-y mission is 74 We. It was found that all three advanced power systems could meet the required end of mission power with two General Purpose Heat Source (GPHS) modules. The RTG required six modules to meet the power requirement. Only the RAMTEC and RTPV met the mass goal of 9.5 kg. The AMTEC has a radiator area more than a factor of 10 lower than the Stirling and RTPV power systems, which simplifies spacecraft integration.

  4. Graphics Expression System Reading Center, Evaluation Period 1975-76.

    ERIC Educational Resources Information Center

    Hopkins, Thomas F.

    This paper describes the Graphics Expression System Reading Center Program, which provided students with limited learning motivation and with below norm achievement in reading, as well as those students reading on and above grade level with the opportunity to use the compact recording studio equipment in the production of television shows. The…

  5. VITELLOGENIN EXPRESSION IN SHEEPSHEAD MINNOWS FROM THE PENSACOLA BAY SYSTEM

    EPA Science Inventory

    Hemmer, M.J., B.L. Hemmer, S.D. Friedman and P.S. Harris. In press. Vitellogenin Expression in Populations of Sheepshead Minnows from the Pensacola Bay System (Abstract). To be presented at the SETAC Fourth World Congress, 14-18 November 2004, Portland, OR. 1 p. (ERL,GB R1015). <...

  6. STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

    PubMed

    Yang, Xuechao; van der Kraan, Peter M; van den Dolder, Juliette; Walboomers, X Frank; Bian, Zhuan; Fan, Mingwen; Jansen, John A

    2007-11-01

    Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium. PMID:17824831

  7. Osteogenic gene regulation and relative acceleration of healing by adenoviral-mediated transfer of human BMP-2 or -6 in equine osteotomy and ostectomy models.

    PubMed

    Ishihara, Akikazu; Shields, Kathleen M; Litsky, Alan S; Mattoon, John S; Weisbrode, Steven E; Bartlett, Jeffrey S; Bertone, Alicia L

    2008-06-01

    This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)-2, Ad-BMP-6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad-BMP-2 or Ad-BMP-6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP-2 than Ad-BMP-6, and both Ad-BMP-2- and Ad-BMP-6-treated osteotomies were greater than Ad-LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta-1, cathepsin H, and gelsolin-like capping protein were greater in Ad-BMP-2- and Ad-BMP-6-treated callus compared to Ad-LacZ-treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad-BMP-2 over Ad-BMP-6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites. PMID:18241059

  8. Differential Type I Interferon-dependent Transgene Silencing of Helper-dependent Adenoviral vs. Adeno-associated Viral Vectors In Vivo

    PubMed Central

    Suzuki, Masataka; Bertin, Terry K; Rogers, Geoffrey L; Cela, Racel G; Zolotukhin, Irene; Palmer, Donna J; Ng, Philip; Herzog, Roland W; Lee, Brendan

    2013-01-01

    We previously dissected the components of the innate immune response to Helper-dependent adenoviral vectors (HDAds) using genetic models, and demonstrated that multiple pattern recognition receptor signaling pathways contribute to this host response to HDAds in vivo. Based on analysis of cytokine expression profiles, type I interferon (IFN) mRNA is induced in host mouse livers at 1 hour post-injection. This type I IFN signaling amplifies cytokine expression in liver independent of the nature of vector DNA sequences after 3 hours post-injection. This type I IFN signaling in response to HDAds administration contributes to transcriptional silencing of both HDAd prokaryotic and eukaryotic DNA in liver. This silencing occurs early and is mediated by epigenetic modification as shown by in vivo chromatin immunoprecipitation (ChIP) with anti-histone deacetylase (HDAC) and promyelocytic leukemia protein (PML). In contrast, self-complementary adeno-associated viral vectors (scAAVs) showed significantly lower induction of type I IFN mRNA in liver compared to HDAds at both early and late time points. These results show that the type I IFN signaling dependent transgene silencing differs between AAV and HDAd vectors after liver-directed gene transfer. PMID:23319058

  9. Impact of Residual Inducer on Titratable Expression Systems

    PubMed Central

    Afroz, Taliman; Luo, Michelle L.; Beisel, Chase L.

    2015-01-01

    Inducible expression systems are widely employed for the titratable control of gene expression, yet molecules inadvertently present in the growth medium or synthesized by the host cells can alter the response profile of some of these systems. Here, we explored the quantitative impact of these residual inducers on the apparent response properties of inducible systems. Using a simple mathematical model, we found that the presence of residual inducer shrinks the apparent dynamic range and causes the apparent Hill coefficient to converge to one. We also found that activating systems were more sensitive than repressing systems to the presence of residual inducer and the response parameters were most heavily dependent on the original Hill coefficient. Experimental interrogation of common titratable systems based on an L-arabinose inducible promoter or a thiamine pyrophosphate-repressing riboswitch in Escherichia coli confirmed the predicted trends. We finally found that residual inducer had a distinct effect on “all-or-none” systems, which exhibited increased sensitivity to the added inducer until becoming fully induced. Our findings indicate that residual inducer or repressor alters the quantitative response properties of titratable systems, impacting their utility for scientific discovery and pathway engineering. PMID:26348036

  10. Impact of Residual Inducer on Titratable Expression Systems.

    PubMed

    Afroz, Taliman; Luo, Michelle L; Beisel, Chase L

    2015-01-01

    Inducible expression systems are widely employed for the titratable control of gene expression, yet molecules inadvertently present in the growth medium or synthesized by the host cells can alter the response profile of some of these systems. Here, we explored the quantitative impact of these residual inducers on the apparent response properties of inducible systems. Using a simple mathematical model, we found that the presence of residual inducer shrinks the apparent dynamic range and causes the apparent Hill coefficient to converge to one. We also found that activating systems were more sensitive than repressing systems to the presence of residual inducer and the response parameters were most heavily dependent on the original Hill coefficient. Experimental interrogation of common titratable systems based on an L-arabinose inducible promoter or a thiamine pyrophosphate-repressing riboswitch in Escherichia coli confirmed the predicted trends. We finally found that residual inducer had a distinct effect on "all-or-none" systems, which exhibited increased sensitivity to the added inducer until becoming fully induced. Our findings indicate that residual inducer or repressor alters the quantitative response properties of titratable systems, impacting their utility for scientific discovery and pathway engineering. PMID:26348036

  11. Development of a System for Automatic Facial Expression Analysis

    NASA Astrophysics Data System (ADS)

    Diago, Luis A.; Kitaoka, Tetsuko; Hagiwara, Ichiro

    Automatic recognition of facial expressions can be an important component of natural human-machine interactions. While a lot of samples are desirable for estimating more accurately the feelings of a person (e.g. likeness) about a machine interface, in real world situation, only a small number of samples must be obtained because the high cost in collecting emotions from observed person. This paper proposes a system that solves this problem conforming to individual differences. A new method is developed for facial expression classification based on the combination of Holographic Neural Networks (HNN) and Type-2 Fuzzy Logic. For the recognition of emotions induced by facial expressions, compared with former HNN and Support Vector Machines (SVM) classifiers, proposed method achieved the best generalization performance using less learning time than SVM classifiers.

  12. Integrative food grade expression system for lactic acid bacteria.

    PubMed

    Douglas, Grace L; Goh, Yong Jun; Klaenhammer, Todd R

    2011-01-01

    Lactobacillus acidophilus NCFM is a probiotic microbe with the ability to survive passage to the -gastrointestinal tract, interact intimately with the host and induce immune responses. The genome of NCFM has been determined and the bacterium is genetically accessible. Therefore, L. acidophilus has excellent potential for use as a vaccine delivery vehicle to express antigens at mucosal surfaces. Plasmids, commonly used to carry antigen encoding genes, are inherently unstable and require constant selection by antibiotics, which can be problematic for in vivo studies and clinical trials. Chromosomal expression of gene cassettes encoding antigens offers enhanced genetic stability by eliminating requirements for marker selection. This work illustrates the integration and inducible expression of the reporter gene gusA3, -encoding a β-glucuronidase (GusA3), in the L. acidophilus chromosome. A previously described upp-counterselectable gene replacement system was used to direct insertion of the gusA3 gene into an intergenic chromosomal location downstream of lacZ (LBA1462), encoding a β-galactosidase. The transcriptional activity of integrated gusA3 was evaluated by GUS activity assays using 4-methyl-umbelliferyl-β-D: -glucuronide (MUG) and was determined to be one to two orders of magnitude higher than the GusA3-negative parent, NCK1909. The successful chromosomal integration and expression of GusA3 demonstrate the potential of this method for higher levels of inducible gene expression in L. acidophilus. PMID:21815104

  13. A baculoviral display system to assay viral entry.

    PubMed

    Iida, Manami; Yoshida, Takeshi; Watari, Akihiro; Yagi, Kiyohito; Hamakubo, Takao; Kondoh, Masuo

    2013-01-01

    In this study, we evaluated a baculoviral display system for analysis of viral entry by using a recombinant adenovirus (Ad) carrying a luciferase gene and budded baculovirus (BV) that displays the adenoviral receptor, coxsackievirus and adenovirus receptor (CAR). CAR-expressing B16 cells (B16-CAR cells) were infected with luciferase-expressing Ad vector in the presence of BV that expressed or lacked CAR (CAR-BV and mock-BV, respectively). Treatment with mock-BV even at doses as high as 5 µg/mL failed to attenuate the luciferase activity of B16-CAR cells. In contrast, treatment with CAR-BV with doses as low as 0.5 µg/mL significantly decreased the luciferase activity of infected cells, which reached 65% reduction at 5 µg/mL. These findings suggest that a receptor-displaying BV system could be used to evaluate viral infection. PMID:24189431

  14. Cloning and Expression of Recombinant Human Endostatin in Periplasm of Escherichia coli Expression System

    PubMed Central

    Mohajeri, Abbas; Pilehvar-Soltanahmadi, Yones; Pourhassan-Moghaddam, Mohammad; Abdolalizadeh, Jalal; Karimi, Pouran; Zarghami, Nosratollah

    2016-01-01

    Purpose: Recombinant human endostatin (rhEs) is an angiogenesis inhibitor which is used as a specific drug in the treatment of non-small-cell lung cancer. In the current research, we developed an efficient method for expressing soluble form of the rhEs protein in the periplasmic space of Escherichia coli via fusing with pelB signal peptide. Methods: The human endostatin (hEs) gene was amplified using synthetic (hEs) gene as a template; then, cloned and expressed under T7 lac promoter. IPTG was used as an inducer for rhEs expression. Next, the osmotic shock was used to extraction of protein from the periplasmic space. The presence of rhEs in the periplasmic space was approved by SDS-PAGE and Western blotting. Results: The results show the applicability of pelB fusion protein system usage for secreting rhEs in the periplasm of E. coli in the laboratory scale. The rhEs represents approximately 35 % (0.83mg/l) of the total cell protein. Conclusion: The present study apparently is the first report of codon-optimized rhEs expression as a fusion with pelB signal peptide. The results presented the successful secretion of soluble rhEs to the periplasmic space. PMID:27478780

  15. Dynamic Visualization of Co-expression in Systems Genetics Data

    SciTech Connect

    New, Joshua Ryan; Huang, Jian; Chesler, Elissa J

    2008-01-01

    Biologists hope to address grand scientific challenges by exploring the abundance of data made available through modern microarray technology and other high-throughput techniques. The impact of this data, however, is limited unless researchers can effectively assimilate such complex information and integrate it into their daily research; interactive visualization tools are called for to support the effort. Specifically, typical studies of gene co-expression require novel visualization tools that enable the dynamic formulation and fine-tuning of hypotheses to aid the process of evaluating sensitivity of key parameters. These tools should allow biologists to develop an intuitive understanding of the structure of biological networks and discover genes which reside in critical positions in networks and pathways. By using a graph as a universal data representation of correlation in gene expression data, our novel visualization tool employs several techniques that when used in an integrated manner provide innovative analytical capabilities. Our tool for interacting with gene co-expression data integrates techniques such as: graph layout, qualitative subgraph extraction through a novel 2D user interface, quantitative subgraph extraction using graph-theoretic algorithms or by querying an optimized b-tree, dynamic level-of-detail graph abstraction, and template-based fuzzy classification using neural networks. We demonstrate our system using a real-world workflow from a large-scale, systems genetics study of mammalian gene co-expression.

  16. A systematic approach for testing expression of human full-length proteins in cell-free expression systems

    PubMed Central

    Langlais, Claudia; Guilleaume, Birgit; Wermke, Nadja; Scheuermann, Tina; Ebert, Lars; LaBaer, Joshua; Korn, Bernhard

    2007-01-01

    Background The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems. Results In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%. Conclusion We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield. PMID:17915018

  17. Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

    PubMed Central

    Ketzer, Patrick; Kaufmann, Johanna K.; Engelhardt, Sarah; Bossow, Sascha; von Kalle, Christof; Hartig, Jörg S.; Ungerechts, Guy; Nettelbeck, Dirk M.

    2014-01-01

    Aptazymes are small, ligand-dependent self-cleaving ribozymes that function independently of transcription factors and can be customized for induction by various small molecules. Here, we introduce these artificial riboswitches for regulation of DNA and RNA viruses. We hypothesize that they represent universally applicable tools for studying viral gene functions and for applications as a safety switch for oncolytic and live vaccine viruses. Our study shows that the insertion of artificial aptazymes into the adenoviral immediate early gene E1A enables small-molecule–triggered, dose-dependent inhibition of gene expression. Aptazyme-mediated shutdown of E1A expression translates into inhibition of adenoviral genome replication, infectious particle production, and cytotoxicity/oncolysis. These results provide proof of concept for the aptazyme approach for effective control of biological outcomes in eukaryotic systems, specifically in virus infections. Importantly, we also demonstrate aptazyme-dependent regulation of measles virus fusion protein expression, translating into potent reduction of progeny infectivity and virus spread. This not only establishes functionality of aptazymes in fully cytoplasmic genetic systems, but also implicates general feasibility of this strategy for application in viruses with either DNA or RNA genomes. Our study implies that gene regulation by artificial riboswitches may be an appealing alternative to Tet- and other protein-dependent gene regulation systems, based on their small size, RNA-intrinsic mode of action, and flexibility of the inducing molecule. Future applications range from gene analysis in basic research to medicine, for example as a safety switch for new generations of efficiency-enhanced oncolytic viruses. PMID:24449891

  18. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    DOE PAGESBeta

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; et al

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle tomore » establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.« less

  19. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    SciTech Connect

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; Kaul, Karen; Brendler, Charles; Iozzo, Renato V.; Seth, Prem

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.

  20. Severe Traumatic Head Injury Affects Systemic Cytokine Expression

    PubMed Central

    LaPar, Damien J; Rosenberger, Laura H; Walters, Dustin M; Hedrick, Traci L; Swenson, Brian R; Young, Jeffrey S; Dossett, Lesly A; May, Addison K; Sawyer, Robert G

    2012-01-01

    Background The neuroimmunologic effect of traumatic head injury remains ill-defined. This study aimed to characterize systemic cytokine profiles among traumatically injured patients to assess the effect of traumatic head injury on the systemic inflammatory response. Study Design Over five years, 1,022 patients were evaluated from a multi-institutional trauma immunomodulatory database (TIMD). Patients were stratified by presence of severe head injury (SHI, Head ISS ≥ 4, n=335) versus non-severe head injury (NHI, Head ISS ≤ 3, n=687). Systemic cytokine expression was quantified by ELISA within 72 hours of admission. Patient factors, outcomes, and cytokine profiles were compared by univariate analyses. Results SHI patients were more severely injured with higher mortality despite similar ICU infection and ventilator associated pneumonia (VAP) rates. Expression of early pro-inflammatory cytokines, IL-6 (p<0.001) and tumor necrosis factor (TNF)-α (p=0.02), were higher among NHI patients, while expression of immunomodulatory cytokines, interferon-γ (p=0.01) and IL-12 (p=0.003), was higher in SHI patients. High TNF-α levels in NHI patients were associated with mortality (p=0.01), increased mechanical ventilation (p=0.02), and development of VAP (p=0.01). Alternatively, among SHI patients, high IL-2 levels were associated with survival, decreased mechanical ventilation, and absence of VAP. Conclusions The presence of severe traumatic head injury significantly alters systemic cytokine expression and exerts an immunomodulatory effect. Early recognition of these profiles may allow for targeted intervention to reduce patient morbidity and mortality. PMID:22342787

  1. Nanobarcode gene expression monitoring system for potential miniaturized space applications

    NASA Astrophysics Data System (ADS)

    Ruan, Weiming; Eastman, P. Scott; Cooke, Patrick A.; Park, Jennifer S.; Chu, Julia S. F.; Gray, Joe W.; Li, Song; Chen, Fanqing Frank

    Manned mission to space has been threatened by various cosmos risks including radiation, mirogravity, vacuum, confinement, etc., which may cause genetic variations of astronauts and eventually lead to damages of their health. Thus, the development of small biomedical devices, which can monitor astronaut gene expression changes, is useful for future long-term space missions. Using magnetic microbeads packed with nanocrystal quantum dots at controlled ratios, we were able to generate highly multiplexed nanobarcodes, which can encode a flexible panel of genes. Also, by using a reporter quantum dot, this nanobarcode platform can monitor and quantify gene expression level with improved speed and sensitivity. As a comparison, we studied TGF-β1 induced transcription changes in human bone marrow mesenchymal stem cells with both the nanobarcode microbead system and the Affymetrix GeneChip ® HTA system, which is currently considered as the industrial standard. Though using only 1/20 of the sample RNA, the nanobarcode system showed sensitivity equivalent to Affymetrix GeneChip ® system. The coefficient of variation, dynamic range, and accuracy of the nanobarcodes measurement is equivalent to that of the GeneChip ® HTA system. Therefore, this newly invented nanobarcode microbead platform is thought to be sensitive, flexible, cost-effective and accurate in a level equivalent to the conventional methods. As an extension of the use of this new platform, spacecrafts may carry this miniaturized system as a diagnostic tool for the astronauts.

  2. Serum adropin level and ENHO gene expression in systemic sclerosis.

    PubMed

    Yolbas, Servet; Kara, Murat; Yilmaz, Musa; Aydin, Suleyman; Koca, Suleyman Serdar

    2016-06-01

    Adropin, a secreted protein, is encoded by the energy homeostasis associated (ENHO) gene. It has been implicated in the several physiological and pathological processes such as angiogenesis and apoptosis. Therefore, the aim of present study was to investigate serum adropin levels and ENHO gene expressions in systemic sclerosis (SSc) characterized by vasculopathy, inflammation, and progressive fibrosis of the skin and internal organs. The study includes 27 patients with SSc, 39 patients with Behçet's disease (BD), and 20 healthy controls (HC). Serum adropin levels and ENHO gene expressions by peripheral blood mononuclear cells were analyzed by ELISA method and by real-time PCR, respectively. The serum adropin levels were higher in the SSc and BD groups than in the HC group (p = 0.023 and p < 0.001, respectively). However, there were no significant differences among the groups in terms of ENHO gene expressions (p ANOVA = 0.149). There was no significant difference between the limited and diffuse cutaneous subtypes of SSc in terms of serum adropin level and ENHO gene expression. Moreover, serum adropin level and ENHO gene expression were not associated with the disease activity and severity indexes. ENHO gene expression was correlated with the triglyceride levels in the BD group (r = -0.426, p = 0.027). The augmented serum adropin levels may be expected in the chronic inflammatory disease and seem not to be characteristic of only SSc. However, further studies are needed to explain the precise role of adropin in SSc. PMID:27079850

  3. Recombinant production of mecasermin in E. coli expression system.

    PubMed

    Jafari, S; Babaeipour, V; Seyedi, H A Eslampanah; Rahaie, M; Mofid, M R; Haddad, L; Namvaran, M M; Fallah, J

    2014-01-01

    Human Insulin-like growth factor 1 (hIGF-1) consists of 70 amino acids in a single chain with three intermolecular disulfide bridges possessing valuable therapeutic effects. To date, numerous variants of specifically engineered hIGF-1 have been produced so as to improve hIGF-1 biological activity, stability and stronger binding to IGF-1 receptor. Mecasermin is one of the modified variants with one amino acid substitution near the N-terminal (T4I) approved for the treatment of growth failure diabetes, wound healing, amyotrophic lateral sclerosis and severe primary IGF-1 deficiency. No scientific report for recombinant production of mecasermin in Escherichia coli (E. coli) expression system has been sofar reported. In the present study, we therefore investigated the overexpression of mecasermin in two different E. coli strains in order to obtain higher yield of recombinant protein. To achieve this goal, mecasermin DNA encoding sequence was designed based on polypeptide sequence, optimized according to E. coli codon preference, and cloned in pET15b. Recombinant vector, pET15-mecasermin, transferred into two E. coli strains rigami B (DE3) and BL21 (DE3) and induced for expression in a small scale. Results revealed the E. coli Origami B (DE3) expression system was a preferable host for mecasermin production due to its high expression level being around twice as much as BL21 (DE3). Large scale mecasermin production was performed in batch culture and produced recombinant protein specifically confirmed by western blotting and mass spectroscopy. Since major part of recombinant mecasermin was expressed as inclusion body, isolation and refolding was accomplished through developed purification procedure, and finally recombinant protein was successfully purified by gel filtration chromatography. PMID:26339260

  4. Recombinant production of mecasermin in E. coli expression system

    PubMed Central

    Jafari, S.; Babaeipour, V.; Seyedi, H.A. Eslampanah; Rahaie, M.; Mofid, M.R.; Haddad, L.; Namvaran, M.M.; Fallah, J.

    2014-01-01

    Human Insulin-like growth factor 1 (hIGF-1) consists of 70 amino acids in a single chain with three intermolecular disulfide bridges possessing valuable therapeutic effects. To date, numerous variants of specifically engineered hIGF-1 have been produced so as to improve hIGF-1 biological activity, stability and stronger binding to IGF-1 receptor. Mecasermin is one of the modified variants with one amino acid substitution near the N-terminal (T4I) approved for the treatment of growth failure diabetes, wound healing, amyotrophic lateral sclerosis and severe primary IGF-1 deficiency. No scientific report for recombinant production of mecasermin in Escherichia coli (E. coli) expression system has been sofar reported. In the present study, we therefore investigated the overexpression of mecasermin in two different E. coli strains in order to obtain higher yield of recombinant protein. To achieve this goal, mecasermin DNA encoding sequence was designed based on polypeptide sequence, optimized according to E. coli codon preference, and cloned in pET15b. Recombinant vector, pET15-mecasermin, transferred into two E. coli strains rigami B (DE3) and BL21 (DE3) and induced for expression in a small scale. Results revealed the E. coli Origami B (DE3) expression system was a preferable host for mecasermin production due to its high expression level being around twice as much as BL21 (DE3). Large scale mecasermin production was performed in batch culture and produced recombinant protein specifically confirmed by western blotting and mass spectroscopy. Since major part of recombinant mecasermin was expressed as inclusion body, isolation and refolding was accomplished through developed purification procedure, and finally recombinant protein was successfully purified by gel filtration chromatography. PMID:26339260

  5. Relating protein adduction to gene expression changes: a systems approach

    PubMed Central

    Zhang, Bing; Shi, Zhiao; Duncan, Dexter T; Prodduturi, Naresh; Marnett, Lawrence J; Liebler, Daniel C

    2013-01-01

    Modification of proteins by reactive electrophiles such as the 4-hydroxy-2-nonenal (HNE) plays a critical role in oxidant-associated human diseases. However, little is known about protein adduction and the mechanism by which protein damage elicits adaptive effects and toxicity. We developed a systems approach for relating protein adduction to gene expression changes through the integration of protein adduction, gene expression, protein-DNA interaction, and protein-protein interaction data. Using a random walk strategy, we expanded a list of responsive transcription factors inferred from gene expression studies to upstream signaling networks, which in turn allowed overlaying protein adduction data on the network for the prediction of stress sensors and their associated regulatory mechanisms. We demonstrated the general applicability of transcription factor-based signaling network inference using 103 known pathways. Applying our workflow on gene expression and protein adduction data from HNE-treatment not only rediscovered known mechanisms of electrophile stress but also generated novel hypotheses regarding protein damage sensors. Although developed for analyzing protein adduction data, the framework can be easily adapted for phosphoproteomics and other types of protein modification data. PMID:21594272

  6. Ion Channel Expression in the Developing Enteric Nervous System

    PubMed Central

    Stamp, Lincon A.; Fegan, Emily; Dent, Stephan; Cooper, Edward C.; Lomax, Alan E.; Anderson, Colin R.; Bornstein, Joel C.; Young, Heather M.; McKeown, Sonja J.

    2015-01-01

    The enteric nervous system arises from neural crest-derived cells (ENCCs) that migrate caudally along the embryonic gut. The expression of ion channels by ENCCs in embryonic mice was investigated using a PCR-based array, RT-PCR and immunohistochemistry. Many ion channels, including chloride, calcium, potassium and sodium channels were already expressed by ENCCs at E11.5. There was an increase in the expression of numerous ion channel genes between E11.5 and E14.5, which coincides with ENCC migration and the first extension of neurites by enteric neurons. Previous studies have shown that a variety of ion channels regulates neurite extension and migration of many cell types. Pharmacological inhibition of a range of chloride or calcium channels had no effect on ENCC migration in cultured explants or neuritogenesis in vitro. The non-selective potassium channel inhibitors, TEA and 4-AP, retarded ENCC migration and neuritogenesis, but only at concentrations that also resulted in cell death. In summary, a large range of ion channels is expressed while ENCCs are colonizing the gut, but we found no evidence that ENCC migration or neuritogenesis requires chloride, calcium or potassium channel activity. Many of the ion channels are likely to be involved in the development of electrical excitability of enteric neurons. PMID:25798587

  7. Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System

    PubMed Central

    Baeshen, Mohammed N.; Bouback, Thamer A. F.; Alzubaidi, Mubarak A.; Alabbas, Omar T. O.; Alshahrani, Sultan M.; Aljohani, Ahmed A. M.; Munshi, Rayan A. A.; Al-Hejin, Ahmed; Redwan, Elrashdy M.; Ramadan, Hassan A. I.; Saini, Kulvinder S.; Baeshen, Nabih A.

    2016-01-01

    Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system. PMID:27579308

  8. Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System.

    PubMed

    Baeshen, Mohammed N; Bouback, Thamer A F; Alzubaidi, Mubarak A; Bora, Roop S; Alotaibi, Mohammed A T; Alabbas, Omar T O; Alshahrani, Sultan M; Aljohani, Ahmed A M; Munshi, Rayan A A; Al-Hejin, Ahmed; Ahmed, Mohamed M M; Redwan, Elrashdy M; Ramadan, Hassan A I; Saini, Kulvinder S; Baeshen, Nabih A

    2016-01-01

    Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system. PMID:27579308

  9. Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2

    PubMed Central

    Neumann, Alexander J.; Gardner, Oliver F. W.; Williams, Rebecca; Alini, Mauro; Archer, Charles W.; Stoddart, Martin J.

    2015-01-01

    Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a

  10. The route of immunization with adenoviral vaccine influences the recruitment of cytotoxic T lymphocytes in the lung that provide potent protection from influenza A virus.

    PubMed

    Suda, Tatsuya; Kawano, Masaaki; Nogi, Yasuhisa; Ohno, Naohito; Akatsuka, Toshitaka; Matsui, Masanori

    2011-09-01

    Virus-specific cytotoxic T lymphocytes (CTLs) in the lung are considered to confer protection from respiratory viruses. Several groups demonstrated that the route of priming was likely to have an implication for the trafficking of antigen-specific CTLs. Therefore, we investigated whether the route of immunization with adenoviral vaccine influenced the recruitment of virus-specific CTLs in the lung that should provide potent protection from influenza A virus. Mice were immunized with recombinant adenovirus expressing the matrix (M1) protein of influenza A virus via various immunization routes involving intraperitoneal, intranasal, intramuscular, or intravenous administration as well as subcutaneous administration in the hind hock. We found that the immunization route dramatically impacted the recruitment of M1-specific IFN-γ(+) CD8(+) T cells both in the lung and the spleen. Surprisingly, hock immunization was most effective for the accumulation in the lung of IFN-γ-producing CD8(+) T cells that possessed M1-specific cytolytic activity. Further, antigen-driven IFN-γ(+) CD8(+) T cells in the lung, but not in the spleen, were likely to be correlated with the resistance to challenge with influenza A virus. These results may improve our ability to design vaccines that target virus-specific CTL responses to respiratory viruses such as influenza A virus. PMID:21722671

  11. Permissive environment in postnatal wounds induced by adenoviral-mediated overexpression of the anti-inflammatory cytokine interleukin-10 prevents scar formation.

    PubMed

    Gordon, Ashley; Kozin, Elliott D; Keswani, Sundeep G; Vaikunth, Sachin S; Katz, Anna B; Zoltick, Philip W; Favata, Michele; Radu, Antoneta P; Soslowsky, Louis J; Herlyn, Meenhard; Crombleholme, Timothy M

    2008-01-01

    Wound healing in the mid-gestation fetus is scarless with minimal inflammation and a unique extracellular matrix. We have previously documented the relative lack of inflammatory cytokines in this environment. We demonstrate that interleukin (IL)-10 is highly expressed in mid-gestation human fetal skin but is absent in postnatal human skin. We hypothesize that overexpression of IL-10 in postnatal skin may replicate a permissive environment for scarless healing. To study the mechanism underlying this process we performed immunohistochemistry for IL-10 in human mid-gestation fetal and postnatal skin. We also determined if adenoviral-mediated overexpression of IL-10 could allow for scarless wound healing in a murine incisional wound model. Wounds were analyzed at 1-90 days postwounding for effects on scar formation, inflammatory response, and biomechanical properties. Ad-IL-10 reconstitutes a permissive environment for scarless healing as shown by reconstitution of a normal dermal reticular collagen pattern and distribution of dermal elements. Compared with controls, Ad-IL-10 treated wounds showed reduced inflammatory response and no difference in biomechanical parameters. Therefore, overexpression of IL-10 in postnatal wounds results in a permissive environment for scarless wound repair, possibly by replicating a fetal wound environment. PMID:18086289

  12. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.

    PubMed

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A F V

    2016-02-18

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  13. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations

    PubMed Central

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M.; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A.F.V.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  14. Silkworm expression system as a platform technology in life science.

    PubMed

    Kato, Tatsuya; Kajikawa, Mizuho; Maenaka, Katsumi; Park, Enoch Y

    2010-01-01

    Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3-6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the "Silkroad," we expect that the "Bioroad" from Asia to Europe will be established by the silkworm expression system. PMID:19830419

  15. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

    PubMed Central

    Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

  16. AMTEC radioisotope power system for the Pluto Express mission

    SciTech Connect

    Ivanenok, J.F. III; Sievers, R.K.

    1995-12-31

    The Alkali Metal Thermal to Electric Converter (AMTEC) technology has made substantial advances in the last 3 years through design improvements and technical innovations. In 1993 programs began to produce an AMTEC cell specifically for the NASA Pluto Express Mission. A set of efficiency goals was established for this series of cells to be developed. According to this plan, cell {number_sign}8 would be 17% efficient but was actually 18% efficient. Achieving this goal, as well as design advances that allow the cell to be compact, has resulted in pushing the cell from an unexciting 2 W/kg and 2% efficiency to very attractive 40 W/kg and 18% measured efficiency. This paper will describe the design and predict the performance of a radioisotope powered AMTEC system for the Pluto Express mission.

  17. FPGA-accelerated algorithm for the regular expression matching system

    NASA Astrophysics Data System (ADS)

    Russek, P.; Wiatr, K.

    2015-01-01

    This article describes an algorithm to support a regular expressions matching system. The goal was to achieve an attractive performance system with low energy consumption. The basic idea of the algorithm comes from a concept of the Bloom filter. It starts from the extraction of static sub-strings for strings of regular expressions. The algorithm is devised to gain from its decomposition into parts which are intended to be executed by custom hardware and the central processing unit (CPU). The pipelined custom processor architecture is proposed and a software algorithm explained accordingly. The software part of the algorithm was coded in C and runs on a processor from the ARM family. The hardware architecture was described in VHDL and implemented in field programmable gate array (FPGA). The performance results and required resources of the above experiments are given. An example of target application for the presented solution is computer and network security systems. The idea was tested on nearly 100,000 body-based viruses from the ClamAV virus database. The solution is intended for the emerging technology of clusters of low-energy computing nodes.

  18. MEASURING AND EXPRESSING THE PERFORMANCE OF ENERGY STORAGE SYSTEMS

    SciTech Connect

    Schoenwald, David; Conover, David R.

    2013-12-03

    Until late 2012, there was no uniform methodology to measure and express the performance of energy storage systems (ESS). A void in this area can affect the acceptance of ESS in the marketplace because different systems cannot be equitably compared and ESS cost-benefit analysis may be challenging due to a lack of verified and relevant ESS performance. The lack of such criteria also furthers the probability that each ESS customer or user will make up their own; necessitating “custom validation” to a unique set of criteria each time an ESS is to be considered or installed. To address this need and foster the acceptance of ESS, the U.S. Department of Energy’s (DOE) Energy Storage Systems Program facilitated the development of a protocol to measure and express ESS performance and is supporting its updating, enhancement and use in formal consensus standards development. Of particular interest is the development of the document through an open and transparent process that saved considerable time.

  19. PCR diagnostics and monitoring of adenoviral infections in hematopoietic stem cell transplantation recipients.

    PubMed

    Bil-Lula, Iwona; Ussowicz, Marek; Rybka, Blanka; Wendycz-Domalewska, Danuta; Ryczan, Renata; Gorczyńska, Ewa; Kałwak, Krzysztof; Woźniak, Mieczysław

    2010-12-01

    After stem cell transplantation, human patients are prone to life-threatening opportunistic infections with a plethora of microorganisms. We report a retrospective study on 116 patients (98 children, 18 adults) who were transplanted in a pediatric bone marrow transplantation unit. Blood, urine and stool samples were collected and monitored for adenovirus (AdV) DNA using polymerase chain reaction (PCR) and real-time PCR (RT-PCR) on a regular basis. AdV DNA was detected in 52 (44.8%) patients, with mortality reaching 19% in this subgroup. Variables associated with adenovirus infection were transplantations from matched unrelated donors and older age of the recipient. An increased seasonal occurrence of adenoviral infections was observed in autumn and winter. Analysis of immune reconstitution showed a higher incidence of AdV infections during periods of low T-lymphocyte count. This study also showed a strong interaction between co-infections of AdV and BK polyomavirus in patients undergoing hematopoietic stem cell transplantations. PMID:20848295

  20. Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1.

    PubMed

    Castello, R; Borzone, R; D'Aria, S; Annunziata, P; Piccolo, P; Brunetti-Pierri, N

    2016-02-01

    Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT), which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate that ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Toward this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared with saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with ethylene glycol, a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy. PMID:26609667

  1. PCR diagnostics and monitoring of adenoviral infections in hematopoietic stem cell transplantation recipients

    PubMed Central

    Ussowicz, Marek; Rybka, Blanka; Wendycz-Domalewska, Danuta; Ryczan, Renata; Gorczyńska, Ewa; Kałwak, Krzysztof; Woźniak, Mieczysław

    2010-01-01

    After stem cell transplantation, human patients are prone to life-threatening opportunistic infections with a plethora of microorganisms. We report a retrospective study on 116 patients (98 children, 18 adults) who were transplanted in a pediatric bone marrow transplantation unit. Blood, urine and stool samples were collected and monitored for adenovirus (AdV) DNA using polymerase chain reaction (PCR) and real-time PCR (RT-PCR) on a regular basis. AdV DNA was detected in 52 (44.8%) patients, with mortality reaching 19% in this subgroup. Variables associated with adenovirus infection were transplantations from matched unrelated donors and older age of the recipient. An increased seasonal occurrence of adenoviral infections was observed in autumn and winter. Analysis of immune reconstitution showed a higher incidence of AdV infections during periods of low T-lymphocyte count. This study also showed a strong interaction between co-infections of AdV and BK polyomavirus in patients undergoing hematopoietic stem cell transplantations. PMID:20848295

  2. Restoration of β -Adrenergic Signaling in Failing Cardiac Ventricular Myocytes via Adenoviral-Mediated Gene Transfer

    NASA Astrophysics Data System (ADS)

    Akhter, Shahab A.; Skaer, Christine A.; Kypson, Alan P.; McDonald, Patricia H.; Peppel, Karsten C.; Glower, Donald D.; Lefkowitz, Robert J.; Koch, Walter J.

    1997-10-01

    Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β -adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β -adrenergic signaling defects including down-regulation of myocardial β -adrenergic receptors (β -ARs), functional β -AR uncoupling, and an upregulation of the β -AR kinase (β ARK1). Adenoviral-mediated gene transfer of the human β 2-AR or an inhibitor of β ARK1 to these failing myocytes led to the restoration of β -AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of β ARK1 activity in the heart.

  3. Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1

    PubMed Central

    Castello, Raffaele; Borzone, Roberta; D’Aria, Stefania; Annunziata, Patrizia; Piccolo, Pasquale; Brunetti-Pierri, Nicola

    2015-01-01

    Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT) which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate which ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared to saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with Ethylene Glycol (EG), a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy. PMID:26609667

  4. Amelioration of carbon tetrachloride-induced cirrhosis and portal hypertension in rat using adenoviral gene transfer of Akt

    PubMed Central

    Deng, Gang; Huang, Xiang-Jun; Luo, Hong-Wu; Huang, Fei-Zhou; Liu, Xun-Yang; Wang, Yong-Heng

    2013-01-01

    AIM: To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride (CCl4). METHODS: Using cre-loxp technique, we created an Ad-myr-HA-Akt virus, in which Akt is labeled by a HA tag and its expression is driven by myr promoter. Further, through measuring enzyme levels and histological structure, we determined the efficacy of this Ad-myr-HA-Akt virus in inhibiting the development of cirrhosis induced by CCl4 in rats. Lastly, using western blotting, we examined the expression levels and/or phosphorylation status of Akt, apoptotic mediators, endothelial nitric oxide synthase (eNOS), and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus. RESULTS: The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment, which was consistent with the sequence reported in the GenBank. The concentrations of Ad-myr-HA-Akt and adenoviral enhanced green fluorescent protein (Ad-EGFP) virus used in the current study were 5.5 × 1011 vp/mL. The portal vein diameter, peak velocity of blood flow, portal blood flow and congestion index were significantly increased in untreated, saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection. In contrast, these parameters in the Akt cirrhosis group were comparable to normal control group. Compared to the normal control, the liver function (Alanine aminotransferase, Aspartate aminotransferase and Albumin) was significantly impaired in the untreated, saline and Ad-EGFP cirrhosis groups. The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated, saline and Ad-EGFP cirrhosis groups. The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups

  5. The Q System: A Repressible Binary System for Transgene Expression, Lineage Tracing and Mosaic Analysis

    PubMed Central

    Potter, Christopher J.; Tasic, Bosiljka; Russler, Emilie V.; Liang, Liang; Luo, Liqun

    2010-01-01

    Summary We describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This ‘Q system’ offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications including: 1) intersectional ‘logic gates’ with the GAL4 system for manipulating transgene expression patterns, 2) GAL4-independent MARCM analysis, 3) coupled MARCM analysis to independently visualize and genetically manipulate siblings from any cell division. We demonstrate the utility of the Q system in determining cell division patterns of a neuronal lineage and gene function in cell growth and proliferation, and in dissecting neurons responsible for olfactory attraction. The Q system can be expanded to other uses in Drosophila, and to any organism conducive to transgenesis. PMID:20434990

  6. 21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system....

  7. 21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system....

  8. 21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system....

  9. 21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system....

  10. 21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system....

  11. A reproducible and quantifiable model of choroidal neovascularization induced by VEGF A165 after subretinal adenoviral gene transfer in the rabbit

    PubMed Central

    Kreppel, Florian; Beck, Susanne; Heiduschka, Peter; Brito, Veronica; Schnichels, Sven; Kochanek, Stefan; Schraermeyer, Ulrich

    2008-01-01

    Purpose To determine the effects of the vascular endothelial growth factor (VEGF)-A165 delivered using a high capacity adenoviral vector (HC Ad.VEGF-A) on vascular growth and pathological changes in the rabbit eye. To combine different detection methods of VEGF-A165 overexpression-induced neovascularization in the rabbit. Methods HC Ad.VEGF-A165 was constructed and injected at 5x106 infectious units (iu) into the subretinal space of rabbit eyes. Two and four weeks postinjection, the development of neovascularization and the expression of HC Ad-transduced VEGF-A165 protein were followed up in vivo by scanning laser ophthalmoscopy, fluorescein and indocyanine green angiographies and ex vivo by electron microscopy and immunohistochemistry Results We observed a choroidal neovascularization (CNV) with leakage in 83% of the rabbit eyes. Our findings present clear indications that there is a significant effect on the endothelial cells of the choriocapillaris after subretinal transduction of the retinal pigment epithelium (RPE) with VEGF-A165 vector. The choroidal endothelial cells were activated, adherent junctions opened, and the fenestration was minimized, while the extracellular matrix localized between the RPE and the endothelium of the choriocapillaris was enlarged toward the lumen of the vessels, inducing a deep invagination of the endothelial cells into the vessel lumen. They also proliferated and formed pathological vessels in the subretinal space. Moreover,there was an increased expression of basic fibroblast growth factor and VEGF-A accompanied by macrophage stimulation, retinal edema, and photoreceptor loss. Conclusions This is the first model of VEGF-induced CNV in the rabbit in which the pathological events following overexpression of VEGF by RPE cells have been described in detail. Many of the features of our experimental CNV resemble those observed clinically in patients having wet age-related macular degeneration. PMID:18682809

  12. Adenoviral gene transfer into the normal and injured spinal cord: enhanced transgene stability by combined administration of temperature-sensitive virus and transient immune blockade.

    PubMed

    Romero, M I; Smith, G M

    1998-12-01

    This study characterized gene transfer into both normal and injured adult rat dorsal spinal cord using first (E1-/E3-) or second (E1-/E2A125/E3-, temperature-sensitive; ts) generation of replication-defective adenoviral (Ad) vectors. A novel immunosuppressive regimen aimed at blocking CD4/CD45 lymphocytic receptors was tested for improving transgene persistence. In addition, the effect of gene transfer on nociception was also evaluated. Seven days after treatment, numerous LacZ-positive cells were observed after transfection with either viral vector. By 21 days after transfection, beta-galactosidase staining was reduced and suggestive of ongoing cytopathology in both Ad-treated groups, despite the fact that the immunogenicity of LacZ/Adts appeared less when compared with that elicited by the LacZ/Ad vector. In contrast, immunosuppressed animals showed a significant (P < or = 0.05) increase in the number of LacZ-positive cells not displaying cytopathology. In these animals, a concomitant reduction in numbers of macrophages/microglia and CD4 and CD8 lymphocytes was observed. Only animals that received LacZ/Adts and immunosuppression showed transgene expression after 60 days. Similar results were observed in animals in which the L4-L5 dorsal roots were lesioned before transfection. Gene transfer into the dorsal spinal cord did not affect nociception, independent of the adenovirus vector. These results indicate that immune blockade of the CD4/CD45 lymphocytic receptors enhanced transgene stability in adult animals with normal or injured spinal cords and that persistent transgene expression in the spinal cord does not interfere with normal neural function. PMID:10023440

  13. Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering.

    PubMed

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  14. Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model

    PubMed Central

    Ahmed, Atique U; Rolle, Cleo E; Tyler, Matthew A; Han, Yu; Sengupta, Sadhak; Wainwright, Derek A; Balyasnikova, Irina V; Ulasov, Ilya V; Lesniak, Maciej S

    2010-01-01

    Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow–derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response. PMID:20588259

  15. Bone marrow mesenchymal stem cells loaded with an oncolytic adenovirus suppress the anti-adenoviral immune response in the cotton rat model.

    PubMed

    Ahmed, Atique U; Rolle, Cleo E; Tyler, Matthew A; Han, Yu; Sengupta, Sadhak; Wainwright, Derek A; Balyasnikova, Irina V; Ulasov, Ilya V; Lesniak, Maciej S

    2010-10-01

    Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow-derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response. PMID:20588259

  16. Expression of Functional Recombinant Human Tissue Transglutaminase (TG2) Using the Bac-to-Bac Baculovirus Expression System

    PubMed Central

    Yazdani, Yaghoub; Azari, Shahram; Kalhor, Hamid Reza

    2016-01-01

    Purpose: Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer’s disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. Methods: pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Results: Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Conclusion: Human TG2 was expressed efficiently in the active biological form in the Bac-to-Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system. PMID:27123417

  17. Connexin32 expression in central and peripheral nervous systems

    SciTech Connect

    Deschenes, S.M.; Scherer, S.S.; Fischbeck, K.H.

    1994-09-01

    Mutations have been identified in the gap junction gene, connexin32 (Cx32), in patients affected with the X-linked form of the demyelinating neuropathy, Charcot-Marie-Tooth disease (CMTX). Gap junctions composed of Cx32 are present and developmentally regulated in a wide variety of tissues. In peripheral nerve, our immunohistochemical analysis localized Cx32 to the noncompacted myelin of the paranodal regions and the Schmidt-Lantermann incisures, where previous studies describe gap junctions. In contrast to the location of Cx32 in peripheral nerve and the usual restriction of clinical manifestations to the peripheral nervous system (PNS) (abstract by Paulson describes an exception), preliminary studies show that Cx32 is present in the compacted myelin of the central nervous system (CNS), as demonstrated by radial staining through the myelin sheath of oligodendrocytes in rat spinal cord. Analysis of Cx32 expression in various regions of rat CNS during development shows that the amount of Cx32 mRNA and protein increases as myelination increases, a pattern observed for other myelin genes. Studies in the PNS provide additional evidence that Cx32 and myelin genes are coordinately regulated at the transcriptional level; Cx32 and peripheral myelin gene PMP-22 mRNAs are expressed in parallel following transient or permanent nerve injury. Differences in post-translational regulation of Cx32 in the CNS and PNS may be indicated by the presence of a faster migrating form of Cs32 in cerebrum versus peripheral nerve. Studies are currently underway to determine the unique role of Cx32 in peripheral nerve.

  18. The visual system discounts emotional deviants when extracting average expression

    PubMed Central

    Haberman, Jason; Whitney, David

    2011-01-01

    There has been a recent surge in the study of ensemble coding, the idea that the visual system represents a set of similar items using summary statistics (Alvarez & Oliva, 2008; Ariely, 2001; Chong & Treisman, 2003; Parkes, Lund, Angelucci, Solomon, & Morgan, 2001). We previously demonstrated that this ability extends to faces and thus requires a high level of object processing (Haberman & Whitney, 2007, 2009). Recent debate has centered on the nature of the summary representation of size (e.g., Myczek & Simons, 2008) and whether the perceived average simply reflects the sampling of a very small subset of the items in a set. In the present study, we explored this further in the context of faces, asking observers to judge the average expressions of sets of faces containing emotional outliers. Our results suggest that the visual system implicitly and unintentionally discounts the emotional outliers, thereby computing a summary representation that encompasses the vast majority of the information present. Additional computational modeling and behavioral results reveal that an intentional, cognitive sampling strategy does not accurately capture observer performance. Observers derive precise ensemble information given a 250-msec exposure, suggesting a rapid and flexible system not bound by the limits of serial attention. PMID:20952781

  19. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    PubMed

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs. PMID:26026665

  20. Comparison of Efficacy of Two Different Topical 0.05% Cyclosporine A Formulations in the Treatment of Adenoviral Keratoconjunctivitis-Related Subepithelial Infiltrates

    PubMed Central

    Bayraktutar, Betül N.; Uçakhan, Ömur Ö.

    2016-01-01

    Subepithelial infiltrates secondary to adenoviral keratoconjunctivitis may persist for years and cause blurred vision, halos, glare, and photophobia. These infiltrates arise from immune reaction against the virus, and few studies have reported topical cyclosporine A to be effective in the treatment of subepithelial infiltrates. Herein, we describe a patient with adenoviral keratoconjunctivitis-related subepithelial infiltrates who did not respond to treatment with a new topical cyclosporine A emulsion prepared with castor oil (Depores 0.05%; Deva İlaç, Kocaeli, Turkey), while the FDA-approved nanoemulsion formulation provided improvement in symptoms and reduced the inflammatory reaction (Restasis 0.05%; Allergan, Irvine, Calif., USA). PMID:27065851

  1. Comparison of Efficacy of Two Different Topical 0.05% Cyclosporine A Formulations in the Treatment of Adenoviral Keratoconjunctivitis-Related Subepithelial Infiltrates.

    PubMed

    Bayraktutar, Betül N; Uçakhan, Ömur Ö

    2016-01-01

    Subepithelial infiltrates secondary to adenoviral keratoconjunctivitis may persist for years and cause blurred vision, halos, glare, and photophobia. These infiltrates arise from immune reaction against the virus, and few studies have reported topical cyclosporine A to be effective in the treatment of subepithelial infiltrates. Herein, we describe a patient with adenoviral keratoconjunctivitis-related subepithelial infiltrates who did not respond to treatment with a new topical cyclosporine A emulsion prepared with castor oil (Depores 0.05%; Deva İlaç, Kocaeli, Turkey), while the FDA-approved nanoemulsion formulation provided improvement in symptoms and reduced the inflammatory reaction (Restasis 0.05%; Allergan, Irvine, Calif., USA). PMID:27065851

  2. Expression of corticosteroid binding globulin in the rat olfactory system.

    PubMed

    Dölz, Wilfried; Eitner, Annett; Caldwell, Jack D; Jirikowski, Gustav F

    2013-05-01

    Glucocorticoids are known to act on the olfactory system although their mode of action is still unclear since nuclear glucocorticoid receptors are mostly absent in the olfactory mucosa. In this study we used immunocytochemistry, in situ hybridization, and RT-PCR to study the expression and distribution of corticosteroid binding globulin (CBG) in the rat olfactory system. Mucosal goblet cells could be immunostained for CBG. Nasal secretion contained measurable amounts of CBG suggesting that CBG is liberated. CBG immunoreactivity was localized in many of the basal cells of the olfactory mucosa, while mature sensory cells contained CBG only in processes as determined by double immunostaining with the olfactory marker protein OMP. This staining was most pronounced in the vomeronasal organ (VNO). The appearance of CBG in the non-sensory and sensory parts of the VNO and in nerve terminals in the accessory bulb indicated axonal transport. Portions of the periglomerular cells, the mitral cells and the tufted cells were also CBG positive. CBG encoding transcripts were confirmed by RT-PCR in homogenates of the olfactory mucosa and VNO. Olfactory CBG may be significant for uptake, accumulation and transport of glucocorticoids, including aerosolic cortisol. PMID:23141917

  3. The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

    PubMed

    Lu, Zhi Hong; Kaliberov, Sergey; Zhang, Jingzhu; Muz, Barbara; Azab, Abdel K; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Curiel, David T; Arbeit, Jeffrey M

    2014-08-01

    Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases. PMID:24955893

  4. Expression of an alternative nitrogen fixation system in Azotobacter vinelandii.

    PubMed Central

    Bishop, P E; Jarlenski, D M; Hetherington, D R

    1982-01-01

    Nitrogenase activities were determined from maximum acetylene reduction rates for mutant strains of Azotobacter vinelandii which are unable to fix N2 in the presence of molybdenum (Nif-) but undergo phenotypic reversal to Nif+ under conditions of Mo deficiency. The system responsible for N2 fixation under these conditions is thought to be an alternative N2 fixation system (Bishop et al., Proc. Natl. Acad. Sci. U.S.A. 77:7342-7346, 1980). Phenotypic reversal of Nif- strains to Nif+ strains was also observed in N-free medium without Mo but with either V or Re. Two protein patterns were found on two-dimensional gels of proteins from the extracts of wild-type cells cultured in N-free medium without Mo and with or without V or Re. The expression of each protein pattern in the wild-type strain of A. vinelandii seemed to depend upon the physiological state of the N2-fixing culture. Electron paramagnetic resonance experiments were conducted on whole cells of A. vinelandii grown under conditions of Mo deprivation in the absence of fixed N. No g = 3.65 signal (an electron paramagnetic resonance signal characteristic of the Mo-containing component of nitrogenase) was detectable in these cells, regardless of whether V or Re was present during growth of these cells, These results are discussed from the perspective that the well-known effect of V on N2 fixation by A. vinelandii may involve an alternative N2 fixation system. Images PMID:6281240

  5. A Multi-Antigenic Adenoviral-Vectored Vaccine Improves BCG-Induced Protection of Goats against Pulmonary Tuberculosis Infection and Prevents Disease Progression

    PubMed Central

    Pérez de Val, Bernat; Vidal, Enric; Villarreal-Ramos, Bernardo; Gilbert, Sarah C.; Andaluz, Anna; Moll, Xavier; Martín, Maite; Nofrarías, Miquel; McShane, Helen; Vordermeier, H. Martin; Domingo, Mariano

    2013-01-01

    The “One world, one health” initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG. PMID:24278420

  6. Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.

    PubMed

    Bjerga, Gro Elin Kjæreng; Lale, Rahmi; Williamson, Adele Kim

    2016-01-01

    Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications. PMID:26710170

  7. Express

    Integrated Risk Information System (IRIS)

    Express ; CASRN 101200 - 48 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effect

  8. Adenoviral-Mediated Glial Cell Line–Derived Neurotrophic Factor Gene Transfer Has a Protective Effect on Sciatic Nerve Following Constriction-Induced Spinal Cord Injury

    PubMed Central

    Chou, An-Kuo; Yang, Ming-Chang; Tsai, Hung-Pei; Chai, Chee-Yin; Tai, Ming-Hong; Kwan, Aij-Li; Hong, Yi-Ren

    2014-01-01

    Neuropathic pain due to peripheral nerve injury may be associated with abnormal central nerve activity. Glial cell-line-derived neurotrophic factor (GDNF) can help attenuate neuropathic pain in different animal models of nerve injury. However, whether GDNF can ameliorate neuropathic pain in the spinal cord dorsal horn (SCDH) in constriction-induced peripheral nerve injury remains unknown. We investigated the therapeutic effects of adenoviral-mediated GDNF on neuropathic pain behaviors, microglial activation, pro-inflammatory cytokine expression and programmed cell death in a chronic constriction injury (CCI) nerve injury animal model. In this study, neuropathic pain was produced by CCI on the ipsilateral SCDH. Mechanical allodynia was examined with von Frey filaments and thermal sensitivity was tested using a plantar test apparatus post-operatively. Target proteins GDNF-1, GDNFRa-1, MMP2, MMP9, p38, phospho-p38, ED1, IL6, IL1β, AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, Beclin-1, PKCσ, PKCγ, iNOS, eNOS and nNOS were detected. Microglial activity was measured by observing changes in immunoreactivity with OX-42. NeuN and TUNEL staining were used to reveal whether apoptosis was attenuated by GDNF. Results showed that administrating GDNF began to attenuate both allodynia and thermal hyperalgesia at day 7. CCI-rats were found to have lower GDNF and GDNFRa-1 expression compared to controls, and GDNF re-activated their expression. Also, GDNF significantly down-regulated CCI-induced protein expression except for MMP2, eNOS and nNOS, indicating that the protective action of GDNF might be associated with anti-inflammation and prohibition of microglia activation. Immunocytochemistry staining showed that GDNF reduced CCI-induced neuronal apoptosis. In sum, GDNF enhanced the neurotrophic effect by inhibiting microglia activation and cytokine production via p38 and PKC signaling. GDNF could be a good

  9. Efficient expression and purification of recombinant human m-calpain using an Escherichia coli expression system at low temperature.

    PubMed

    Hata, Shoji; Ueno, Mika; Kitamura, Fujiko; Sorimachi, Hiroyuki

    2012-04-01

    Calpain belongs to the superfamily of Ca(2+)-regulated cysteine proteases, which are indispensable to the regulation of various cellular functions. Of the 15 mammalian calpain isoforms, µ- and m-calpains are the best characterized. Both µ- and m-calpain are ubiquitously expressed and exist as heterodimers, containing a distinct 80-kDa catalytic subunit (CAPN1 and CAPN2, respectively) and the common, 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for use in structural and physiological studies, however Escherichia coli systems have proven incompatible with large-scale preparation of calpain, with the exception of rat m-calpain. Here, we have established a highly efficient method to purify active recombinant human m-calpain using an E. coli expression system at low temperature (22°C). This was achieved by co-expressing CAPN2 with a C-terminal histidine-tag, and CAPNS1, lacking the first Gly-repeated region at the N-terminal. After three sequential passes through a chromatographic column, ~5 mg of human m-calpain was homogenously purified from 1 l of E. coli culture. Proteins were stable for several months. This is the first report of efficient, large-scale purification of recombinant human m-calpain using an E. coli expression system. PMID:22232565

  10. STANDARDIZATION AND VALIDATION OF ADENOVIRAL TRANSDUCTION OF AN ANDROGEN RECEPTOR POSITIVE CELL LINE WITH AN MMTV-LUC REPORTER FOR ENDOCRINE SCREENING

    EPA Science Inventory

    Standardization and Validation of Adenoviral Transduction of an Androgen Receptor Positive Cell Line with an MMTV-Luc Reporter for Endocrine Screening P. Hartig, K . Bobseine,
    M. Cardon, C. Lambright and L. E. Gray, Jr. USEPA, Reproductive Toxicology Division, NHEERL, RTP, NC...

  11. Development of a Cold-Adapted Pseudoalteromonas Expression System for the Pseudoalteromonas Proteins Intractable for the Escherichia coli System.

    PubMed

    Yu, Zi-Chao; Tang, Bai-Lu; Zhao, Dian-Li; Pang, Xiuhua; Qin, Qi-Long; Zhou, Bai-Cheng; Zhang, Xi-Ying; Chen, Xiu-Lan; Zhang, Yu-Zhong

    2015-01-01

    Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psychrophilic strain Pseudoalteromonas sp. SM20429 was established. Based on the shuttle vector, three reporter vectors were constructed to compare the strength of the cloned promoters at low temperature. The promoter of xylanase gene from Pseudoalteromonas sp. BSi20429 was chosen due to its high activity at 10-15°C. An expression vector pEV containing the chosen promoter, multiple cloning sites and a His tag was constructed for protein expression and purification. With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h. Recombinant pseudoalterin purified from the culture by Ni affinity chromatography had identical N-terminal sequence, similar molecular mass and substrate specificity as the native pseudoalterin. In addition, another two cold-adapted enzymes were also successfully expressed by this system. Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system. PMID:26333173

  12. The Physcomitrella patens System for Transient Gene Expression Assays.

    PubMed

    Thévenin, Johanne; Xu, Wenjia; Vaisman, Louise; Lepiniec, Loïc; Dubreucq, Bertrand; Dubos, Christian

    2016-01-01

    Transient expression assays are valuable techniques to study in vivo the transcriptional regulation of gene expression. These methods allow to assess the transcriptional properties of a given transcription factor (TF) or a complex of regulatory proteins against specific DNA motifs, called cis-regulatory elements. Here, we describe a fast, efficient, and reliable method based on the use of Physcomitrella patens protoplasts that allows the study of gene expression in a qualitative and quantitative manner by combining the advantage of GFP (green fluorescent protein) as a marker of promoter activity with flow cytometry for accurate measurement of fluorescence in individual cells. PMID:27557766

  13. Production of recombinant botulism antigens: a review of expression systems.

    PubMed

    Moreira, G M S G; Cunha, C E P; Salvarani, F M; Gonçalves, L A; Pires, P S; Conceição, F R; Lobato, F C F

    2014-08-01

    Botulism is a paralytic disease caused by intoxication with neurotoxins produced by Clostridium botulinum. Despite their similar mechanism of action, the botulinum neurotoxins (BoNT) are classified in eight serotypes (A to H). As to veterinary medicine, the impact of this disease is essentially economic, since different species of production animals can be affected, especially by BoNT/C and D. In human health, botulism is feared in a possible biological warfare, what would involve mainly the BoNT/A, B, E and F. In both cases, the most effective way to deal with botulism is through prevention, which involves vaccination. However, the current vaccines against this disease have several drawbacks on their process of production and, besides this, can be dangerous to producers since it requires certain level of biosafety. This way, recombinant vaccines have been shown to be a great alternative for the development of vaccines against both animal and human botulism. All BoNTs have a 50-kDa light chain (LC) and a 100-kDa heavy chain (HC). The latter one presents two domains of 50 kDa, called the N-terminal (HN) and C-terminal (HC) halves. Among these regions, the HC alone seem to confer the proper immune response against intoxication. Since innumerous studies describe the expression of these distinct regions using different systems, strategies, and protocols, it is difficult to define the best option for a viable vaccine production. Thereby, the present review describes the problematic of botulism and discusses the main advances for the viable production of vaccines for both human and veterinary medicine using recombinant antigens. PMID:24930432

  14. Genetic immunization in the lung induces potent local and systemic immune responses.

    PubMed

    Song, Kaimei; Bolton, Diane L; Wei, Chih-Jen; Wilson, Robert L; Camp, Jeremy V; Bao, Saran; Mattapallil, Joseph J; Herzenberg, Leonore A; Herzenberg, Leonard A; Andrews, Charla A; Sadoff, Jerald C; Goudsmit, Jaap; Pau, Maria Grazia; Seder, Robert A; Kozlowski, Pamela A; Nabel, Gary J; Roederer, Mario; Rao, Srinivas S

    2010-12-21

    Successful vaccination against respiratory infections requires elicitation of high levels of potent and durable humoral and cellular responses in the lower airways. To accomplish this goal, we used a fine aerosol that targets the entire lung surface through normal respiration to deliver replication-incompetent recombinant adenoviral vectors expressing gene products from several infectious pathogens. We show that this regimen induced remarkably high and stable lung T-cell responses in nonhuman primates and that it also generated systemic and respiratory tract humoral responses of both IgA and IgG isotypes. Moreover, strong immunogenicity was achieved even in animals with preexisting antiadenoviral immunity, overcoming a critical hurdle to the use of these vectors in humans, who commonly are immune to adenoviruses. The immunogenicity profile elicited with this regimen, which is distinct from either intramuscular or intranasal delivery, has highly desirable properties for protection against respiratory pathogens. We show that it can be used repeatedly to generate mucosal humoral, CD4, and CD8 T-cell responses and as such may be applicable to other mucosally transmitted pathogens such as HIV. Indeed, in a lethal challenge model, we show that aerosolized recombinant adenoviral immunization completely protects ferrets against H5N1 highly pathogenic avian influenza virus. Thus, genetic immunization in the lung offers a powerful platform approach to generating protective immune responses against respiratory pathogens. PMID:21135247

  15. Construction of a host-independent T7 expression system with small RNA regulation.

    PubMed

    Wang, Gang; Li, Qiang; Xu, Dikai; Cui, Mingxin; Sun, Xiao; Xu, Yanyan; Wang, Wenya

    2014-11-10

    It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572. PMID:25193711

  16. Potent antitumor activity of oncolytic adenovirus expressing Beclin-1 via induction of autophagic cell death in leukemia

    PubMed Central

    Liu, Hui; Li, Lu; Meng, Haitao; Qian, Qijun

    2013-01-01

    An attractive strategy among adenovirus-based oncolytic systems is to design adenoviral vectors to express pro-apoptotic genes, in which this gene-virotherapy approach significantly enhances tumor cell death by activating apoptotic pathways. However, the existence of cancer cells with apoptotic defects is one of the major obstacles in gene-virotherapy. Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia. A Beclin-1 cDNA was cloned in an oncolytic adenovirus with chimeric Ad5/11 fiber (SG511-BECN). SG511-BECN treatment induced significant autophagic cell death, and resulted in enhanced cell killing in a variety of leukemic cell lines and primary leukemic blasts. SG511-BECN effects were seen in chronic myeloid leukemia and acute myeloid leukemia with resistance to imatinib or chemotherapy, but exhibited much less cytotoxicity on normal cells. The SG511-BECN-induced autophagic cell death could be partially reversed by RNA interference knockdown of UVRAG, ATG5, and ATG7. We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro. In murine leukemia models, SG511-BECN prolonged the survival and decreased the xenograft tumor size by inducing autophagic cell death. Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis. PMID:23765161

  17. Regulation of gene expression in the nervous system

    SciTech Connect

    Stella, A.M.G. ); de Vellis, J. ); Perez-Polo, J.R. 62230.

    1990-01-01

    This book covers subjects under the following topics: Plenary Lecture; Growth factors; Regulation of gene expression in neurons; Cell adhesion molecules and development; Nervous tissue reaction to injury-aging; and Poster presentation.

  18. Standard free droplet digital polymerase chain reaction as a new tool for the quality control of high-capacity adenoviral vectors in small-scale preparations.

    PubMed

    Boehme, Philip; Stellberger, Thorsten; Solanki, Manish; Zhang, Wenli; Schulz, Eric; Bergmann, Thorsten; Liu, Jing; Doerner, Johannes; Baiker, Armin E; Ehrhardt, Anja

    2015-02-01

    High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy. PMID:25640117

  19. Standard Free Droplet Digital Polymerase Chain Reaction as a New Tool for the Quality Control of High-Capacity Adenoviral Vectors in Small-Scale Preparations

    PubMed Central

    Boehme, Philip; Stellberger, Thorsten; Solanki, Manish; Zhang, Wenli; Schulz, Eric; Bergmann, Thorsten; Liu, Jing; Doerner, Johannes; Baiker, Armin E.

    2015-01-01

    Abstract High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy. PMID:25640117

  20. Expression of trisomic proteins in Down syndrome model systems.

    PubMed

    Spellman, Claire; Ahmed, Md Mahiuddin; Dubach, Daphne; Gardiner, Katheleen J

    2013-01-10

    Down syndrome (DS) is the most common genetic aberration leading to intellectual disability. DS results from an extra copy of the long arm of human chromosome 21 (HSA21) and the increased expression of trisomic genes due to gene dosage. While expression in DS and DS models has been studied extensively at the RNA level, much less is known about expression of trisomic genes at the protein level. We have used quantitative Western blotting with antibodies to 20 proteins encoded by HSA21 to assess trisomic protein expression in lymphoblastoid cell lines (LCLs) from patients with DS and in brains from two mouse models of DS. These antibodies have recently become available and the 20 proteins largely have not been investigated previously for their potential contributions to the phenotypic features of DS. Twelve proteins had detectable expression in LCLs and three, CCT8, MX1 and PWP2, showed elevated levels in LCLs derived from patients with DS compared with controls. Antibodies against 15 proteins detected bands of appropriate sizes in lysates from mouse brain cortex. Genes for 12 of these proteins are trisomic in the Tc1 mouse model of DS, but only SIM2 and ZNF295 showed elevated expression in Tc1 cortex when compared with controls. Genes for eight of the 15 proteins are trisomic in the Ts65Dn mouse model of DS, but only ZNF294 was over expressed in cortex. Comparison of trisomic gene expression at the protein level with previous reports at the mRNA level showed many inconsistencies. These may be caused by natural inter-individual variability, differences in the age of mice analyzed, or post-transcriptional regulation of gene dosage effects. These antibodies provide resources for further investigation of the molecular basis of intellectual disability in DS. PMID:23103828

  1. Expression of transgenes in midbrain dopamine neurons using the tyrosine hydroxylase promoter

    PubMed Central

    Oh, Myung Sook; Hong, Seok Jong; Huh, Youngbuhm; Kim, Kwang-Soo

    2009-01-01

    Billions of neurons are interconnected in the central nervous system (CNS). Identification of specific neuronal circuit is indispensable for understanding the relationship between structure and function in the CNS. The midbrain dopamine (DA) neuron system consists of the retrorubral area (A8), the substantia nigra (SN; A9), and the ventral tegmental area (VTA; A10). We hypothesized that genetic methods using cell-type specific promoters may offer the possibility to express tracer molecules in DA neurons to facilitate neuronal tracing. To address this, we used the 2.5 kb rat tyrosine hydroxylase (TH) promoter in adenovirus or adeno-associated virus (AAV) to express tracers specifically in DA neurons. We found that stereotaxic injection of TH promoter containing adenoviral construct resulted in cell type-specific transgene expression in the noradrenaline (NA) neurons of the locus coeruleus (LC). However, it caused a significant toxicity to DA neurons in the SN. In contrast, stereotaxic injection of TH promoter containing AAV to the SN resulted in cell type-specific transgene expression in DA neurons with no detectable toxicity. Taken together, our results demonstrate that it is possible to selectively trace DA neuronal circuits in rodent brains using the TH promoter in the context of AAV. PMID:18800154

  2. An adaptable system for improving transposon-based gene expression in vivo via transient transgene repression

    PubMed Central

    Doherty, Joseph E.; Woodard, Lauren E.; Bear, Adham S.; Foster, Aaron E.; Wilson, Matthew H.

    2013-01-01

    Transposons permit permanent cellular genome engineering in vivo. However, transgene expression falls rapidly postdelivery due to a variety of mechanisms, including immune responses. We hypothesized that delaying initial transgene expression would improve long-term transgene expression by using an engineered piggyBac transposon system that can regulate expression. We found that a 2-part nonviral Tet-KRAB inducible expression system repressed expression of a luciferase reporter in vitro. However, we also observed nonspecific promoter-independent repression. Thus, to achieve temporary transgene repression after gene delivery in vivo, we utilized a nonintegrating version of the repressor plasmid while the gene of interest was delivered in an integrating piggyBac transposon vector. When we delivered the luciferase transposon and repressor to immunocompetent mice by hydrodynamic injection, initial luciferase expression was repressed by 2 orders of magnitude. When luciferase expression was followed long term in vivo, we found that expression was increased >200-fold compared to mice that received only the luciferase transposon and piggyBac transposase. We found that repression of early transgene expression could prevent the priming of luciferase-specific T cells in vivo. Therefore, transient transgene repression postgene delivery is an effective strategy for inhibiting the antitransgene immune response and improving long-term expression in vivo without using immunosuppression.—Doherty, J. E., Woodard, L. E., Bear, A. S., Foster, A. E., Wilson, M. H. An adaptable system for improving transposon-based gene expression in vivo via transient transgene repression. PMID:23752206

  3. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

    PubMed Central

    Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

    2015-01-01

    Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

  4. [Homologous expression of Burkholderia cepacia G63 lipase gene based on T7 RNA polymerase expression system].

    PubMed

    Jia, Bin; Yang, Jiangke; Yan, Yunjun

    2009-02-01

    In order to realize over-expression of Burkholderia cepacia (B. cepacia) lipase, we introduced the widely used T7 RAN polymerase expression system into B. cepacia G63 to over-express the lipase gene. By using PCR technique, we amplified the T7 RNA polymerase gene (T7 RNAP) from the BL21 (DE3) and cloned it into the suicide plasmid pJQ200SK. After that, we flanked T7 RNAP with two 500 bp homologous fragments and integrated it into the genomes of B. cepacia by tri-parental mating, so that T7 RNAP was under-controlled by lipase gene (lipA) promoter. Then, we cloned the lipA and its partner gene lipB into the vector pUCPCM and pBBR22b both or separately. Therefore, we got 7 expression plasmids pBBR22blipAB, pBBR22blipA, pUCPCMlipAB, pUCPCMlipA, pUCPCMdeltalipAlipB, pUCPCMdeltalipA, pUCPCMdeltalipB, and then electroporated them into B. cepacia containing T7 RNA. After shake flask culture, we found B. cepacia containing pUCPCMlipAB produced the most quantity of lipase, and lipase activity was up to 607.2 U/mg, 2.8-folds higher than that of the wild strain. Moreover, lipase activities of all engineering strains except the one containing pUCPCMdeltalipB were enhanced to some extent. The specific activities of wild type B. cepacia and B. cepacia containing pUCPCMlipAB were respectively 29 984 U/mg and 30 875 U/mg after ammonium sulfate precipitation and gel filtration chromatography. The T7 RNA polymerase expression system could effectively enhanced lipase expression in B. cepacia, and secretion signal PelB and ribosome-binding site may promote lipase expression in engineering strain. PMID:19459326

  5. Osteopontin Expression in the Brain Triggers Localized Inflammation and Cell Death When Immune Cells Are Activated by Pertussis Toxin

    PubMed Central

    Marcondes, Maria Cecilia Garibaldi; Ojakian, Ryan; Bortell, Nikki; Flynn, Claudia; Conti, Bruno; Fox, Howard S.

    2014-01-01

    Upregulation of osteopontin (OPN) is a characteristic of central nervous system pathologies. However, the role of OPN in inflammation is still controversial, since it can both prevent cell death and induce the migration of potentially damaging inflammatory cells. To understand the role of OPN in inflammation and cell survival, we expressed OPN, utilizing an adenoviral vector, in the caudoputamen of mice deficient in OPN, using beta-galactosidase- (β-gal-) expressing vector as control. The tissue pathology and the expression of proinflammatory genes were compared in both treatments. Interestingly, inflammatory infiltrate was only found when the OPN-vector was combined with a peripheral treatment with pertussis toxin (Ptx), which activated peripheral cells to express the OPN receptor CD44v6. Relative to β-gal, OPN increased the levels of inflammatory markers, including IL13Rα1, CXCR3, and CD40L. In Ptx-treated OPN KOs, apoptotic TUNEL+ cells surrounding the OPN expression site increased, compared to β-gal. Together, these results show that local OPN expression combined with a peripheral inflammatory stimulus, such as Ptx, may be implicated in the development of brain inflammation and induction of cell death, by driving a molecular pattern characteristic of cytotoxicity. These are characteristics of inflammatory pathologies of the CNS in which OPN upregulation is a hallmark. PMID:25525298

  6. Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

    PubMed

    Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L

    2016-07-01

    The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases. PMID:27245510

  7. Immunogenicity without Efficacy of an Adenoviral Tuberculosis Vaccine in a Stringent Mouse Model for Immunotherapy during Treatment.

    PubMed

    Alyahya, S Anisah; Nolan, Scott T; Smith, Cara M R; Bishai, William R; Sadoff, Jerald; Lamichhane, Gyanu

    2015-01-01

    To investigate if bacterial persistence during TB drug treatment could be overcome by modulation of host immunity, we adapted a clinically-relevant model developed for the evaluation of new drugs and examined if immunotherapy with two adenoviral vaccines, Ad35-TBS (AERAS-402) and Ad26-TBS, could shorten therapy in mice. Even though immunotherapy resulted in strong splenic IFN-γ responses, no effect on bacterial replication in the lungs was seen. Multiplex assay analysis of lung samples revealed the absence of cytokine augmentation such as IFN-γ, TNF-α and IL-2, suggesting that immunization failed to induce immunity in the lungs. In this model, we show that IFN-γ levels were not associated with protection against disease relapse. The results obtained from our study raise questions regarding the traits of protective TB immunity that are relevant for the development of future immunotherapeutic and post-exposure vaccination strategies. PMID:25996375

  8. Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system.

    PubMed

    Zhang, Ziguo; Yang, Jing; Barford, David

    2016-02-15

    The capacity to reconstitute complex biological processes in vitro is a crucial step in providing a quantitative understanding of these systems. It provides material for structural, biochemical and biophysical analyses and allows the testing of biological hypotheses and the introduction of chemical probes and tags for single molecule analysis. Reconstitution of these systems requires access to homogenous components, usually through their over-production in heterologous over-expression systems. Here we describe the application of the USER (Uracil-Specific Excision Reagent) ligation-free cloning method to assemble recombinant MultiBac transfer vectors for the generation of recombinant baculovirus suitable for the expression of multi-protein complexes in insect cells. PMID:26454197

  9. Clinical and Antiviral Efficacy of an Ophthalmic Formulation of Dexamethasone Povidone-Iodine in a Rabbit Model of Adenoviral Keratoconjunctivitis

    PubMed Central

    Clement, Christian; Capriotti, Joseph A.; Kumar, Manish; Hobden, Jeffery A.; Foster, Timothy P.; Bhattacharjee, Partha S.; Thompson, Hilary W.; Mahmud, Rashed; Liang, Bo

    2011-01-01

    Purpose. To determine the efficacy of a new formulation of topical dexamethasone 0.1%/povidone-iodine 0.4% (FST-100) in reducing clinical symptoms and infectious viral titers in a rabbit model of adenoviral keratoconjunctivitis. Methods. Rabbit corneas were inoculated bilaterally with 2 × 106 plaque-forming-units (PFU) of adenovirus type 5 (Ad5) after corneal scarification. Animals were randomized 1:1:1:1 (five rabbits per group) to FST-100, 0.5% cidofovir, tobramycin/dexamethasone (Tobradex; Alcon Laboratories, Fort Worth, TX) ophthalmic suspension, and balanced salt solution (BSS; Alcon Laboratories). Treatment began 12 hours after viral inoculation and continued for 7 consecutive days. The eyes were clinically scored daily for scleral inflammation (injection), ocular neovascularization, eyelid inflammation (redness), friability of vasculature, inflammatory discharge (pus), and epiphora (excessive tearing). Eye swabs were collected daily before treatment for the duration of the study. Virus was eluted from the swabs and PFU determined by titration on human A549 cells, according to standard procedures. Results. The FST-100 treatment resulted in significantly lower clinical scores (P < 0.05) than did the other treatments. The 0.5% cidofovir exhibited the most ocular toxicity compared with FST-100, tobramycin/dexamethasone, and balanced salt solution treatments. FST-100 and 0.5% cidofovir significantly (P < 0.05) reduced viral titers compared with tobramycin/dexamethasone or balanced salt solution. Conclusions. FST-100 was the most efficacious in minimizing the clinical symptoms of adenovirus infection in rabbit eyes. FST-100 and 0.5% cidofovir were both equally effective in reducing viral titers and decreasing the duration of viral shedding. By providing symptomatic relief in addition to reducing infectious virus titers, FST-100 should be a valuable addition to treatment of epidemic adenoviral keratoconjunctivitis. PMID:20702820

  10. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    SciTech Connect

    Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  11. Cardiac angiotensin-(1-12) expression and systemic hypertension in rats expressing the human angiotensinogen gene.

    PubMed

    Ferrario, Carlos M; VonCannon, Jessica; Jiao, Yan; Ahmad, Sarfaraz; Bader, Michael; Dell'Italia, Louis J; Groban, Leanne; Varagic, Jasmina

    2016-04-15

    Angiotensin-(1-12) [ANG-(1-12)] is processed into ANG II by chymase in rodent and human heart tissue. Differences in the amino acid sequence of rat and human ANG-(1-12) render the human angiotensinogen (hAGT) protein refractory to cleavage by renin. We used transgenic rats harboring the hAGT gene [TGR(hAGT)L1623] to assess the non-renin-dependent effects of increased hAGT expression on heart function and arterial pressure. Compared with Sprague-Dawley (SD) control rats (n= 11), male homozygous TGR(hAGT)L1623 (n= 9) demonstrated sustained daytime and nighttime hypertension associated with no changes in heart rate but increased heart rate lability. Increased heart weight/tibial length ratio and echocardiographic indexes of cardiac hypertrophy were associated with modest reduction of systolic function in hAGT rats. Robust human ANG-(1-12) immunofluorescence within myocytes of TGR(hAGT)L1623 rats was associated with a fourfold increase in cardiac ANG II content. Chymase enzymatic activity, using the rat or human ANG-(1-12) as a substrate, was not different in the cardiac tissue of SD and hAGT rats. Since both cardiac angiotensin-converting enzyme (ACE) and ACE2 activities were not different among the two strains, the changes in cardiac structure and function, blood pressure, and left ventricular ANG II content might be a product of an increased cardiac expression of ANG II generated through a non-renin-dependent mechanism. The data also underscore the existence in the rat of alternate enzymes capable of acting on hAGT protein. Homozygous transgenic rats expressing the hAGT gene represent a novel tool to investigate the contribution of human relevant renin-independent cardiac ANG II formation and function. PMID:26873967

  12. Glucose induces FGF21 mRNA expression through ChREBP activation in rat hepatocytes.

    PubMed

    Iizuka, Katsumi; Takeda, Jun; Horikawa, Yukio

    2009-09-01

    Fibroblast growth factor 21 (FGF21) has beneficial effects of improving the plasma glucose and lipid profiles in diabetic rodents. Here, we investigated carbohydrate response element binding protein (ChREBP) involvement in the regulation of FGF21 mRNA expression in liver. Glucose stimulation and adenoviral overexpression of dominant active ChREBP increased FGF21 mRNA. Consistently, adenoviral expression of dominant negative Mlx inhibited glucose induction of FGF21 mRNA. Furthermore, deletion studies of mouse FGF21 gene promoter (-2000 to +65 bp) revealed a glucose responsive region between -74 and -52 bp. These findings suggest that FGF21 expression is regulated by ChREBP. PMID:19660458

  13. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  14. Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system.

    PubMed

    Sguazza, Guillermo H; Fuentealba, Nadia A; Tizzano, Marco A; Galosi, Cecilia M; Pecoraro, Marcelo R

    2013-01-01

    Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests. PMID:24401775

  15. Adenoviral-based foot-and-mouth disease virus vaccine: evaluation of new vectors expressing serotype O in bovines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease virus (FMDV), an antigenically variable virus, is considered the most important infectious disease of cloven-hoofed animals. Recently serotypes A and O have been the cause of major outbreaks. We previously demonstrated that an adenovirus-based FMDV serotype A24 subunit vaccine...

  16. Transient Expression Systems in Plants: Potentialities and Constraints.

    PubMed

    Canto, Tomas

    2016-01-01

    Plants have been used from old to extract and isolate by different means the products of interest that they store. In recent years new techniques have emerged that allow the use of plants as factories to overexpress transiently and often efficiently, specific genes of interest, either endogenous or foreign, in their native form or modified. These techniques allow and facilitate the targeted purification of gene products for research and commercial purposes without resorting to lengthy, time-consuming and sometimes challenging plant stable transformations, while avoiding some of their associated regulatory constraints. In this chapter we describe the main strategies available for the transient expression of gene sequences and their encoded products in plants. We discuss biological issues affecting transient expression, including resistance responses elicited by the plant against sequences that it recognizes naturally as foreign, and ways to neutralize them. We also discuss the relative advantages of each expression strategy as well as their inherent drawbacks and technical limitations, and how to partially prevent or overcome them, whenever possible. PMID:27165332

  17. DISOSS--Distributed Office Support System. An Electronic Messaging System Training Program for American Express TRS Company Employees.

    ERIC Educational Resources Information Center

    Rowell, Mary Emily

    An instructional system design model was developed for training new employees of the American Express TRS (Travel Related Services) Company, Inc. on the Distributed Office Support System (DISOSS), an electronic mail system. The project used the Interservice Procedures for Instructional Systems Development, which consists of five phases: analysis,…

  18. An optogenetic gene expression system with rapid activation and deactivation kinetics

    PubMed Central

    Motta-Mena, Laura B.; Reade, Anna; Mallory, Michael J.; Glantz, Spencer; Weiner, Orion D.; Lynch, Kristen W.; Gardner, Kevin H.

    2013-01-01

    Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range, or slow activation/deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach utilizes an engineered version of EL222, a bacterial Light-Oxygen-Voltage (LOV) protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (< 10 s) and deactivation kinetics (< 50 s), and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time. PMID:24413462

  19. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase.

    PubMed

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

  20. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase

    PubMed Central

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

  1. Dictyostelium discoideum--a promising expression system for the production of eukaryotic proteins.

    PubMed

    Arya, Ranjana; Bhattacharya, Alok; Saini, Kulvinder Singh

    2008-12-01

    In general, four different expression systems, namely, bacterial, yeast, baculovirus, and mammalian, are widely used for the overproduction of biochemical enzymes and therapeutic proteins. Clearly, bacterial expression systems offer ease of maneuverability with respect to large-scale production of recombinant proteins, while, a baculovirus expression system ensures proper protein modifications, processing, and refolding of complex proteins. Despite these advantages, mammalian cells remain the preferred host for many eukaryotic proteins of pharmaceutical importance, particularly, those requiring post-translational modifications. Recently, the single-celled slime mold, Dictyostelium discoideum (Dd), has emerged as a promising eukaryotic host for the expression of a variety of heterologous recombinant eukaryotic proteins. This organism possesses the complex cellular machinery required for orchestrating post-translational modifications similar to the one observed in higher eukaryotes. This review summarizes the advantages and disadvantages of Dictyostelium as an alternate system compared to other well-established expression systems. The key lessons learned from the expression of human recombinant proteins in this system are reviewed. Also, the strengths, weaknesses, and challenges associated with industrial-scale production of proteins in Dd expression system are discussed. PMID:18714070

  2. Energy expressions for n=3 and 4 systems in a single-j shell

    SciTech Connect

    Qi Chong

    2010-03-15

    For systems with three and four fermions within a single-j shell, analytical expressions for the state energies are presented from a decomposition of the angular momentum. In some important cases the expressions acquire a very simple form. The expression may help us in understanding the structure of isomeric states. The decomposition also makes it possible to construct the algebraic condition for conservation of seniority.

  3. Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

    NASA Technical Reports Server (NTRS)

    Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

  4. Implications of the innate immune response to adenovirus and adenoviral vectors

    PubMed Central

    Gregory, Seth M; Nazir, Shoab A; Metcalf, Jordan P

    2011-01-01

    Adenovirus (AdV) is a common cause of respiratory illness in both children and adults. Respiratory symptoms can range from those of the common cold to severe pneumonia. Infection can also cause significant disease in the immunocompromised and among immunocompetent subjects in close quarters. Fortunately, infection with AdV in the normal host is generally mild. This is one reason why its initial use as a gene-therapy vector appeared to be so promising. Unfortunately, both innate and adaptive responses to the virus have limited the development of AdV vectors as a tool of gene therapy by increasing toxicity and limiting duration of transgene expression. This article will focus on the innate immune response to infection with wild-type AdV and exposure to AdV gene-therapy vectors. As much of the known information relates to the pulmonary inflammatory response, this organ system will be emphasized. This article will also discuss how that understanding has led to the creation of new vectors for use in gene therapy. PMID:21738557

  5. Increased caspase-3 expression and activity contribute to reduced CD3zeta expression in systemic lupus erythematosus T cells.

    PubMed

    Krishnan, Sandeep; Kiang, Juliann G; Fisher, Carolyn U; Nambiar, Madhusoodana P; Nguyen, Hang T; Kyttaris, Vasileios C; Chowdhury, Bhabadeb; Rus, Violeta; Tsokos, George C

    2005-09-01

    T cells isolated from patients with systemic lupus erythematosus (SLE) express low levels of CD3zeta-chain, a critical molecule involved in TCR-mediated signaling, but the involved mechanisms are not fully understood. In this study we examined caspase-3 as a candidate for cleaving CD3zeta in SLE T cells. We demonstrate that SLE T cells display increased expression and activity of caspase-3. Treatment of SLE T cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-FMK reduced proteolysis of CD3zeta and enhanced its expression. In addition, Z-Asp-Glu-Val-Asp-FMK treatment increased the association of CD3zeta with lipid rafts and simultaneously reversed the abnormal lipid raft preclustering, heightened TCR-induced calcium responses, and reduced the expression of FcRgamma-chain exclusively in SLE T cells. We conclude that caspase-3 inhibitors can normalize SLE T cell function by limiting the excessive digestion of CD3zeta-chain and suggest that such molecules can be considered in the treatment of this disease. PMID:16116236

  6. The Effect of an Intelligent Tutoring System (ITS) on Student Achievement in Algebraic Expression

    ERIC Educational Resources Information Center

    Chien, Tsai Chen; Md. Yunus, Aida Suraya; Ali, Wan Zah Wan; Bakar, Ab. Rahim

    2008-01-01

    In this experimental study, use of Computer Assisted Instruction (CAI) followed by use of an Intelligent Tutoring System (CAI+ITS) was compared to the use of CAI (CAI only) in tutoring students on the topic of Algebraic Expression. Two groups of students participated in the study. One group of 32 students studied algebraic expression in a CAI…

  7. Comparative Analysis of the Magnitude, Quality, Phenotype and Protective Capacity of SIV Gag-Specific CD8+ T Cells Following Human-, Simian- and Chimpanzee-Derived Recombinant Adenoviral Vector Immunisation

    PubMed Central

    Quinn, Kylie M.; Costa, Andreia Da; Yamamoto, Ayako; Berry, Dana; Lindsay, Ross W.B.; Darrah, Patricia A.; Wang, Lingshu; Cheng, Cheng; Kong, Wing-Pui; Gall, Jason G.D.; Nicosia, Alfredo; Folgori, Antonella; Colloca, Stefano; Cortese, Riccardo; Gostick, Emma; Price, David A.; Gomez, Carmen E.; Esteban, Mariano; Wyatt, Linda S.; Moss, Bernard; Morgan, Cecilia; Roederer, Mario; Bailer, Robert T.; Nabel, Gary J.; Koup, Richard A.; Seder, Robert A.

    2013-01-01

    Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8+ T cell-mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. Here we show low seroreactivity in humans against simian- (sAd11, sAd16), or chimpanzee-derived (chAd3, chAd63) compared to human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype and protective capacity of CD8+ T cell responses in mice vaccinated with rAds encoding SIV Gag. Using a dose range (1 × 107 to 109 PU), we defined a hierarchy among rAd vectors based on the magnitude and protective capacity of CD8+ T cell responses, from most to least as: rAd5 and chAd3, rAd28 and sAd11, chAd63, sAd16, and rAd35. Selection of rAd vector or dose could modulate the proportion and/or frequency of IFNγ+TNFα+IL-2+ and KLRG1+CD127- CD8+ T cells, but strikingly ~30–80% of memory CD8+ T cells co-expressed CD127 and KLRG1. To further optimise CD8+ T cell responses, we assessed rAds as part of prime-boost regimens. Mice primed with rAds and boosted with NYVAC generated Gag-specific responses that approached ~60% of total CD8+ T cells at peak. Alternatively, priming with DNA or rAd28 and boosting with rAd5 or chAd3 induced robust and equivalent CD8+ T cell responses compared to prime or boost alone. Collectively, these data provide the immunologic basis for using specific rAd vectors alone or as part of prime-boost regimens to induce CD8+ T cells for rapid effector function or robust long-term memory, respectively. PMID:23390298

  8. Expression of Hepatoma-derived growth factor family members in the adult central nervous system

    PubMed Central

    El-Tahir, Heba M; Dietz, Frank; Dringen, Ralf; Schwabe, Kerstin; Strenge, Karen; Kelm, Sørge; Abouzied, Mekky M; Gieselmann, Volkmar; Franken, Sebastian

    2006-01-01

    Background Hepatoma-derived growth factor (HDGF) belongs to a polypeptide family containing five additional members called HDGF related proteins 1–4 (HRP-1 to -4) and Lens epithelial derived growth factor. Whereas some family members such as HDGF and HRP-2 are expressed in a wide range of tissues, the expression of others is very restricted. HRP-1 and -4 are only expressed in testis, HRP-3 only in the nervous system. Here we investigated the expression of HDGF, HRP-2 and HRP-3 in the central nervous system of adult mice on the cellular level by immunohistochemistry. In addition we performed Western blot analysis of various brain regions as well as neuronal and glial cell cultures. Results HDGF was rather evenly expressed throughout all brain regions tested with the lowest expression in the substantia nigra. HRP-2 was strongly expressed in the thalamus, prefrontal and parietal cortex, neurohypophysis, and the cerebellum, HRP-3 in the bulbus olfactorius, piriform cortex and amygdala complex. HDGF and HRP-2 were found to be expressed by neurons, astrocytes and oligodendrocytes. In contrast, strong expression of HRP-3 in the adult nervous system is restricted to neurons, except for very weak expression in oligodendrocytes in the brain stem. Although the majority of neurons are HRP-3 positive, some like cerebellar granule cells are negative. Conclusion The coexpression of HDGF and HRP-2 in glia and neurons as well as the coexpression of all three proteins in many neurons suggests different functions of members of the HDGF protein family in cells of the central nervous system that might include proliferation as well as cell survival. In addition the restricted expression of HRP-3 point to a special function of this family member for neuronal cells. PMID:16430771

  9. Construction of an effective protein expression system using the tpl promoter in Escherichia coli.

    PubMed

    Koyanagi, Takashi; Katayama, Takane; Hirao, Ai; Suzuki, Hideyuki; Kumagai, Hidehiko

    2005-09-01

    An effective protein expression system was constructed in Escherichia coli using the promoter of the tyrosine phenol-lyase (tpl) gene of Erwinia herbicola. This system involves a mutant form of the TyrR protein with an enhanced ability to activate tpl and the TutB protein with an ability to transport L-tyrosine (an inducer of Tpl). The highest expression level obtained for this system was more than twice that obtained for the tac system, although it was lower than the level obtained for the T7 system, as revealed with the lac-reporter assay and SDS-polyacrylamide gel electrophoresis. PMID:16215823

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

    PubMed Central

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

    2015-01-01

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

  11. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation.

    PubMed

    Binder, Dennis; Probst, Christopher; Grünberger, Alexander; Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  12. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation

    PubMed Central

    Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  13. An Efficient System for Heterologous Expression of Secondary Metabolite Genes in Aspergillus nidulans

    PubMed Central

    Chiang, Yi-Ming; Oakley, C. Elizabeth; Ahuja, Manmeet; Entwistle, Ruth; Schultz, Aric; Chang, Shu-Lin; Sung, Calvin T.; Wang, Clay C. C.; Oakley, Berl R.

    2013-01-01

    Fungal secondary metabolites (SMs) are an important source of medically valuable compounds. Genome projects have revealed that fungi have many SM biosynthetic gene clusters that are not normally expressed. To access these potentially valuable, cryptic clusters, we have developed a heterologous expression system in Aspergillus nidulans. We have developed an efficient system for amplifying genes from a target fungus, placing them under control of a regulatable promoter, transferring them into A. nidulans and expressing them. We have validated this system by expressing non-reducing polyketide synthases of Aspergillus terreus and additional genes required for compound production and release. We have obtained compound production and release from six of these NR-PKSs and have identified the products. To demonstrate that the procedure allows transfer and expression of entire secondary metabolite biosynthetic pathways, we have expressed all the genes of a silent A. terreus cluster and demonstrate that it produces asperfuranone. Further, by expressing the genes of this pathway in various combinations, we have clarified the asperfuranone biosynthetic pathway. We have also developed procedures for deleting entire A. nidulans SM clusters. This allows us to remove clusters that might interfere with analyses of heterologously expressed genes and to eliminate unwanted toxins. PMID:23621425

  14. Parents' Cultural Belief Systems: Their Origins, Expressions, and Consequences.

    ERIC Educational Resources Information Center

    Harkness, Sara, Ed.; Super, Charles M., Ed.

    This volume presents observations and thinking of scholars from a variety of disciplines about parental cultural belief systems. The chapters are concerned with the sources and consequences of parental ethnotheories in a number of societies. The following chapters are included: (1) "Introduction" (Sara Harkness and Charles M. Super); (2) "Parents'…

  15. The Body Action Coding System II: muscle activations during the perception and expression of emotion

    PubMed Central

    Huis In ‘t Veld, Elisabeth M. J.; van Boxtel, Geert J. M.; de Gelder, Beatrice

    2014-01-01

    Research into the expression and perception of emotions has mostly focused on facial expressions. Recently, body postures have become increasingly important in research, but knowledge on muscle activity during the perception or expression of emotion is lacking. The current study continues the development of a Body Action Coding System (BACS), which was initiated in a previous study, and described the involvement of muscles in the neck, shoulders and arms during expression of fear and anger. The current study expands the BACS by assessing the activity patterns of three additional muscles. Surface electromyography of muscles in the neck (upper trapezius descendens), forearms (extensor carpi ulnaris), lower back (erector spinae longissimus) and calves (peroneus longus) were measured during active expression and passive viewing of fearful and angry body expressions. The muscles in the forearm were strongly active for anger expression and to a lesser extent for fear expression. In contrast, muscles in the calves were recruited slightly more for fearful expressions. It was also found that muscles automatically responded to the perception of emotion, without any overt movement. The observer's forearms responded to the perception of fear, while the muscles used for leaning backwards were activated when faced with an angry adversary. Lastly, the calf responded immediately when a fearful person was seen, but responded slower to anger. There is increasing interest in developing systems that are able to create or recognize emotional body language for the development of avatars, robots, and online environments. To that end, multiple coding systems have been developed that can either interpret or create bodily expressions based on static postures, motion capture data or videos. However, the BACS is the first coding system based on muscle activity. PMID:25294993

  16. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae

    PubMed Central

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

  17. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae.

    PubMed

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja; Jäntti, Jussi; Mojzita, Dominik

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

  18. NLRP3 inflammasome expression is driven by NF-κB in cultured hepatocytes.

    PubMed

    Boaru, Sorina Georgiana; Borkham-Kamphorst, Erawan; Van de Leur, Eddy; Lehnen, Eric; Liedtke, Christian; Weiskirchen, Ralf

    2015-03-13

    The inflammasomes are cytoplasmic multiprotein complexes that are responsible for activation of inflammatory reactions. In principle, there are four individual inflammasome branches (NLRP1, NLRP3, NLRC4/NALP4, and AIM2) that mediate the cleavage and activation of Caspase-1 and IL-1β that in turn lead to a complex network of cellular reactions initiating local and systemic inflammatory reactions. We have recently shown that NLRP3 expression is virtually absent in primary cultured hepatocytes and that in vitro the stimulation of hepatocytes with lipopolysaccharides results in strong activation of NLRP3 expression. We here demonstrate that this activation can be blocked by the NF-κB activation inhibitor QNZ or by infection with an adenoviral expression vector constitutively expressing a superrepressor of NF-κB. We show that QNZ blocks NF-κB-dependent expression of TNF-α, IL-1β and NLRP3. Likewise, the superrepressor of NF-κB prevents expression of NLRP3 and significantly reduces expression of inflammatory marker genes in liver cells. In a primary murine hepatoma cells, the concomitant depletion of NEMO and Caspase-8 resulted in a significant suppression of NLRP3 expression after Lipopolysaccharide challenge. Moreover, we demonstrate that a 1.3-kbp fragment located in close proximity of the most upstream transcriptional start site of the human NLRP3 gene that harbours one putative octamer NF-κB binding site renders LPS sensitivity in reporter gene assay. We conclude that NF-κB signalling is a necessary prerequisite for proper activation of the NLRP3 inflammasome in primary hepatocytes. PMID:25686493

  19. A Bacillus subtilis dipeptide transport system expressed early during sporulation.

    PubMed

    Mathiopoulos, C; Mueller, J P; Slack, F J; Murphy, C G; Patankar, S; Bukusoglu, G; Sonenshein, A L

    1991-08-01

    Two previously identified Bacillus subtilis DNA segments, dciA and dciB, whose transcripts accumulate very rapidly after induction of sporulation, were found in the same 6.2 kb transcription unit, now known as the dciA operon. Analysis of the sequence of the dciA operon showed that its putative products are homologous to bacterial peptide transport systems. The product of the fifth gene, DciAE, is similar to peptide-binding proteins from Escherichia coli and Salmonella typhimurium (DppA and OppA) and B. subtilis (OppA). A null mutation in dciAE abolished the ability of a proline auxotroph to grow in a medium containing the dipeptide Pro-Gly as sole proline source, suggesting that the dciA operon encodes a dipeptide transport system. PMID:1766370

  20. SimCheck: An Expressive Type System for Simulink

    NASA Technical Reports Server (NTRS)

    Roy, Pritam; Shankar, Natarajan

    2010-01-01

    MATLAB Simulink is a member of a class of visual languages that are used for modeling and simulating physical and cyber-physical systems. A Simulink model consists of blocks with input and output ports connected using links that carry signals. We extend the type system of Simulink with annotations and dimensions/units associated with ports and links. These types can capture invariants on signals as well as relations between signals. We define a type-checker that checks the wellformedness of Simulink blocks with respect to these type annotations. The type checker generates proof obligations that are solved by SRI's Yices solver for satisfiability modulo theories (SMT). This translation can be used to detect type errors, demonstrate counterexamples, generate test cases, or prove the absence of type errors. Our work is an initial step toward the symbolic analysis of MATLAB Simulink models.

  1. Analytical expressions for the nonlinear interference in dispersion managed transmission coherent optical systems

    NASA Astrophysics Data System (ADS)

    Qiao, Yaojun; Li, Ming; Yang, Qiuhong; Xu, Yanfei; Ji, Yuefeng

    2015-01-01

    Closed-form expressions of nonlinear interference of dense wavelength-division-multiplexed (WDM) systems with dispersion managed transmission (DMT) are derived. We carry out a simulative validation by addressing an ample and significant set of the Nyquist-WDM systems based on polarization multiplexed quadrature phase-shift keying (PM-QPSK) subcarriers at a baud rate of 32 Gbaud per channel. Simulation results show the simple closed-form analytical expressions can provide an effective tool for the quick and accurate prediction of system performance in DMT coherent optical systems.

  2. A self-inducible heterologous protein expression system in Escherichia coli

    PubMed Central

    Briand, L.; Marcion, G.; Kriznik, A.; Heydel, J. M.; Artur, Y.; Garrido, C.; Seigneuric, R.; Neiers, F.

    2016-01-01

    Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter’s transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale. PMID:27611846

  3. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

    PubMed Central

    Terrón-González, L.; Medina, C.; Limón-Mortés, M. C.; Santero, E.

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance. PMID:23346364

  4. The activity of the TRP-like channel depends on its expression system

    PubMed Central

    Lev, Shaya; Katz, Ben; Minke, Baruch

    2012-01-01

    The Drosophila light activated TRP and TRPL channels have been a model for TRPC channel gating. Several gating mechanisms have been proposed following experiments conducted on photoreceptor and tissue cultured cells. However, conclusive evidence for any mechanism is still lacking. Here, we show that the Drosophila TRPL channel expressed in tissue cultured cells is constitutively active in S2 cells but is silent in HEK cells. Modulations of TRPL channel activity in different expression system by pharmacology or specific enzymes, which change the lipid content of the plasma membrane, resulted in conflicting effects. These findings demonstrate the difficulty in elucidating TRPC gating, as channel behavior is expression system dependent. However, clues on the gating mechanism may arise from understanding how different expression systems affect TRPC channel activation. PMID:22627924

  5. A self-inducible heterologous protein expression system in Escherichia coli.

    PubMed

    Briand, L; Marcion, G; Kriznik, A; Heydel, J M; Artur, Y; Garrido, C; Seigneuric, R; Neiers, F

    2016-01-01

    Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter's transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale. PMID:27611846

  6. Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

    PubMed Central

    Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

    2013-01-01

    Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

  7. Systemic Sclerosis Patients Present Alterations in the Expression of Molecules Involved in B-Cell Regulation

    PubMed Central

    Soto, Lilian; Ferrier, Ashley; Aravena, Octavio; Fonseca, Elianet; Berendsen, Jorge; Biere, Andrea; Bueno, Daniel; Ramos, Verónica; Aguillón, Juan Carlos; Catalán, Diego

    2015-01-01

    The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naive, and memory B-cell subpopulations from systemic sclerosis patients. To achieve this, blood samples were drawn from 31 systemic sclerosis patients and 53 healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcγRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naive B cells related to memory B cells compared with healthy controls. Transitional and naive B cells from patients express higher levels of CD86 and FcγRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, whereas memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate with different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B-cell regulation. These abnormalities may be determinant in the B-cell hyperactivation observed in systemic sclerosis. PMID:26483788

  8. Regulated Expression Systems for Mycobacteria and Their Applications

    PubMed Central

    Schnappinger, Dirk; Ehrt, Sabine

    2014-01-01

    For bacterial model organisms like Escherichia coli and Bacillus subtilis genetic tools to experimentally manipulate the activity of individual genes existed for decades. But for genetically less tractable yet medically important bacteria such as M. tuberculosis such tools have rarely been available. More recently several groups developed genetic switches that function efficiently in M. tuberculosis and other mycobacteria. Together these systems utilize six different transcription factors, eight different regulated promoters, and three different regulatory principles. Here we describe their design features, review their main applications, and discuss advantages and disadvantages of regulating transcription, translation, or protein stability for controlling gene activities in bacteria. PMID:25485177

  9. WeGET: predicting new genes for molecular systems by weighted co-expression.

    PubMed

    Szklarczyk, Radek; Megchelenbrink, Wout; Cizek, Pavel; Ledent, Marie; Velemans, Gonny; Szklarczyk, Damian; Huynen, Martijn A

    2016-01-01

    We have developed the Weighted Gene Expression Tool and database (WeGET, http://weget.cmbi.umcn.nl) for the prediction of new genes of a molecular system by correlated gene expression. WeGET utilizes a compendium of 465 human and 560 murine gene expression datasets that have been collected from multiple tissues under a wide range of experimental conditions. It exploits this abundance of expression data by assigning a high weight to datasets in which the known genes of a molecular system are harmoniously up- and down-regulated. WeGET ranks new candidate genes by calculating their weighted co-expression with that system. A weighted rank is calculated for human genes and their mouse orthologs. Then, an integrated gene rank and p-value is computed using a rank-order statistic. We applied our method to predict novel genes that have a high degree of co-expression with Gene Ontology terms and pathways from KEGG and Reactome. For each query set we provide a list of predicted novel genes, computed weights for transcription datasets used and cell and tissue types that contributed to the final predictions. The performance for each query set is assessed by 10-fold cross-validation. Finally, users can use the WeGET to predict novel genes that co-express with a custom query set. PMID:26582928

  10. WeGET: predicting new genes for molecular systems by weighted co-expression

    PubMed Central

    Szklarczyk, Radek; Megchelenbrink, Wout; Cizek, Pavel; Ledent, Marie; Velemans, Gonny; Szklarczyk, Damian; Huynen, Martijn A.

    2016-01-01

    We have developed the Weighted Gene Expression Tool and database (WeGET, http://weget.cmbi.umcn.nl) for the prediction of new genes of a molecular system by correlated gene expression. WeGET utilizes a compendium of 465 human and 560 murine gene expression datasets that have been collected from multiple tissues under a wide range of experimental conditions. It exploits this abundance of expression data by assigning a high weight to datasets in which the known genes of a molecular system are harmoniously up- and down-regulated. WeGET ranks new candidate genes by calculating their weighted co-expression with that system. A weighted rank is calculated for human genes and their mouse orthologs. Then, an integrated gene rank and p-value is computed using a rank-order statistic. We applied our method to predict novel genes that have a high degree of co-expression with Gene Ontology terms and pathways from KEGG and Reactome. For each query set we provide a list of predicted novel genes, computed weights for transcription datasets used and cell and tissue types that contributed to the final predictions. The performance for each query set is assessed by 10-fold cross-validation. Finally, users can use the WeGET to predict novel genes that co-express with a custom query set. PMID:26582928

  11. Systematic expression analysis of Hox genes at adulthood reveals novel patterns in the central nervous system.

    PubMed

    Hutlet, Bertrand; Theys, Nicolas; Coste, Cécile; Ahn, Marie-Thérèse; Doshishti-Agolli, Konstantin; Lizen, Benoît; Gofflot, Françoise

    2016-04-01

    Hox proteins are key regulators of animal development, providing positional identity and patterning information to cells along the rostrocaudal axis of the embryo. Although their embryonic expression and function are well characterized, their presence and biological importance in adulthood remains poorly investigated. We provide here the first detailed quantitative and neuroanatomical characterization of the expression of the 39 Hox genes in the adult mouse brain. Using RT-qPCR we determined the expression of 24 Hox genes mainly in the brainstem of the adult brain, with low expression of a few genes in the cerebellum and the forebrain. Using in situ hybridization (ISH) we have demonstrated that expression of Hox genes is maintained in territories derived from the early segmental Hox expression domains in the hindbrain. Indeed, we show that expression of genes belonging to paralogy groups PG2-8 is maintained in the hindbrain derivatives at adulthood. The spatial colinearity, which characterizes the early embryonic expression of Hox genes, is still observed in sequential antero-posterior boundaries of expression. Moreover, the main mossy and climbing fibres precerebellar nuclei express PG2-8 Hox genes according to their migration origins. Second, ISH confirms the presence of Hox gene transcripts in territories where they are not detected during development, suggesting neo-expression in these territories in adulthood. Within the forebrain, we have mapped Hoxb1, Hoxb3, Hoxb4, Hoxd3 and Hoxa5 expression in restricted areas of the sensory cerebral cortices as well as in specific thalamic relay nuclei. Our data thus suggest a requirement of Hox genes beyond their role of patterning genes, providing a new dimension to their functional relevance in the central nervous system. PMID:25527350

  12. Adenoviral Vector Vaccination Induces a Conserved Program of CD8+ T Cell Memory Differentiation in Mouse and Man

    PubMed Central

    Bolinger, Beatrice; Sims, Stuart; Swadling, Leo; O’Hara, Geraldine; de Lara, Catherine; Baban, Dilair; Saghal, Natasha; Lee, Lian Ni; Marchi, Emanuele; Davis, Mark; Newell, Evan; Capone, Stefania; Folgori, Antonella; Barnes, Ellie; Klenerman, Paul

    2015-01-01

    Summary Following exposure to vaccines, antigen-specific CD8+ T cell responses develop as long-term memory pools. Vaccine strategies based on adenoviral vectors, e.g., those developed for HCV, are able to induce and sustain substantial CD8+ T cell populations. How such populations evolve following vaccination remains to be defined at a transcriptional level. We addressed the transcriptional regulation of divergent CD8+ T cell memory pools induced by an adenovector encoding a model antigen (beta-galactosidase). We observe transcriptional profiles that mimic those following infection with persistent pathogens, murine and human cytomegalovirus (CMV). Key transcriptional hallmarks include upregulation of homing receptors and anti-apoptotic pathways, driven by conserved networks of transcription factors, including T-bet. In humans, an adenovirus vaccine induced similar CMV-like phenotypes and transcription factor regulation. These data clarify the core features of CD8+ T cell memory following vaccination with adenovectors and indicate a conserved pathway for memory development shared with persistent herpesviruses. PMID:26586434

  13. Adenoviral p53 gene transfer and gemcitabine in three patients with liver metastases due to advanced pancreatic carcinoma

    PubMed Central

    Thiede, Christian; Fischer, Rainer; Ehninger, Gerhard; Haag, Cornelie

    2007-01-01

    Background. Current therapies for adenocarcinoma of the pancreas do not improve the life expectancy of patients. Methods. In a non-randomized pilot trail we tested whether a local therapy based upon an adenoviral gene transfer of wild type p53 in combination with gemcitabine administration would be safe in patients with liver metastases due to pancreatic carcinoma. We report on the clinical course of three patients with respect to safety, tolerability and tumor response. Results. Transient grade III toxicities occurred with fever, leucopenia, elevation of AP, ALT, AST, GGT, while grade IV toxicity occurred for bilirubin only. Laboratory tests suggested disseminated intravascular coagulation in all three patients, but fine needle biopsies of liver did not show any histological evidence of thrombus or clot formation. Progression of liver metastases was documented in one and stable disease in another patient two months after treatment. However, a major improvement with regression of the indexed lesion by 80% occurred in a third patient after a single administration of 7.5×1012 viral particles, and time to progression was extended to six months. Conclusion. The combination therapy of viral gene transfer and chemotherapy temporarily controls and diminishes tumor burden. Improvement of the toxicity profile is necessary. Further trials are warranted to improve treatment and life expectancy of patients suffering from fatal diseases such as pancreatic carcinoma. PMID:18333108

  14. Emphysematous lung destruction by cigarette smoke. The effects of latent adenoviral infection on the lung inflammatory response.

    PubMed

    Meshi, Bernard; Vitalis, Timothy Z; Ionescu, Diana; Elliott, W Mark; Liu, Chun; Wang, Xiang-Dong; Hayashi, Shizu; Hogg, James C

    2002-01-01

    This study was designed to test the hypothesis that cigarette smoke-induced inflammation and emphysema are amplified by the presence of latent adenoviral (Ad) infection, and to determine whether this emphysematous process can be reversed by all-trans-retinoic acid (RA) treatment. The results confirm that in guinea pigs, chronic cigarette-smoke exposure caused lesions similar to human centrilobular emphysema. They also show that latent Ad infection combined with cigarette-smoke exposure caused an excess increase in lung volume (P < 0.001), air-space volume (P < 0.001), and lung weight (P < 0.01), and further decrease in surface-to-volume ratio (P < 0.001) compared with smoke exposure alone. RA treatment failed to reverse these emphysematous changes. Analysis of inflammatory response in parenchymal and airway tissue showed that smoking caused an increase of polymorphonuclear leukocytes (PMNs) (P < 0.0002), macrophages (P < 0.001), and CD4 cells (P < 0.0009), and that latent Ad infection independently increased PMNs (P < 0.001), macrophages (P = 0.003), and CD8 cells (P < 0.001). We conclude that latent Ad infection amplifies the emphysematous lung destruction and increases the inflammatory response produced by cigarette-smoke exposure. In this study, the increase in CD4 was associated with cigarette smoke and the increase in CD8 cells with latent Ad infection. PMID:11751203

  15. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients.

    PubMed

    Dasari, Vijayendra; Schuessler, Andrea; Smith, Corey; Wong, Yide; Miles, John J; Smyth, Mark J; Ambalathingal, George; Francis, Ross; Campbell, Scott; Chambers, Daniel; Khanna, Rajiv

    2016-01-01

    Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as "off-the-shelf" therapeutics as well as autologous T-cell therapies for transplant patients. PMID:27606351

  16. Prolonged mechanical support in children with severe adenoviral infections: a case series and review of the literature.

    PubMed

    Gupta, Punkaj; Tobias, Joseph D; Goyal, Sunali; Hervie, Peter; Harris, Jason B; Sadot, Efraim; Noviski, Natan

    2011-01-01

    Adenovirus infections occur primarily in infants and children less than 5 years of age, accounting for 2% to 5% of respiratory illnesses in the pediatric population and 4% to 10% of childhood pneumonias. Although the majority of children with adenovirus disease develop mild upper respiratory tract disease, more severe disease may occur with involvement of the lower respiratory tract characterized by pneumonitis and/or small airways disease. The authors present a case series of 3 high-risk children with severe lower respiratory tract adenoviral infections. These cases demonstrate the potential for the development of severe respiratory involvement from adenovirus in infants and children with comorbid conditions and illustrate that there may be a rapid progression of the disease as well as the need, in selected circumstances, for prolonged mechanical support. We review the role of adenovirus in lower respiratory tract infections in infants and children, its potential to result in life-threatening complications in pediatric patients with comorbid conditions, and the potential life-saving role of mechanical ventilation and extracorporeal life support (ECLS) in these children. PMID:21320864

  17. Short-term Correction of Arginase Deficiency in a Neonatal Murine Model With a Helper-dependent Adenoviral Vector

    PubMed Central

    Gau, Chia-Ling; Rosenblatt, Robin A; Cerullo, Vincenzo; Lay, Fides D; Dow, Adrienne C; Livesay, Justin; Brunetti-Pierri, Nicola; Lee, Brendan; Cederbaum, Stephen D; Grody, Wayne W; Lipshutz, Gerald S

    2009-01-01

    Neonatal gene therapy has the potential to ameliorate abnormalities before disease onset. Our gene knockout of arginase I (AI) deficiency is characterized by increasing hyperammonemia, neurological deterioration, and early death. We constructed a helper-dependent adenoviral vector (HDV) carrying AI and examined for correction of this defect. Neonates were administered 5 × 109 viral particles/g and analyzed for survival, arginase activity, and ammonia and amino acids levels. The life expectancy of arg−/− mice increased to 27 days while controls died at 14 days with hyperammonemia and in extremis. Death correlated with a decrease in viral DNA/RNA per cell as liver mass increased. Arginase assays demonstrated that vector-injected hepatocytes had ~20% activity of heterozygotes at 2 weeks of age. Hepatic arginine and ornithine in treated mice were similar to those of saline-injected heterozygotes at 2 weeks, whereas ammonia was normal. By 26 days, arginase activity in the treated arg−/− livers declined to <10%, and arginine and ornithine increased. Ammonia levels began increasing by day 25, suggesting the cause of death to be similar to that of uninjected arg−/− mice, albeit at a later time. These studies demonstrate that the AI deficient newborn mouse can be temporarily corrected and rescued using a HDV. PMID:19367256

  18. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients

    PubMed Central

    Dasari, Vijayendra; Schuessler, Andrea; Smith, Corey; Wong, Yide; Miles, John J; Smyth, Mark J; Ambalathingal, George; Francis, Ross; Campbell, Scott; Chambers, Daniel; Khanna, Rajiv

    2016-01-01

    Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as “off-the-shelf” therapeutics as well as autologous T-cell therapies for transplant patients. PMID:27606351

  19. Evaluation of excipients for enhanced thermal stabilization of a human type 5 adenoviral vector through spray drying.

    PubMed

    LeClair, Daniel A; Cranston, Emily D; Xing, Zhou; Thompson, Michael R

    2016-06-15

    We have produced a thermally stable recombinant human type 5 adenoviral vector (AdHu5) through spray drying with three excipient formulations (l-leucine, lactose/trehalose and mannitol/dextran). Spray drying leads to immobilization of the viral vector which is believed to prevent viral protein unfolding, aggregation and inactivation. The spray dried powders were characterized by scanning electron microscopy, differential scanning calorimetry, Karl Fischer titrations, and X-ray diffraction to identify the effects of temperature and atmospheric moisture on the immobilizing matrix. Thermal stability of the viral vector was confirmed in vitro by infection of A549 lung epithelial cells. Mannitol/dextran powders showed the greatest improvement in thermal stability with almost no viral activity loss after storage at 20°C for 90days (0.7±0.3 log TCID50) which is a significant improvement over the current -80°C storage protocol. Furthermore, viral activity was retained over short term exposure (72h) to temperatures as high as 55°C. Conversely, all powders exhibited activity loss when subjected to moisture due to amplified molecular motion of the matrix. Overall, a straightforward method ideal for the production of thermally stable vaccines has been demonstrated through spray drying AdHu5 with a blend of mannitol and dextran and storing the powder under low humidity conditions. PMID:27130366

  20. A novel baculovirus-derived promoter with high activity in the baculovirus expression system.

    PubMed

    Martínez-Solís, María; Gómez-Sebastián, Silvia; Escribano, José M; Jakubowska, Agata K; Herrero, Salvador

    2016-01-01

    The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS. PMID:27375973

  1. A novel baculovirus-derived promoter with high activity in the baculovirus expression system

    PubMed Central

    Martínez-Solís, María; Gómez-Sebastián, Silvia; Escribano, José M.; Jakubowska, Agata K.

    2016-01-01

    The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS. PMID:27375973

  2. Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

    PubMed

    Liu, Li; Spurrier, Joshua; Butt, Tauseef R; Strickler, James E

    2008-11-01

    Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function. PMID:18713650

  3. A Coursewriter II Function (FCALC) For the Manipulation of Numerical and Algebraic Expressions. Systems Memo Number One.

    ERIC Educational Resources Information Center

    Smith, Authella; And Others

    Documentation of the Coursewriter II Function FCALC is provided. The function is designed for use on the IBM 1500 instructional system and has three major applications: 1) comparison of a numeric expression in buffer 5 with a numeric expression in buffer 0; 2) comparison of an algebraic expression in buffer 5 with an algebraic expression in buffer…

  4. Transgenic expression of interleukin 6 in the central nervous system regulates brain metallothionein-I and -III expression in mice.

    PubMed

    Hernández, J; Molinero, A; Campbell, I L; Hidalgo, J

    1997-08-01

    The metallothionein (MT) gene family consists of several members (MT-I-IV) that are tightly regulated during development. MT-I and MT-II are expressed in many tissues, including the brain, whereas MT-III is expressed mainly in the central nervous system. However, the physiological roles of these isoforms in the brain and their regulation are poorly characterized. In this report, we have studied the putative role of IL-6 in the regulation of brain MT. The present results demonstrated that transgenic mice expressing IL-6 under the regulatory control of the glial fibrillary acidic protein gene promoter (GFAP-IL6 mice), and which develop chronic progressive neurodegenerative disease, show significantly increased MT-I + II protein levels in specific brain areas. Thus, the MT-I + II levels of 1- and 3-month-old GFAP-IL6 mice (G16 and/or G36 lines) were not altered in hippocampus but they were elevated in the cerebellum (highest induction), medulla plus pons, hypothalamus and remaining brain (lowest induction). The effect of the transgenic expression of IL-6 was more dramatic for MT-I + II protein than for MT-I mRNA levels, with the latter only marginally elevated in the G16 line at 3 months but not at 6 months of age where there was a tendency to decreased levels. Brain MT-I mRNA levels also tended to decrease in the higher expressor G36 line in 3-month-old mice despite the strongly elevated MT-I + II protein levels at this age. Therefore, in addition to increasing MT gene transcription, these results suggest a post-transcriptional effect of IL-6 or of a IL-6-dependent factor, in this chronic situation. The up-regulated brain MT-I + II protein levels in the GFAP-IL6 mice was comparable to the expression of the acute-phase response gene EB22/5, suggesting that these MT isoforms could be considered acute-phase response proteins in the brain. Brain MT-III mRNA levels followed a somewhat similar pattern that those of MT-I mRNA but the decreasing effect of IL-6 transgene

  5. Adenoviral transduction of naive CD4 T cells to study Treg differentiation.

    PubMed

    Warth, Sebastian C; Heissmeyer, Vigo

    2013-01-01

    Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGFβ and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown. Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44(low), CD62L(high)) and resting (CD25(-), CD69(-)) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation. PMID:23979424

  6. Construction of a tunable multi-enzyme-coordinate expression system for biosynthesis of chiral drug intermediates

    PubMed Central

    Jiang, Wei; Fang, Baishan

    2016-01-01

    Systems that can regulate and coordinate the expression of multiple enzymes for metabolic regulation and synthesis of important drug intermediates are poorly explored. In this work, a strategy for constructing a tunable multi-enzyme-coordinate expression system for biosynthesis of chiral drug intermediates was developed and evaluated by connecting protein-protein expressions, regulating the strength of ribosome binding sites (RBS) and detecting the system capacity for producing chiral amino acid. Results demonstrated that the dual-enzyme system had good enantioselectivity, low cost, high stability, high conversion rate and approximately 100% substrate conversion. This study has paved a new way of exploring metabolic mechanism of functional genes and engineering whole cell-catalysts for synthesis of chiral α-hydroxy acids or chiral amino acids. PMID:27456301

  7. Expression and functional characterization of intrafollicular GH-IGF system in the zebrafish ovary.

    PubMed

    Zhou, Rui; Yu, Susana Man Ying; Ge, Wei

    2016-06-01

    The somatotrophic axis plays important roles in influencing reproduction. All key members of this axis including growth hormone (GH, gh), GH receptors (ghra and ghrb), insulin-like growth factors (IGFs, igf1, igf2 and igf3) and IGF receptors (igf1ra and igf1rb) were detected in the zebrafish ovary. GH was exclusively expressed in the full-grown oocytes, while its receptors were detectable in both the follicle cells and oocytes. The IGFs and their receptors were all expressed in both compartments except igf3, which was expressed in the follicle cells only. During folliculogenesis, there was a sharp decrease of gh expression at follicle activation; however, the expression of its receptors increased significantly. The expression profiles of igf1, igf2a, and igf2b were similar to that of fshr, whereas igf3 expression was close to lhcgr, suggesting differential roles for different forms of IGFs in follicle development. To examine if the ovarian GH-IGF system is regulated by gonadotropins (e.g., hCG) and GH, we performed in vitro experiments using cultured zebrafish follicle cells. The expression of igf1 and igf1ra, but not others, was down-regulated by hCG (LH analog), whereas recombinant zebrafish GH stimulated igf1 expression. In addition, GH also increased the expression of activin βA subunit (inhbaa). In agreement with this, the stimulatory effect of GH but not IGF-I on oocyte maturation could be abolished by follistatin. In conclusion, the present study revealed an intrafollicular network involving GH-IGF mini-axis in the zebrafish ovary; however, it might not work in the same way as that of the systemic somatotrophic axis. PMID:26654745

  8. THE CRE-LOXP RECOMBINATION-BASED REPORTER SYSTEM FOR PLANT TRANSCRIPTIONAL EXPRESSION STUDIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To facilitate the characterization of plant genes, the Cre-loxP site-specific recombination system was adapted to make reporter vectors for plant expression studies. This system allows promoter fragments to be cloned into a small vector (univector) and subsequently recombined in vitro with binary ve...

  9. Understanding the Earth Systems: Expressions of Dynamic and Cyclic Thinking among University Students

    ERIC Educational Resources Information Center

    Batzri, Or; Ben Zvi Assaraf, Orit; Cohen, Carmit; Orion, Nir

    2015-01-01

    In this two-part study, we examine undergraduate university students' expression of two important system thinking characteristics--dynamic thinking and cyclic thinking--focusing particularly on students of geology. The study was conducted using an Earth systems questionnaire designed to elicit and reflect either dynamic or cyclic thinking. The…

  10. A New Motivation for Learning: The Graphics Expression System. Manual for Administrators.

    ERIC Educational Resources Information Center

    New York State Education Dept., Albany. Div. of Educational Communications.

    This manual describes how the Uniondale, New York, school district uses a technological system, the Graphics Expression System (GES), to produce dramatic reading improvement on the part of elementary school underachievers. It shows how GES, based upon the assumption that all youngsters want to communicate, employs a television mini-studio and an…

  11. Viral transduction of primary Schwann cells using a Cre-lox system to regulate GDNF expression.

    PubMed

    Wu-Fienberg, Yuewei; Moore, Amy M; Marquardt, Laura M; Newton, Piyaraj; Johnson, Philip J; Mackinnon, Susan E; Sakiyama-Elbert, Shelly E; Wood, Matthew D

    2014-09-01

    Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to enhance motor nerve regeneration following its delivery. However, recent studies have determined that extended GDNF delivery to regenerating axons can entrap motor axons at the site of GDNF delivery. This entrapment leads to reduced motor axons available to reinnervate muscle. To address this issue, we designed a cell-based GDNF expression system that can temporally regulate protein expression using an inducible gene excision mechanism to prevent entrapment at the site of expression. To design this system for regulation of GDNF expression, we transduced two lentiviral vectors, one containing a constitutively active GDNF transgene flanked by two loxP sites, and the other containing a tetracycline-inducible cre transgene along with its constitutively active transactivator, into Schwann cells (SCs). These SCs over-express GDNF, but expression can be suppressed through the administration of tetracycline family antibiotics, such as doxycycline. The engineered SCs produced significantly more GDNF as compared to untransduced controls, as measured by enzyme-linked immunosorbent assay (ELISA). Following doxycycline treatment, these SCs produced significantly lower levels of GDNF and induced less neurite extension as compared to untreated SCs. Engineered SCs treated with doxycycline showed a marked increase in Cre recombinase expression, as visualized by immunohistochemistry (IHC), providing evidence of a mechanism for the observed changes in GDNF expression levels and biological activity. This cell-based GDNF expression system could have potential for future in vivo studies to provide a temporally controlled GDNF source to promote axon growth. PMID:24728940

  12. Viral transduction of primary Schwann cells using a Cre-lox system to regulate GDNF expression

    PubMed Central

    Wu-Fienberg, Yuewei; Moore, Amy M.; Marquardt, Laura M.; Newton, Piyaraj; Johnson, Philip J.; Mackinnon, Susan E.; Sakiyama-Elbert, Shelly E.; Wood, Matthew D.

    2014-01-01

    Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to enhance motor nerve regeneration following its delivery. However, recent studies have determined that extended GDNF delivery to regenerating axons can entrap motor axons at the site of GDNF delivery. This entrapment leads to reduced motor axons available to reinnervate muscle. To address this issue, we designed a cell-based GDNF expression system that can temporally regulate protein expression using an inducible gene excision mechanism to prevent entrapment at the site of expression. To design this system for regulation of GDNF expression, we transduced two lentiviral vectors, one containing a constitutively active GDNF transgene flanked by two loxP sites, and the other containing a tetracycline-inducible cre transgene along with its constitutively active transactivator, into Schwann cells (SCs). These SCs over-express GDNF, but expression can be suppressed through the administration of tetracycline family antibiotics, such as doxycycline. The engineered SCs produced significantly more GDNF as compared to untransduced controls, as measured by enzyme-linked immunosorbent assay (ELISA). Following doxycycline treatment, these SCs produced significantly lower levels of GDNF and induced less neurite extension as compared to untreated SCs. Engineered SCs treated with doxycycline showed a marked increase in Cre recombinase expression, as visualized by immunohistochemistry (IHC), providing evidence of a mechanism for the observed changes in GDNF expression levels and biological activity. This cell-based GDNF expression system could have potential for future in vivo studies to provide a temporally-controlled GDNF source to promote axon growth. PMID:24728940

  13. Expression of K2P channels in sensory and motor neurons of the autonomic nervous system.

    PubMed

    Cadaveira-Mosquera, Alba; Pérez, Montse; Reboreda, Antonio; Rivas-Ramírez, Paula; Fernández-Fernández, Diego; Lamas, J Antonio

    2012-09-01

    Several types of neurons within the central and peripheral somatic nervous system express two-pore-domain potassium (K2P) channels, providing them with resting potassium conductances. We demonstrate that these channels are also expressed in the autonomic nervous system where they might be important modulators of neuronal excitability. We observed strong mRNA expression of members of the TRESK and TREK subfamilies in both the mouse superior cervical ganglion (mSCG) and the mouse nodose ganglion (mNG). Motor mSCG neurons strongly expressed mRNA transcripts for TRESK and TREK-2 subunits, whereas TASK-1 and TASK-2 subunits were only moderately expressed, with only few or very few transcripts for TREK-1 and TRAAK (TRESK ≈ TREK-2 > TASK-2 ≈ TASK-1 > TREK-1 > TRAAK). Similarly, the TRESK and TREK-1 subunits were the most strongly expressed in sensorial mNG neurons, while TASK-1 and TASK-2 mRNAs were moderately expressed, and fewer TREK-2 and TRAAK transcripts were detected (TRESK ≈ TREK-1 > TASK-1 ≈ TASK-2 > TREK-2 > TRAAK). Moreover, cell-attached single-channel recordings showed a major contribution of TRESK and TREK-1 channels in mNG. As the level of TRESK mRNA expression was not statistically different between the ganglia analysed, the distinct expression of TREK-1 and TREK-2 subunits was the main difference observed between these structures. Our results strongly suggest that TRESK and TREK channels are important modulators of the sensorial and motor information flowing through the autonomic nervous system, probably exerting a strong influence on vagal reflexes. PMID:22544515

  14. Development of an enhanced chromosomal expression system based on porin synthesis operon for halophile Halomonas sp.

    PubMed

    Yin, Jin; Fu, Xiao-Zhi; Wu, Qiong; Chen, Jin-Chun; Chen, Guo-Qiang

    2014-11-01

    Since halophile Halomonas spp. can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low-cost chemicals in an economic way. Yet, metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was proven functional by some examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaC Re from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaC Re was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, an inducible expression system was constructed in phaC-deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp. was established. PMID:25070598

  15. Coordinated regulation of natural killer receptor expression in the maturing human immune system

    PubMed Central

    Strauss-Albee, Dara M.; Horowitz, Amir; Parham, Peter; Blish, Catherine A.

    2014-01-01

    Natural killer (NK) cells are responsible for recognizing and killing transformed, stressed, and infected cells. They recognize a set of non-antigen-specific features termed “altered self” through combinatorial signals from activating and inhibitory receptors. These natural killer cell receptors (NKR) are also expressed on CD4+ and CD8+ T cells, B cells, and monocytes, though a comprehensive inventory of NKR expression patterns across leukocyte lineages has never been performed. Using mass cytometry, we found that NKR expression patterns distinguish cell lineages in human peripheral blood. In individuals with high levels of CD57, indicative of a mature immune repertoire, NKR are more likely to be expressed on non-NK cells, especially CD8+ T cells. Mature NK and CD8+ T cell populations show increased diversity of NKR surface expression patterns, but with distinct determinants: mature NK cells acquire primarily inhibitory receptors, while CD8+ T cells attain a specific subset of both activating and inhibitory receptors, potentially imbuing them with a distinct functional role. Concurrently, monocytes show decreased expression of the generalized inhibitory receptor LILRB1, consistent with an increased activation threshold. Therefore, NKR expression is coordinately regulated as the immune system matures, resulting in the transfer of “altered self” recognition potential among leukocyte lineages. This likely reduces antigen specificity in the mature human immune system, and implies that vaccines and therapeutics that engage both its innate and adaptive branches may be more effective in the settings of aging and chronic infection. PMID:25288567

  16. A model system for the study of gene expression in the undergraduate laboratory.

    PubMed

    Hargadon, Kristian M

    2016-07-01

    The flow of genetic information from DNA to RNA to protein, otherwise known as the "central dogma" of biology, is one of the most basic and overarching concepts in the biological sciences. Nevertheless, numerous studies have reported student misconceptions at the undergraduate level of this fundamental process of gene expression. This study reports on the efficacy of a model system for teaching gene expression in the undergraduate laboratory. A student-centered investigation of Tgfb1 gene expression in two murine melanoma cell lines was used to emphasize not only the process of gene expression but also various research methods for studying this phenomenon. Traditional RT-PCR, quantitative real-time RT-PCR, and flow cytometry-based in situ hybridization assays were employed to study expression of this immunosuppressive cytokine gene in the highly tumorigenic B16-F1 melanoma cell line and the poorly tumorigenic D5.1G4 melanoma cell line, both at the population and single-cell levels. A pre- and post-laboratory assessment instrument demonstrated the utility of this model system in enhancing student learning both of content related to gene expression and of research methods and data analysis skills. The pedagogical approach described in this study is therefore an effective way to improve the teaching and learning of gene expression at the undergraduate level. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(4):397-404, 2016. PMID:26898783

  17. COUP-TFII Stimulates Dentin Sialophosphoprotein Expression and Mineralization in Odontoblasts.

    PubMed

    Hur, S-W; Oh, S-H; Jeong, B-C; Choi, H; Kim, J-W; Lee, K-N; Hwang, Y-C; Ryu, J-H; Kim, S-H; Koh, J-T

    2015-08-01

    Chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), an orphan nuclear receptor belonging to the steroid-thyroid hormone receptor superfamily, plays an important role in cell fate determination of various tissues. However, the specific role of COUP-TFII in tooth development has not yet been elucidated. In the present study, we aimed to explore the role of COUP-TFII in dentin sialophosphoprotein (DSPP) expression and matrix mineralization in odontoblast-lineage cells. In primary human dental pulp cells (HDPCs) and murine dental papilla-derived cells (MDPC-23) cultured in a mineralizing medium, the expression of COUP-TFII was induced along with the increased odontoblast-specific dentin matrix protein-1 (DMP-1) and DSPP expression. Endogenous expression of COUP-TFII in maxillary second molar germs of rats showed an increasing tendency as development of the tooth progressed. Also, COUP-TFII protein was detected in greater quantity in the odontoblastic layer of second molar germs than in that of third molar germs of rats. Overexpression of COUP-TFII using an adenoviral system upregulated the expression of odontoblast-specific genes with increased alkaline phosphatase activity and matrix mineralization in odontoblast-lineage cells. In contrast, knockdown of COUP-TFII using small interfering RNA decreased the expression of odontoblast-specific genes, which reduced matrix mineralization. Mechanistic studies revealed that COUP-TFII increased DSPP transcription by direct binding on the DSPP promoter. In addition, COUP-TFII physically interacted with the homeodomain transcription factor Msx2 and antagonistically regulated the Msx2 effect on DSPP promoter activity. Taken together, these results suggest that COUP-TFII has a stimulatory role in DSPP expression and matrix mineralization in odontoblast-lineage cells. PMID:25940145

  18. Intersectional Gene Expression in Zebrafish Using the Split KalTA4 System

    PubMed Central

    2015-01-01

    Abstract In this study, we describe the adaptation of the split Gal4 system for zebrafish. The Gal4-UAS system is widely used for expression of genes-of-interest by crossing driver lines expressing the transcription factor Gal4 (under the control of the promoter of interest) with reporter lines where upstream activating sequence (UAS) repeats (recognized by Gal4) drive expression of the genes-of-interest. In the Split Gal4 system, hemi-drivers separately encode the DNA-binding domain (DBD) and the activation domain (AD) of Gal4. When encoded under two different promoters, only those cells in the intersection of the promoters' expression pattern and in which both promoters are active reconstitute a functional Gal4 and activate expression from a UAS-driven transgene. We split the zebrafish-optimized version of Gal4, KalTA4, and generated a hemi-driver encoding the KalTA4 DBD and a hemi-driver encoding KalTA4's AD. We show that split KalTA4 domains can assemble in vivo and transactivate a UAS reporter transgene and that each hemi-driver alone cannot transactivate the reporter. Also, transactivation can happen in several cell types, with similar efficiency to intact KalTA4. Finally, in transient mosaic expression assays, we show that when hemi-drivers are preceded by two distinct promoters, they restrict the expression of an UAS-driven reporter from a broader pattern (sox10) to its constituent smaller neuronal pattern. The Split KalTA4 system should be useful for expression of genes-of-interest in an intersectional manner, allowing for more refined manipulations of cell populations in zebrafish. PMID:26485616

  19. Integration Method of Emphatic Motions and Adverbial Expressions with Scalar Parameters for Robotic Motion Coaching System

    NASA Astrophysics Data System (ADS)

    Okuno, Keisuke; Inamura, Tetsunari

    A robotic coaching system can improve humans' learning performance of motions by intelligent usage of emphatic motions and adverbial expressions according to user reactions. In robotics, however, method to control both the motions and the expressions and how to bind them had not been adequately discussed from an engineering point of view. In this paper, we propose a method for controlling and binding emphatic motions and adverbial expressions by using two scalar parameters in a phase space. In the phase space, variety of motion patterns and verbal expressions are connected and can be expressed as static points. We show the feasibility of the proposing method through experiments of actual sport coaching tasks for beginners. From the results of participants' improvements in motion learning, we confirmed the feasibility of the methods to control and bind emphatic motions and adverbial expressions, as well as confirmed contribution of the emphatic motions and positive correlation of adverbial expressions for participants' improvements in motion learning. Based on the results, we introduce a hypothesis that individually optimized method for binding adverbial expression is required.

  20. Bioprocess applications of a Sindbis virus-based temperature-inducible expression system.

    PubMed

    Boorsma, M; Hoenke, S; Marrero, A; Fischer, R; Bailey, J E; Renner, W A; Bachmann, M F

    2002-09-20

    The production and study of toxic proteins requires inducible expression systems with low basal level expression and high inducibility. Here, we describe bioprocess applications of the pCytTS temperature-regulatable Sindbis virus replicon-based expression system. We used green fluorescent protein as a marker protein to optimize the selection of stable transfected clones with increased expression levels. Using the optimized protocol, clones were constructed that produced the growth-inhibiting, anti-viral protein interferon beta (beta-IFN). Selected clones were analyzed for temperature-dependent beta-IFN production in adherent and suspension cultures in serum free medium. Specific expression levels were around 1.0 x 10(5) IU/10(6) cells/day (0.5 microg/10(6) cells/day) in suspension cultures and over 1.5 x 10(6) IU/mL/day (7.5 microg/mL/day) in hollow fiber reactors using adherent cells. Hexahistidine-tagged beta-IFN purified from T-flask cultures was highly glycosylated and showed high specific activity. beta-IFN mRNA amplified by the viral replicase for 10 days did not show an accumulation of mutations. These data suggest the applicability of the pCytTS-inducible expression system for the production of high-quality glycoproteins in different reactors. PMID:12209807

  1. The expression patterns of Reg IV gene in normal rat reproduction system.

    PubMed

    Du, Fang; Yao, Zhen-Wei

    2013-01-01

    Reg IV, the latest member of the regenerating gene family, has been documented in different tissues of human and rat, such as the colon, small intestine, stomach, and pancreas. Expression of Reg IV gene in distinct cell types has been correlated with its various functions in regeneration, cell growth and survival, proliferation and differentiation, cell adhesion, and resistance to apoptosis. However, there was no evidence to show whether the Reg IV protein is present in the reproductive system of normal rat. The aim of this study was to reveal the expression patterns of Reg IV in rat ovary and uterus. The expression of Reg IV was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot at mRNA and protein levels, respectively. The localization of Reg IV protein within rat ovary and uterus was investigated by immunohistochemistry (IHC). Our results showed that the expression of Reg IV in ovary was significantly higher than that in the uterus. The strong immunoreactive signals of Reg IV was observed in granulosa cells and oocytes of ovarian follicles, corpus luteum, and interstitial cells in rat ovary; only weak signals were detected in luminal and gland epithelium of rat endometrium. These findings first demonstrate the expression of Reg IV in ovary and uterus of the healthy rat at both mRNA and protein levels. It provides an evidence of Reg IV expression in rat reproductive system, which may help elucidate a potential role in cell growth and proliferation of reproductive system. PMID:23203400

  2. Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila.

    PubMed

    McGuire, Sean E; Mao, Zhengmei; Davis, Ronald L

    2004-02-17

    Targeted gene expression has become a standard technique for the study of biological questions in Drosophila. Until recently, transgene expression could be targeted in the dimension of either time or space, but not both. Several new systems have recently been developed to direct transgene expression simultaneously in both time and space. We describe here two such systems that we developed in our laboratory. The first system provides a general method for temporal and regional gene expression targeting (TARGET) with the conventional GAL4-upstream activator sequence (UAS) system and a temperature-sensitive GAL80 molecule, which represses GAL4 transcriptional activity at permissive temperatures. The second system, termed Gene-Switch, is based on a GAL4-progesterone receptor chimera that is hormone-inducible. We have used both systems for simultaneous spatial and temporal rescue of memory dysfunction in the rutabaga (rut) memory mutant of Drosophila. In this protocol, we provide guidelines for the use of these two novel systems, which should have general utility in studying Drosophila biology and in using the fly as a model for human disease. PMID:14970377

  3. Efficient microbial production of stylopine using a Pichia pastoris expression system

    PubMed Central

    Hori, Kentaro; Okano, Shunsuke; Sato, Fumihiko

    2016-01-01

    Stylopine is a protoberberine-type alkaloid that has potential biological activities. Based on the successful microbial production of (S)-reticuline, we attempted to produce stylopine from (S)-reticuline by the reaction of berberine bridge enzyme, cheilanthifoline synthase (CYP719A5), and stylopine synthase (CYP719A2). Biosynthetic enzyme expression was examined in a methanol-utilizing yeast (Pichia pastoris), and both a “consolidated” system with all genes expressed in one cell and a “co-culture” system with three cell lines that each express a single gene were examined. Although both systems efficiently converted reticuline to stylopine, the consolidated system was more rapid and efficient than the co-culture system. However, substrate-feeding experiments revealed a decrease in the conversion efficiency in the consolidated system during successive cultures, whereas the conversion efficiency in the co-culture system remained constant. Thus, the final amount of stylopine produced from reticuline after successive feedings in the co-culture system was more than 150 nmoles from 750 nmoles of (R, S)-reticuline (375 nmoles of (S)-reticuline). The advantages and drawbacks of the “consolidated” system and the “co-culture” system are discussed. PMID:26923560

  4. Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system.

    PubMed

    DiFranco, Marino; Neco, Patricia; Capote, Joana; Meera, Pratap; Vergara, Julio L

    2006-05-01

    The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement. PMID:16325422

  5. Systemic Inflammation Affects Human Osteocyte-Specific Protein and Cytokine Expression.

    PubMed

    Pathak, Janak L; Bakker, Astrid D; Luyten, Frank P; Verschueren, Patrick; Lems, Willem F; Klein-Nulend, Jenneke; Bravenboer, Nathalie

    2016-06-01

    Bone remodeling can be disturbed in active rheumatoid arthritis (RA), possibly as a result of elevated levels of circulating inflammatory cytokines. Osteocyte-specific proteins and cytokines play a vital role in bone remodeling by orchestrating bone formation and/or bone resorption. Therefore, we aimed to investigate the effect of RA-serum or inflammatory cytokines on expression of human osteocyte-specific proteins and cytokines. Human trabecular bone chips were cultured with RA-serum or inflammatory cytokines for 7-days. Live-dead staining was performed to assess cell viability. Gene expression of osteocyte-specific proteins and cytokines was analyzed by qPCR. Immuno-staining was performed for osteocyte-specific markers. Approximately 60 % of the osteocytes on the bone chips were alive at day-7. Cells in or on the bone chips did express the gene for osteocyte markers SOST, FGF23, DMP1, and MEPE, and the cytokines IL-1β, IL-6, and TNFα at day 0 and 7. Active RA-serum treatment enhanced IL-1β, TNFα, SOST, and DKK1 gene expression. IL-1β treatment enhanced IL-1β, TNFα, IL-6, IL-8, FGF23, and SOST gene expression. TNFα treatment enhanced IL-1β, TNFα, IL-6, IL-8, and FGF23 gene expression. IL-8 treatment enhanced TNFα, IL-8, and FGF23 gene expression. A combination of IL-1β, IL-6, and TNFα treatment synergistically upregulated IL-1β, IL-6, and IL-8 gene expression, as well as enhanced TNFα, OPG, SOST, and FGF23, and inhibited DKK1 gene expression. In conclusion, gene expression of human osteocyte-specific proteins and cytokines was affected by RA-serum, and exogenous recombinant cytokines treatment suggesting that osteocytes could provide a new target to prevent systemic inflammation-induced bone loss in RA. PMID:26887974

  6. An animal model allowing controlled receptor expression for molecular ultrasound imaging.

    PubMed

    Saini, Reshu; Sorace, Anna G; Warram, Jason M; Mahoney, Marshall J; Zinn, Kurt R; Hoyt, Kenneth

    2013-01-01

    Reported in this study is an animal model system for evaluating targeted ultrasound (US) contrast agents binding using adenoviral (Ad) vectors to modulate cellular receptor expression. An Ad vector encoding an extracellular hemagglutinin (HA) epitope tag and a green fluorescent protein (GFP) reporter was used to regulate receptor expression. A low and high receptor density (in breast cancer tumor bearing mice) was achieved by varying the Ad dose with a low plaque forming unit (PFU) on day 1 and high PFU on day 2 of experimentation. Targeted US contrast agents, or microbubbles (MB), were created by conjugating either biotinylated anti-HA or IgG isotype control antibodies to the MB surface with biotin-streptavidin linkage. Targeted and control MBs were administered on both days of experimentation and contrast-enhanced US (CEUS) was performed on each mouse using MB flash destruction technique. Signal intensities from MBs retained within tumor vasculature were analyzed through a custom Matlab program. Results showed intratumoral enhancement attributable to targeted MB accumulation was significantly increased from the low Ad vector dosing and the high Ad vector dosing (p = 0.001). Control MBs showed no significant differences between day 1 and day 2 imaging (p = 0.96). Additionally, targeted MBs showed a 10.5-fold increase in intratumoral image intensity on day 1 and an 18.8-fold increase in image intensity on day 2 compared with their control MB counterparts. PMID:23122640

  7. Global gene expression profiling of the polyamine system in suicide completers.

    PubMed

    Fiori, Laura M; Bureau, Alexandre; Labbe, Aurélie; Croteau, Jordie; Noël, Simon; Mérette, Chantal; Turecki, Gustavo

    2011-06-01

    In recent years, gene expression, genetic association, and metabolic studies have implicated the polyamine system in psychiatric conditions, including suicide. Given the extensive regulation of genes involved in polyamine metabolism, as well as their interconnections with the metabolism of other amino acids, we were interested in further investigating the expression of polyamine-related genes across the brain in order to obtain a more comprehensive view of the dysregulation of this system in suicide. To this end, we examined the expression of genes related to polyamine metabolism across 22 brain regions in a sample of 29 mood-disordered suicide completers and 16 controls, and identified 14 genes displaying differential expression. Among these, altered expression of spermidine/spermine N1-acetyltransferase, spermine oxidase, and spermine synthase, has previously been observed in brains of suicide completers, while the remainder of the genes represent novel findings. In addition to genes with direct involvement in polyamine metabolism, including S-adenosylmethionine decarboxylase, ornithine decarboxylase antizymes 1 and 2, and arginase II, we identified altered expression of several more distally related genes, including aldehyde dehydrogenase 3 family, member A2, brain creatine kinase, mitochondrial creatine kinase 1, glycine amidinotransferase, glutamic-oxaloacetic transaminase 1, and arginyl-tRNA synthetase-like. Many of these genes displayed altered expression across several brain regions, strongly implying that dysregulated polyamine metabolism is a widespread phenomenon in the brains of suicide completers. This study provides a broader view of the nature and extent of the dysregulation of the polyamine system in suicide, and highlights the importance of this system in the neurobiology of suicide. PMID:21208503

  8. Development of an inducible gene expression system for primary murine keratinocytes

    PubMed Central

    Nagarajan, Priyadharsini

    2008-01-01

    Background The tetracycline (Tet) responsive system is a valuable tool that is routinely used in a wide variety of mammalian cells for regulatable expression of gene products. However, technical difficulties such as harsh selection conditions and extensive screening processes to identify suitably responsive clones limit the generation of stable cell lines. Hence, application of this system in mammalian cells with relatively slow growth rates and / or the capacity to undergo terminal differentiation such as primary mouse keratinocytes is particularly challenging. Objective To our knowledge, no Tet-responsive stable cell lines have been generated from mouse keratinocytes, presumably due to their sensitivity to selection conditions. Our goal was to utilize a modified and robust Tet-expression system to generate a stable primary mouse keratinocyte cell line. These cells could be then utilized for conditional expression of potentially toxic proteins in an inducible fashion. Methods We utilized a eukaryotic promoter instead of a viral promoter to express a modified reverse tetracycline transactivator in mouse keratinocytes and optimized the selection process for generating stable cell lines. Results Here, we report the generation of a stable mouse keratinocyte cell line for Tet-regulated gene expression with minimal leakiness and high degree of Tet responsivity. This mouse keratinocyte cell line was further engineered for generation of a double stable cell line, which expresses the transcription factor AP-2α in an inducible manner. Importantly, the selected cells retain their inherent keratinocyte morphology, respond to differentiation signals and exhibit a persistent and highly tunable Tet inducibility upon continuous culturing. Conclusion We have generated a tetracycline inducible gene expression model system in mouse epidermal keratinocytes. Such inducible cell lines will serve as valuable in vitro models for future gain-of-function and loss-of-function studies. PMID

  9. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

    PubMed Central

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B.; Patel, Trushar R.; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems. PMID:27029048

  10. Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

    PubMed Central

    Dingermann, T; Werner, H; Schütz, A; Zündorf, I; Nerke, K; Knecht, D; Marschalek, R

    1992-01-01

    We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal. Images PMID:1508201

  11. Complex regulation of nucleoside transporter expression in epithelial and immune system cells.

    PubMed

    Pastor-Anglada, M; Casado, F J; Valdés, R; Mata, J; García-Manteiga, J; Molina, M

    2001-01-01

    Nucleoside transporters have a variety of functions in the cell, such as the provision of substrates for nucleic acid synthesis and the modulation of purine receptors by determining agonist availability. They also transport a wide range of nucleoside-derived antiviral and anticancer drugs. Most mammalian cells co-express several nucleoside transporter isoforms at the plasma membrane, which are differentially regulated. This paper reviews studies on nucleoside transporter regulation, which has been extensively characterized in the laboratory in several model systems: the hepatocyte, an epithelial cell type, and immune system cells, in particular B cells, which are non-polarized and highly specialized. The hepatocyte co-expresses at least two Na+-dependent nucleoside transporters, CNT1 and CNT2, which are up-regulated during cell proliferation but may undergo selective loss in certain experimental models of hepatocarcinomas. This feature is consistent with evidence that CNT expression also depends on the differentiation status of the hepatocyte. Moreover, substrate availability also modulates CNT expression in epithelial cells, as reported for hepatocytes and jejunum epithelia from rats fed nucleotide-deprived diets. In human B cell lines, CNT and ENT transporters are co-expressed but differentially regulated after B cell activation triggered by cytokines or phorbol esters, as described for murine bone marrow macrophages induced either to activate or to proliferate. The complex regulation of the expression and activity of nucleoside transporters hints at their relevance in cell physiology. PMID:11396615

  12. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems. PMID:27029048

  13. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    SciTech Connect

    Kajikawa, Mizuho; Sasaki, Kaori; Wakimoto, Yoshitaro; Toyooka, Masaru; Motohashi, Tomoko; Shimojima, Tsukasa; Takeda, Shigeki; Park, Enoch Y.; Maenaka, Katsumi

    2009-07-31

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  14. In vivo imaging of inducible tyrosinase gene expression with an ultrasound array-based photoacoustic system

    NASA Astrophysics Data System (ADS)

    Harrison, Tyler; Paproski, Robert J.; Zemp, Roger J.

    2012-02-01

    Tyrosinase, a key enzyme in the production of melanin, has shown promise as a reporter of genetic activity. While green fluorescent protein has been used extensively in this capacity, it is limited in its ability to provide information deep in tissue at a reasonable resolution. As melanin is a strong absorber of light, it is possible to image gene expression using tyrosinase with photoacoustic imaging technologies, resulting in excellent resolutions at multiple-centimeter depths. While our previous work has focused on creating and imaging MCF-7 cells with doxycycline-controlled tyrosinase expression, we have now established the viability of these cells in a murine model. Using an array-based photoacoustic imaging system with 5 MHz center frequency, we capture interleaved ultrasound and photoacoustic images of tyrosinase-expressing MCF-7 tumors both in a tissue mimicking phantom, and in vivo. Images of both the tyrosinase-expressing tumor and a control tumor are presented as both coregistered ultrasound-photoacoustic B-scan images and 3-dimensional photoacoustic volumes created by mechanically scanning the transducer. We find that the tyrosinase-expressing tumor is visible with a signal level 12dB greater than that of the control tumor in vivo. Phantom studies with excised tumors show that the tyrosinase-expressing tumor is visible at depths in excess of 2cm, and have suggested that our imaging system is sensitive to a transfection rate of less than 1%.

  15. Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system

    PubMed Central

    Caldwell, James T.; Melkani, Girish C.; Huxford, Tom; Bernstein, Sanford I.

    2011-01-01

    Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization. PMID:22178692

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

    PubMed Central

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

    2008-01-01

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

  17. Cell-Specific Survival Motor Neuron Gene Expression during Human Development of the Central Nervous System

    PubMed Central

    Tizzano, Eduardo F.; Cabot, Carmen; Baiget, Montserrat

    1998-01-01

    Spinal muscular atrophy is an autosomal recessive disorder characterized by the progressive loss or degeneration of the motor neurons. To investigate the expression of survival motor neuron (SMN), the spinal muscular atrophy-determining gene, and its relationship with the pathogenesis of the disease, we analyzed by means of in situ hybridization the location of SMN mRNA in fetal, newborn, infant, and adult human central nervous system tissues. The large motor neurons of the spinal cord are the main cells that express SMN together with the neurons of the medulla oblongata, the pyramidal cells of the cortex, and the Purkinje cells of the cerebellum. Some sensory neurons from the posterior horn and dorsal root ganglia express SMN to a lesser degree. Furthermore, strong SMN expression is detected in the ependymal cells of the central canal. The expression is present in the spinal cord at 8 weeks of fetal life throughout postnatal and adult life. The sharp expression of SMN in the motor neurons of the human spinal cord, the target cells in spinal muscular atrophy, suggests that this gene is implicated in neuronal development and in the pathogenesis of the disease. The location of the SMN gene expression in other neuronal structures not clearly or directly associated with clinical manifestations or pathological findings of spinal muscular atrophy may indicate a varying sensitivity to the absence or dysfunction of the SMN gene in motor neurons. PMID:9708795

  18. CD123 immunostaining patterns in systemic mastocytosis: differential expression in disease subgroups and potential prognostic value.

    PubMed

    Pardanani, A; Reichard, K K; Zblewski, D; Abdelrahman, R A; Wassie, E A; Morice Ii, W G; Brooks, C; Grogg, K L; Hanson, C A; Tefferi, A; Chen, D

    2016-04-01

    CD123 is the α-subunit of the interleukin-3 receptor; it represents a potential therapeutic target in systemic mastocytosis (SM) given its absent expression on normal/reactive mast cells (MCs) and aberrant expression on neoplastic MCs. We studied 58 SM patients to define CD123 expression patterns by immunohistochemistry and its clinical significance. Two hematopathologists independently scored bone marrow slides using predefined histologic parameters. In all, 23 patients had indolent SM (ISM), 10 aggressive SM (ASM), 23 SM with associated hematological neoplasm (SM-AHN) and 2 had mast cell leukemia (MCL). MC_CD123 expression was demonstrable in 37 (64%) cases; expression rates were 100%, 61%, 57% and 0% in ASM, ISM, SM-AHN and MCL, respectively (P=0.02). Focal proliferation of plasmacytoid dendritic cells (PDCs) around MC aggregates, suggesting a tumor-promoting role for PDCs, was noted in 44 (76%) cases, and was significantly higher in CD123-positive versus -negative cases (87% versus 50%, P=0.005). CD123 expression and its staining intensity had prognostic value in SM-chronic myelomonocytic leukemia and nonindolent SM patients, respectively. These observations suggest that targeting CD123 in SM may have direct (via MCs) and indirect (via PDCs) antitumor effects and clinical trials to that effect require laboratory correlative studies to address the observed target expression heterogeneity. PMID:26678095

  19. Expression of the mouse PR domain protein Prdm8 in the developing central nervous system.

    PubMed

    Komai, Tae; Iwanari, Hiroko; Mochizuki, Yasuhiro; Hamakubo, Takao; Shinkai, Yoichi

    2009-10-01

    It was first shown in the PR (PRDI-BF1 and RIZ homology) domain family proteins that the PR domain has homology to the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain, a catalytic domain of the histone lysine methyltransferases. Recently, there are many reports that the PR domain proteins have important roles in development and/or cell differentiation. In this report, we show the expression patterns of one of the mouse PR domain proteins, Prdm8, in the developing central nervous system. In the developing retina, Prdm8 expression was detected in postmitotic neurons in the inner nuclear layer and the ganglion cell layer, and its expression became restricted predominantly to the rod bipolar cells when retinogenesis was completed. In the developing spinal cord, Prdm8 was expressed first in the progenitor populations of ventral interneurons and motor neurons, and later in a subpopulation of interneurons. In the developing brain, Prdm8 expression was observed in postmitotic neurons in the intermediate zone and the cortical plate. In the postnatal brain, Prdm8 was expressed mainly in layer 4 neurons of the cerebral cortex. These results show that Prdm8 expression is tightly regulated in a spatio-temporal manner during neural development and mainly restricted to postmitotic neurons, except in the spinal cord. PMID:19616129

  20. Expression and function of Neuregulin 1 and its signaling system ERBB2/3 in the enteric nervous system

    PubMed Central

    Barrenschee, Martina; Lange, Christina; Cossais, François; Egberts, Jan-Hendrik; Becker, Thomas; Wedel, Thilo; Böttner, Martina

    2015-01-01

    Neuregulin 1 (NRG1) is suggested to promote the survival and maintenance of the enteric nervous system (ENS). As deficiency in its corresponding receptor signaling complex ERBB2/ERBB3 leads to postnatal colonic hypo/aganglionosis we assessed the distributional and expressional pattern of the NRG1-ERBB2/ERBB3 system in the human colon and explored the neurotrophic capacity of NRG1 on cultured enteric neurons. Site-specific mRNA expression of the NRG1-ERBB2/3 system was determined in microdissected samples harvested from enteric musculature and ganglia. Localization of NRG1, ERBB2 and ERBB3 was determined by dual-label-immunohistochemistry using pan-neuronal and pan-glial markers. Morphometric analysis was performed on NRG1-stimulated rat enteric nerve cultures to evaluate neurotrophic effects. mRNA expression of the NRG1-ERBB2/3 system was determined by qPCR. Co-localization of NRG1 with neuronal or synaptic markers was analyzed in enteric nerve cultures stimulated with glial cell line-derived neurotrophic factor (GDNF). The NRG1 system was expressed in both neurons and glial cells of enteric ganglia and in nerve fibers. NRG1 significantly enhanced growth parameters in enteric nerve cell cultures and ErB3 mRNA expression was down-regulated upon NRG1 stimulation. GDNF negatively regulates ErbB2 and ErbB3 mRNA expression. The NRG1-ERBB2/3 system is physiologically present in the human ENS and NRG1 acts as a neurotrophic factor for the ENS. The down-regulation of ErbB3/ErbB2 in GDNF stimulated nerve cell cultures points to an interaction of both neurotrophic factors. Thus, the data may provide a basis to assess disturbed signaling components of the NRG1 system in enteric neuropathies. PMID:26441531

  1. Characterization of infectivity of knob-modified adenoviral vectors in glioma.

    PubMed

    Paul, C P L; Everts, M; Glasgow, J N; Dent, P; Fisher, P B; Ulasov, I V; Lesniak, M S; Stoff-Khalili, M A; Roth, J C; Preuss, M A; Dirven, C M F; Lamfers, M L M; Siegal, G P; Zhu, Z B; Curiel, D T

    2008-05-01

    Malignant 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 from serotype 5 adenoviruses (Ad5). This results from limited cell surface expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on tumor cells. To circumvent this problem, Ad fiber pseudotyping,the genetic replacement of either the entire fiber or fiber knob domain with its structural counterpart from another human Ad serotype that recognizes a cellular receptor other than CAR, has been shown to enhance Ad infectivity in a variety of tumor types,including human glioma. Here, we have extended the paradigm of genetic pseudotyping to include fiber domains from non-human or"xenotype" Ads for infectivity enhancement of human glioma cell populations. In this study, we evaluated the gene transfer efficiency of a panel of Ad vectors which express one of five different "xenotype"fiber knob domains, including those derived from murine,ovine, porcine and canine species, in both human glioma cell lines as well as primary glioma tumor cells from patients. Adenovirus vectors displaying either canine Ad or porcine Ad fiber elements had the highest gene transfer to both glioma cell lines and primary tumor cells. The correlation between the viral infectivity of modified adenovirus vectors and expression of human CAR and CD46(an adenovirus type B receptor) on the surfaces of tumor cells was also analyzed. Taken together, human adenovirus vectors modified with "xenotype" fiber elements could be excellent candidates to target human glioma. PMID:18756624

  2. Prodegenerative IκBα expression in oligodendroglial α-synuclein models of multiple system atrophy

    PubMed Central

    Kragh, Christine L.; Gysbers, Amanda M.; Rockenstein, Edward; Murphy, Karen; Halliday, Glenda M.; Masliah, Eliezer; Jensen, Poul Henning

    2016-01-01

    Multiple system atrophy is a progressive, neurodegenerative disease characterized by parkinsonism, ataxia, autonomic dysfunction, and accumulation of α-synuclein in oligodendrocytes. To understand how α-synuclein aggregates impact oligodendroglial homeostasis, we investigated an oligodendroglial cell model of α-synuclein dependent degeneration and identified responses linked to the NF-κB transcription factor stress system. Coexpression of human α-synuclein and the oligodendroglial protein p25α increased the expression of IκBα mRNA and protein early during the degenerative process and this was dependent on both aggregation and Ser129 phosphorylation of α-synuclein. This response was prodegenerative because blocking IκBα expression by siRNA rescued the cells. IκBα is an inhibitor of NF-κB and acts by binding and retaining NF-κB p65 in the cytoplasm. The protection obtained by silencing IκBα was accompanied by a strong increase in nuclear p65 translocation indicating that NF-κB activation protects against α-synuclein aggregate stress. In the cellular model, two different phenotypes were observed; degenerating cells retracting their microtubules and resilient cells tolerating the coexpression of α-synuclein and p25α. The resilient cells displayed a significant higher nuclear translocation of p65 and activation of the NF-κB system relied on stress elicited by aggregated and Ser129 phosphorylated α-synuclein. To validate the relationship between oligodendroglial α-synuclein expression and IκBα, we analyzed two different lines of transgenic mice expressing human α-synuclein under the control of the oligodendrocytic MBP promotor (intermediate-expresser line 1 and high-expresser line 29). IκBα mRNA expression was increased in both lines and immunofluorescence microscopy and in situ hybridization revealed that IκBα mRNA and protein is expressed in oligodendrocytes. IκBα mRNA expression was demonstrated prior to activation of microglia and

  3. Adenoviral Delivery of VEGF121 Early in Pregnancy Prevents Spontaneous Development of Preeclampsia in BPH/5 Mice

    PubMed Central

    Woods, Ashley K.; Hoffmann, Darren S.; Weydert, Christine J.; Butler, Scott D.; Zhou, Yi; Sharma, Ram V.; Davisson, Robin L.

    2011-01-01

    An imbalance in circulating pro-angiogenic and anti-angiogenic factors is postulated to play a causal role in pre-eclampsia (PE). We have described an inbred mouse strain, BPH/5, which spontaneously develops a PE-like syndrome including late-gestational hypertension, proteinuria, and poor feto-placental outcomes. Here we tested the hypothesis that an angiogenic imbalance during pregnancy in BPH/5 mice leads to the development of PE-like phenotypes in this model. Similar to clinical findings, plasma from pregnant BPH/5 showed reduced levels of free vascular endothelial growth factor (VEGF) and placental growth factor (PGF) compared to C57BL/6 controls. This was paralleled by a marked decrease in VEGF protein and Pgf mRNA in BPH/5 placentae. Surprisingly, antagonism by the soluble form of the FLT1 receptor (sFLT1) did not appear to be the cause of this reduction, as sFLT1 levels were unchanged or even reduced in BPH/5 compared to controls. Adenoviral-mediated delivery of VEGF121 (Ad-VEGF) via tail vein at e7.5 normalized both the plasma free VEGF levels in BPH/5 and restored the in vitro angiogenic capacity of serum from these mice. Ad-VEGF also reduced the incidence of fetal resorptions and prevented the late-gestational spike in blood pressure and proteinuria observed in BPH/5. These data underscore the importance of dysregulation of angiogenic factors in the pathogenesis of PE, and suggest the potential utility of early pro-angiogenic therapies in treating this disease. PMID:21079047

  4. Expression of the Wnt Receptor Frizzled-4 in the Human Enteric Nervous System of Infants

    PubMed Central

    Nothelfer, Katharina; Obermayr, Florian; Belz, Nadine; Reinartz, Ellen; Bareiss, Petra M.; Bühring, Hans-Jörg; Beschorner, Rudi; Just, Lothar

    2016-01-01

    The Wnt signalling pathway plays a crucial role in the development of the nervous system. This signalling cascade is initiated upon binding of the secreted Wnt ligand to a member of the family of frizzled receptors. In the present study, we analysed the presence of frizzled-4 in the enteric nervous system of human infants. Frizzled-4 could be identified by immunohistochemistry in a subpopulation of enteric neuronal and glial cells in the small and large intestine. Detection of frizzled-4 in the tunica muscularis by RT-PCR confirmed this receptor's expression on the mRNA level. Interestingly, we observed distinct cell populations that co-expressed frizzled-4 with the intermediate filament protein nestin and the neurotrophin receptor p75NTR, which have been reported to be expressed in neural progenitor cells. Flow cytometry analysis revealed that 60% of p75NTR positive cells of the tunica muscularis were positive for frizzled-4. Additionally, in pathological samples of Hirschsprung's disease, the expression of this Wnt receptor correlated with the number of myenteric ganglion cells and decreased from normoganglionic to aganglionic areas of large intestine. The expression pattern of frizzled-4 indicates that this Wnt receptor could be involved in postnatal development and/or function of the enteric nervous system. PMID:26697080

  5. A tetracycline expression system in combination with Sox9 for cartilage tissue engineering.

    PubMed

    Yao, Yi; He, Yu; Guan, Qian; Wu, Qiong

    2014-02-01

    Cartilage tissue engineering using controllable transcriptional therapy together with synthetic biopolymer scaffolds shows higher potential for overcoming chondrocyte degradation and constructing artificial cartilages both in vivo and in vitro. Here, the potential regulating tetracycline expression (Tet-on) system was used to express Sox9 both in vivo and in vitro. Chondrocyte degradation was measured in vitro and overcome by Soxf9 expression. Experiments confirmed the feasibility of the combined use of Sox9 and Tet-on system in cartilage tissue engineering. Engineered poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffolds were seeded with recombinant chondrocytes which were transfected with Tet-induced Sox9 expression; the scaffolds were implanted under the skin of 8-week-old rats. The experimental group was injected with Dox in the abdomen, while the control group was injected with normal saline. After 4 or 8 days of implantation in vivo, the newly formed pieces of articular chondrocytes were taken out and measured. Dox injection in vivo showed positive effect on recombinant chondrocytes, in which Sox9 expression was up-regulated by an inducible system with specific matrix proteins. The results demonstrate this controllable transcriptional therapy is a potential approach for tissue engineering. PMID:24321708

  6. Data set for mass spectrometric analysis of recombinant human serum albumin from various expression systems.

    PubMed

    Smith, Daryl G S; Frahm, Grant E; Kane, Anita; Lorbetskie, Barry; Girard, Michel; Johnston, Michael J W; Cyr, Terry D

    2015-09-01

    Human serum albumin (HSA) is a versatile and important protein for the pharmaceutical industry (Fanali et al., Mol. Aspects Med. 33(3) (2012) 209-290). Due to the potential transmission of pathogens from plasma sourced albumin, numerous expression systems have been developed to produce recombinant HSA (rHSA) (Chen et al., Biochim. Biophys. Acta (BBA)-Gen. Subj. 1830(12) (2013) 5515-5525; Kobayashi, Biologicals 34(1) (2006) 55-59). Based on our previous study showing increased glycation of rHSA expressed in Asian rice (Frahm et al., J. Phys. Chem. B 116(15) (2012) 4661-4670), both supplier-to-supplier and lot-to-lot variability of rHSAs from a number of expression systems were evaluated using reversed phase liquid chromatography linked with MS and MS/MS analyses. The data are associated with the research article 'Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa' where further analysis of rHSA samples with additional biophysical methods can be found (Frahm et al., PLoS ONE 10(9) (2014) e109893). We determined that all rHSA samples expressed in rice showed elevated levels of arginine and lysine hexose glycation compared to rHSA expressed in yeast, suggesting that the extensive glycation of the recombinant proteins is a by-product of either the expression system or purification process and not a random occurrence. PMID:26322323

  7. Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO.

    PubMed

    Marblestone, Jeffrey G; Edavettal, Suzanne C; Lim, Yiting; Lim, Peter; Zuo, Xun; Butt, Tauseef R

    2006-01-01

    Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green fluorescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar KM values, but SUMO protease had a 25-fold higher kcat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences. PMID:16322573

  8. Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.

    PubMed

    Sumiya, Nobuko; Fujiwara, Takayuki; Kobayashi, Yusuke; Misumi, Osami; Miyagishima, Shin-ya

    2014-01-01

    The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage. PMID:25337786

  9. Production of small antibacterial peptides using silkworm-baculovirus protein expression system.

    PubMed

    Fukushima, Mai; Iiyama, Kazuhiro; Yamashita, Jun; Furue, Masutaka; Tsuji, Gaku; Imanishi, Shigeo; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2013-01-01

    The recombinant proteins with strong antimicrobial activity are known to be very difficult to express using bacterial expression system. Here, human β-defensin (DEFB) 1, DEFB2, and DEFB3 were successfully produced using a silkworm-baculovirus protein expression system. We have generated four baculoviruses for each DEFB protein to compare the effect of different peptide tags in secretion into silkworm larval hemolymph. Interestingly, the best performing peptide tags for the secretion were different among DEFBs: C-terminal GST-H8 tag for DEFB1, N-terminal H8 tag for DEFB2, and C-terminal H8 tag for DEFB3, respectively. In addition, the colony count assay demonstrated that the recombinant DEFB2 s showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus thiaminolyticus. PMID:23742088

  10. Interspecies Systems Biology Uncovers Metabolites Affecting C. elegans Gene Expression and Life History Traits

    PubMed Central

    Watson, Emma; MacNeil, Lesley T.; Ritter, Ashlyn D.; Yilmaz, L. Safak; Rosebrock, Adam P.; Caudy, Amy A.; Walhout, Albertha J. M.

    2014-01-01

    SUMMARY Diet greatly influences gene expression and physiology. In mammals, elucidating the effects and mechanisms of individual nutrients is challenging due to the complexity of both the animal and its diet. Here we used an interspecies systems biology approach with Caenorhabditis elegans and two if its bacterial diets, Escherichia coli and Comamonas aquatica, to identify metabolites that affect the animal’s gene expression and physiology. We identify vitamin B12 as the major dilutable metabolite provided by Comamonas aq. that regulates gene expression, accelerates development and reduces fertility, but does not affect lifespan. We find that vitamin B12 has a dual role in the animal: it affects development and fertility via the methionine/S-Adenosylmethionine (SAM) cycle and breaks down the short-chain fatty acid propionic acid preventing its toxic buildup. Our interspecies systems biology approach provides a paradigm for understanding complex interactions between diet and physiology. PMID:24529378

  11. Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems

    SciTech Connect

    Conover, David R.; Crawford, Aladsair J.; Viswanathan, Vilayanur V.; Ferreira, Summer; Schoenwald, David

    2014-06-01

    The Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems (PNNL-22010) was first issued in November 2012 as a first step toward providing a foundational basis for developing an initial standard for the uniform measurement and expression of energy storage system (ESS) performance. Its subsequent use in the field and review by the protocol working group and most importantly the users’ subgroup and the thermal subgroup has led to the fundamental modifications reflected in this update of the 2012 Protocol. As an update of the 2012 Protocol, this document (the June 2014 Protocol) is intended to supersede its predecessor and be used as the basis for measuring and expressing ESS performance. The foreword provides general and specific details about what additions, revisions, and enhancements have been made to the 2012 Protocol and the rationale for them in arriving at the June 2014 Protocol.

  12. EXPRESSION SYSTEM-DEPENDENT MODULATION OF HIV-1 ENVELOPE GLYCOPROTEIN ANTIGENICITY AND IMMUNOGENICITY

    PubMed Central

    Kong, Leopold; Sheppard, Neil C.; Stewart-Jones, Guillaume B.E.; Robson, Cynthia L.; Chen, Hongying; Xu, Xiaodong; Krashias, George; Bonomelli, Camille; Scanlan, Christopher N.; Kwong, Peter D.; Jeffs, Simon A.; Jones, Ian M.; Sattentau, Quentin J.

    2010-01-01

    Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the Human Immunodeficiency Virus Type-1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly-related Env surface (gp120) antigens produced in different systems: a) mammalian (293F) cells in the presence of kifunensine which impart only high mannose glycans; b) insect (Spodoptera frugiperda, Sf9) cells, which confer mainly paucimannosidic glycans; c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high mannose, hybrid and complex glycans without sialic acid; d) 293F cells, which impart high mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9- and wild-type mammalian cell-derived material that is predicted to impact upon ligand binding sites proximal to glycans. Modelling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4-binding and CD4–induced sites compared to those expressed in the system that does, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic acid-containing gp120 was significantly more immunogenic than the sialyated version when administered in two different adjuvants, and induced higher titres of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic acid imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations which should be considered when selecting expression systems for glycosylated antigens to be used

  13. Analysis of the structure and function of EMRE in a yeast expression system.

    PubMed

    Yamamoto, Takenori; Yamagoshi, Ryohei; Harada, Kazuki; Kawano, Mayu; Minami, Naoki; Ido, Yusuke; Kuwahara, Kana; Fujita, Atsushi; Ozono, Mizune; Watanabe, Akira; Yamada, Akiko; Terada, Hiroshi; Shinohara, Yasuo

    2016-06-01

    The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore. PMID:27001609

  14. Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

    NASA Technical Reports Server (NTRS)

    Grill, Mischala A.; Bales, Mark A.; Fought, Amber N.; Rosburg, Kristopher C.; Munger, Stephanie J.; Antin, Parker B.

    2003-01-01

    Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

  15. A Novel Tightly Regulated Gene Expression System for the Human Intestinal Symbiont Bacteroides thetaiotaomicron

    PubMed Central

    Horn, Nikki; Carvalho, Ana L.; Overweg, Karin; Wegmann, Udo; Carding, Simon R.; Stentz, Régis

    2016-01-01

    There is considerable interest in studying the function of Bacteroides species resident in the human gastrointestinal (GI)-tract and the contribution they make to host health. Reverse genetics and protein expression techniques, such as those developed for well-characterized Escherichia coli cannot be applied to Bacteroides species as they and other members of the Bacteriodetes phylum have unique promoter structures. The availability of useful Bacteroides-specific genetic tools is therefore limited. Here we describe the development of an effective mannan-controlled gene expression system for Bacteroides thetaiotaomicron containing the mannan-inducible promoter–region of an α-1,2-mannosidase gene (BT_3784), a ribosomal binding site designed to modulate expression, a multiple cloning site to facilitate the cloning of genes of interest, and a transcriptional terminator. Using the Lactobacillus pepI as a reporter gene, mannan induction resulted in an increase of reporter activity in a time- and concentration-dependent manner with a wide range of activity. The endogenous BtcepA cephalosporinase gene was used to demonstrate the suitability of this novel expression system, enabling the isolation of a His-tagged version of BtCepA. We have also shown with experiments performed in mice that the system can be induced in vivo in the presence of an exogenous source of mannan. By enabling the controlled expression of endogenous and exogenous genes in B. thetaiotaomicron this novel inducer-dependent expression system will aid in defining the physiological role of individual genes and the functional analyses of their products. PMID:27468280

  16. Expression of Streptococcus pneumoniae Bacteriocins Is Induced by Antibiotics via Regulatory Interplay with the Competence System

    PubMed Central

    Slager, Jelle; Lake, Frank B.; Gericke, Oliver; Roberts, Ian S.; Rozen, Daniel E.; Veening, Jan-Willem

    2016-01-01

    Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have

  17. Establishment of tetracycline-inducible gene expression systems in the silkworm, Bombyx mori.

    PubMed

    Karasaki, Noriko; Mon, Hiroaki; Takahashi, Masateru; Lee, Jae Man; Koga, Katsumi; Kawaguchi, Yutaka; Kusakabe, Takahiro

    2009-04-01

    Tetracycline-inducible gene expression (Tet-on) system has become one of the first choices for the control of transgenes expression in mammal and drosophila. However, the Tet-on systems that have been established in mammalian system or tuned into drosophila do not function in the silkworm, Bombyx mori. To construct a functional Tet-on system in B. mori, we modified rtTA by introducing a transcription activation domain of immediate-early gene 1 of Autographa californica nuclear polyhedrosis virus and nuclear localization signal of SV40 large T-antigen. The modified rtTA can activate the transcription from 9 x tetO promoter in the silkworm cells up to 250-fold in the presence of doxycycline. PMID:19066730

  18. SYSTEMIC BIOMARKERS AND CARDIAC GENE EXPRESSION PROFILES OF RAT DISEASE MODELS EMPLOYED IN AIR POLLUTION STUDIES

    EPA Science Inventory

    Cardiovascular disease (CVD) models are used for identification of mechanisms of susceptibility to air pollution. We hypothesized that baseline systemic biomarkers and cardiac gene expression in CVD rat models will have influence on their ozone-induced lung inflammation. Male 12-...

  19. A viral over-expression system for the major malaria mosquito Anopheles gambiae

    PubMed Central

    Suzuki, Yasutsugu; Niu, Guodong; Hughes, Grant L.; Rasgon, Jason L.

    2014-01-01

    Understanding pathogen/mosquito interactions is essential for developing novel strategies to control mosquito-borne diseases. Technical advances in reverse-genetics, such as RNA interference (RNAi), have facilitated elucidation of components of the mosquito immune system that are antagonistic to pathogen development, and host proteins essential for parasite development. Forward genetic approaches, however, are limited to generation of transgenic insects, and while powerful, mosquito transgenesis is a resource- and time-intensive technique that is not broadly available to most laboratories. The ability to easily “over-express” genes would enhance molecular studies in vector biology and expedite elucidation of pathogen-refractory genes without the need to make transgenic insects. We developed and characterized an efficient Anopheles gambiae densovirus (AgDNV) over-expression system for the major malaria vector Anopheles gambiae. High-levels of gene expression were detected at 3 days post-infection and increased over time, suggesting this is an effective system for gene induction. Strong expression was observed in the fat body and ovaries. We validated multiple short promoters for gene induction studies. Finally, we developed a polycistronic system to simultaneously express multiple genes of interest. This AgDNV-based toolset allows for consistent transduction of genes of interest and will be a powerful molecular tool for research in Anopheles gambiae mosquitoes. PMID:24875042

  20. 21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control...

  1. 21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control...

  2. 21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control...

  3. 21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control...

  4. 21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control...

  5. Systems for the expression of orthogonal translation components in eubacterial host cells

    DOEpatents

    Ryu, Youngha; Schultz, Peter G.

    2013-01-22

    The invention related to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural acids at genetically-programmed positions.

  6. Systems for the expression of orthogonal translation components eubacterial host cells

    DOEpatents

    Ryu, Youngha; Schultz, Peter G.

    2012-06-12

    The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

  7. Systems for the expression of orthogonal translation components in eubacterial host cells

    DOEpatents

    Ryu, Youngha; Schultz, Peter G.

    2011-06-14

    The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

  8. The infectious BAC genomic DNA expression library: a high capacity vector system for functional genomics.

    PubMed

    Lufino, Michele M P; Edser, Pauline A H; Quail, Michael A; Rice, Stephen; Adams, David J; Wade-Martins, Richard

    2016-01-01

    Gene dosage plays a critical role in a range of cellular phenotypes, yet most cellular expression systems use heterologous cDNA-based vectors which express proteins well above physiological levels. In contrast, genomic DNA expression vectors generate physiologically-relevant levels of gene expression by carrying the whole genomic DNA locus of a gene including its regulatory elements. Here we describe the first genomic DNA expression library generated using the high-capacity herpes simplex virus-1 amplicon technology to deliver bacterial artificial chromosomes (BACs) into cells by viral transduction. The infectious BAC (iBAC) library contains 184,320 clones with an average insert size of 134.5 kb. We show in a Chinese hamster ovary (CHO) disease model cell line and mouse embryonic stem (ES) cells that this library can be used for genetic rescue studies in a range of contexts including the physiological restoration of Ldlr deficiency, and viral receptor expression. The iBAC library represents an important new genetic analysis tool openly available to the research community. PMID:27353647

  9. Contactin-5 expression during development and wiring of the thalamocortical system.

    PubMed

    Kleijer, K T E; Zuko, A; Shimoda, Y; Watanabe, K; Burbach, J P H

    2015-12-01

    The gene encoding the neural cell adhesion molecule Cntn5 (a.k.a. NB-2) has been put forward as a candidate in neurodevelopmental disorders, like autism spectrum disorder (ASD), by recent genetic findings. Little is known about the expression pattern and function of the gene, and its functional involvement in brain development has remained elusive. So far, most research has focused on its early postnatal expression in the auditory system, where the absence of Cntn5 causes abnormal responses to acoustic stimuli and a decrease in fiber density. The current study shows that the Cntn5 gene is expressed in forebrain structures during embryonic development, starting at E15.5, and that it continues to be expressed into adulthood. Sites of strong expression included the thalamus, the caudate putamen (CPu) and to a lesser extent layer Va of the cerebral cortex. Cntn5-positive thalamic nuclei include the laterodorsal (LD), ventrolateral (VL) and posterior group (Po), which contain glutamatergic neurons. Visualization of the expression pattern through the Tau-LacZ fusion protein coded by an insert in the Cntn5 gene, demonstrated that Cntn5-positive nuclei of the thalamus project to the cortex, based on co-localization with thalamocortical markers L1 and Calretinin. These results indicate that the cell adhesion functions of Cntn5 are exploited for circuit formation and connectivity in early development and for synaptic maintenance during adulthood. Subtle alterations in the formation of the thalamocortical circuit may contribute to neurodevelopmental disorders, such as ASD. PMID:26391921

  10. Expression of RYamide in the nervous and endocrine system of Bombyx mori.

    PubMed

    Roller, Ladislav; Čižmár, Daniel; Bednár, Branislav; Žitňan, Dušan

    2016-06-01

    RYamides are neuropeptides encoded by a gene whose precise expression and function have not yet been determined. We identified the RYamide gene transcript (fmgV1g15f, SilkBase database) and predicted two candidates for G-protein coupled RYamide receptors (A19-BAG68418 and A22-BAG68421) in the silkworm Bombyx mori. We cloned the RYamide transcript and described its spatial expression using in situ hybridisation. In the larval central nervous system (CNS) expression of RYamide was restricted to 12-14 small neurons in the brain and two posterior neurons in the terminal abdominal ganglion. During metamorphosis their number decreased to eight protocerebral neurons in the adults. Multiple staining, using various insect neuropeptide antibodies, revealed that neurons expressing RYamide are different from other peptidergic cells in the CNS. We also found RYamide expression in the enteroendocrine cells (EC) of the anterior midgut of larvae, pupae and adults. Two minor subpopulations of these EC were also immunoreactive to antibodies against tachykinin and myosupressin. This expression pattern suggests RYamides may play a role in the regulation of feeding and digestion. PMID:26896568

  11. Expression pattern of neuregulin-1 type III during the development of the peripheral nervous system.

    PubMed

    Huang, Liang-Liang; Liu, Zhong-Yang; Huang, Jing-Hui; Luo, Zhuo-Jing

    2015-01-01

    Neuregulin-1 type III is a key regulator in Schwann cell proliferation, committing to a myelinating fate and regulating myelin sheath thickness. However, the expression pattern of neuregulin-1 type III in the peripheral nervous system during developmental periods (such as the premyelinating stage, myelinating stage and postmyelinating stage) has rarely been studied. In this study, dorsal root ganglia were isolated from rats between postnatal day 1 and postnatal day 56. The expression pattern of neuregulin-1 type III in dorsal root ganglia neurons at various developmental stages were compared by quantitative real-time polymerase chain reaction, western blot assay and immunofluorescent staining. The expression of neuregulin-1 type III mRNA reached its peak at postnatal day 3 and then stabilized at a relative high expression level from postnatal day 3 to postnatal day 56. The expression of neuregulin-1 type III protein increased gradually from postnatal day 1, reached a peak at postnatal day 28, and then decreased at postnatal day 56. Immunofluorescent staining results showed a similar tendency to western blot assay results. Experimental findings indicate that the expression of neuregulin-1 type III in rat dorsal root ganglion was increased during the premyelinating (from postnatal day 2 to postnatal day 5) and myelinating stage (from postnatal day 5 to postnatal day 10), but remained at a high level in the postmyelinating stage (after postnatal day 10). PMID:25788922

  12. The infectious BAC genomic DNA expression library: a high capacity vector system for functional genomics

    PubMed Central

    Lufino, Michele M. P.; Edser, Pauline A. H.; Quail, Michael A.; Rice, Stephen; Adams, David J.; Wade-Martins, Richard

    2016-01-01

    Gene dosage plays a critical role in a range of cellular phenotypes, yet most cellular expression systems use heterologous cDNA-based vectors which express proteins well above physiological levels. In contrast, genomic DNA expression vectors generate physiologically-relevant levels of gene expression by carrying the whole genomic DNA locus of a gene including its regulatory elements. Here we describe the first genomic DNA expression library generated using the high-capacity herpes simplex virus-1 amplicon technology to deliver bacterial artificial chromosomes (BACs) into cells by viral transduction. The infectious BAC (iBAC) library contains 184,320 clones with an average insert size of 134.5 kb. We show in a Chinese hamster ovary (CHO) disease model cell line and mouse embryonic stem (ES) cells that this library can be used for genetic rescue studies in a range of contexts including the physiological restoration of Ldlr deficiency, and viral receptor expression. The iBAC library represents an important new genetic analysis tool openly available to the research community. PMID:27353647

  13. Organ distribution of transgene expression following intranasal mucosal delivery of recombinant replication-defective adenovirus gene transfer vector

    PubMed Central

    Damjanovic, Daniela; Zhang, Xizhong; Mu, Jingyu; Fe Medina, Maria; Xing, Zhou

    2008-01-01

    It is believed that respiratory mucosal immunization triggers more effective immune protection than parenteral immunization against respiratory infection caused by viruses and intracellular bacteria. Such understanding has led to the successful implementation of intranasal immunization in humans with a live cold-adapted flu virus vaccine. Furthermore there has been an interest in developing effective mucosal-deliverable genetic vaccines against other infectious diseases. However, there is a concern that intranasally delivered recombinant viral-based vaccines may disseminate to the CNS via the olfactory tissue. Initial experimental evidence suggests that intranasally delivered recombinant adenoviral gene transfer vector may transport to the olfactory bulb. However, there is a lack of quantitative studies to compare the relative amounts of transgene products in the respiratory tract, lung, olfactory bulb and brain after intranasal mucosal delivery of viral gene transfer vector. To address this issue, we have used fluorescence macroscopic imaging, luciferase quantification and PCR approaches to compare the relative distribution of transgene products or adenoviral gene sequences in the respiratory tract, lung, draining lymph nodes, olfactory bulb, brain and spleen. Intranasal mucosal delivery of replication-defective recombinant adenoviral vector results in gene transfer predominantly in the respiratory system including the lung while it does lead to a moderate level of gene transfer in the olfactory bulb. However, intranasal inoculation of adenoviral vector leads to little or no viral dissemination to the major region of the CNS, the brain. These experimental findings support the efficaciousness of intranasal adenoviral-mediated gene transfer for the purpose of mucosal immunization and suggest that it may not be of significant safety concern. PMID:18261231

  14. A score system for quality evaluation of RNA sequence tags: an improvement for gene expression profiling

    PubMed Central

    Pinheiro, Daniel G; Galante, Pedro AF; de Souza, Sandro J; Zago, Marco A; Silva, Wilson A

    2009-01-01

    Background High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) or Sequencing-by-Synthesis (SBS) represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. Results This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. Conclusion These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at . S3T source code and datasets can also be downloaded from the aforementioned website. PMID:19500384

  15. Automated Facial Action Coding System for Dynamic Analysis of Facial Expressions in Neuropsychiatric Disorders

    PubMed Central

    Hamm, Jihun; Kohler, Christian G.; Gur, Ruben C.; Verma, Ragini

    2011-01-01

    Facial expression is widely used to evaluate emotional impairment in neuropsychiatric disorders. Ekman and Friesen’s Facial Action Coding System (FACS) encodes movements of individual facial muscles from distinct momentary changes in facial appearance. Unlike facial expression ratings based on categorization of expressions into prototypical emotions (happiness, sadness, anger, fear, disgust, etc.), FACS can encode ambiguous and subtle expressions, and therefore is potentially more suitable for analyzing the small differences in facial affect. However, FACS rating requires extensive training, and is time consuming and subjective thus prone to bias. To overcome these limitations, we developed an automated FACS based on advanced computer science technology. The system automatically tracks faces in a video, extracts geometric and texture features, and produces temporal profiles of each facial muscle movement. These profiles are quantified to compute frequencies of single and combined Action Units (AUs) in videos, which can facilitate statistical study of large populations in disorders affecting facial expression. We derived quantitative measures of flat and inappropriate facial affect automatically from temporal AU profiles. Applicability of the automated FACS was illustrated in a pilot study, by applying it to data of videos from eight schizophrenia patients and controls. We created temporal AU profiles that provided rich information on the dynamics of facial muscle movements for each subject. The quantitative measures of flatness and inappropriateness showed clear differences between patients and the controls, highlighting their potential in automatic and objective quantification of symptom severity. PMID:21741407

  16. Central nervous system gene expression changes in a transgenic mouse model for bovine spongiform encephalopathy

    PubMed Central

    2011-01-01

    Gene expression analysis has proven to be a very useful tool to gain knowledge of the factors involved in the pathogenesis of diseases, particularly in the initial or preclinical stages. With the aim of finding new data on the events occurring in the Central Nervous System in animals affected with Bovine Spongiform Encephalopathy, a comprehensive genome wide gene expression study was conducted at different time points of the disease on mice genetically modified to model the bovine species brain in terms of cellular prion protein. An accurate analysis of the information generated by microarray technique was the key point to assess the biological relevance of the data obtained in terms of Transmissible Spongiform Encephalopathy pathogenesis. Validation of the microarray technique was achieved by RT-PCR confirming the RNA change and immunohistochemistry techniques that verified that expression changes were translated into variable levels of protein for selected genes. Our study reveals changes in the expression of genes, some of them not previously associated with prion diseases, at early stages of the disease previous to the detection of the pathological prion protein, that might have a role in neuronal degeneration and several transcriptional changes showing an important imbalance in the Central Nervous System homeostasis in advanced stages of the disease. Genes whose expression is altered at early stages of the disease should be considered as possible therapeutic targets and potential disease markers in preclinical diagnostic tool development. Genes non-previously related to prion diseases should be taken into consideration for further investigations. PMID:22035425

  17. Growth Arrest Specific 1 (GAS1) Is Abundantly Expressed in the Adult Mouse Central Nervous System

    PubMed Central

    Zarco, Natanael; Bautista, Elizabeth; Cuéllar, Manola; Vergara, Paula; Flores-Rodriguez, Paola; Aguilar-Roblero, Raúl

    2013-01-01

    Growth arrest specific 1 (GAS1) is a pleiotropic protein that induces apoptosis and cell arrest in different tumors, but it is also involved in the development of the nervous system and other tissues and organs. This dual ability is likely caused by its capacity to interact both by inhibiting the intracellular signaling cascade induced by glial cell-line derived neurotrophic factor and by facilitating the activity of the sonic hedgehog pathway. The presence of GAS1 mRNA has been described in adult mouse brain, and here we corroborated this observation. We then proceeded to determine the distribution of the protein in the adult central nervous system (CNS). We detected, by western blot analysis, expression of GAS1 in olfactory bulb, caudate-putamen, cerebral cortex, hippocampus, mesencephalon, medulla oblongata, cerebellum, and cervical spinal cord. To more carefully map the expression of GAS1, we performed double-label immunohistochemistry and noticed expression of GAS1 in neurons in all brain areas examined. We also observed expression of GAS1 in astroglial cells, albeit the pattern of expression was more restricted than that seen in neurons. Briefly, in the present article, we report the widespread distribution and cellular localization of the GAS1 native protein in adult mammalian CNS. PMID:23813868

  18. Distribution and expression of the Ade multidrug efflux systems in Acinetobacter baumannii clinical isolates.

    PubMed

    Pagdepanichkit, Sirawit; Tribuddharat, Chanwit; Chuanchuen, Rungtip

    2016-09-01

    One hundred Acinetobacter baumannii clinical isolates were examined for inhibitory effect of reserpine and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the antimicrobial susceptibility and expression of 4 resistant-nodulation-cell division (RND)-type multidrug efflux systems, including AdeABC, AdeDE, AdeIJK, and AdeFGH, using RT-PCR. Ten A. baumannii isolates expressing AdeABC, AdeIJK, or AdeFGH were randomly selected for determination of transcription level and regulatory mutations. While all the isolates were resistant to multiple drugs, the reserpine and CCCP experiment showed that the multidrug resistance phenotype in most A. baumannii isolates was associated with efflux pumps. Most isolates expressed at least one of the RND-type efflux pumps tested (97%). AdeIJK expression was most common (97%), but none of the isolates produced AdeDE. Fifty-two percent of the A. baumannii isolates simultaneously produced up to 3 RND-type efflux systems (i.e., AdeABC, AdeFGH, and AdeIJK). No good correlation between the expression of RND-type efflux pumps and the type of antimicrobial resistance was observed. Overexpression of AdeABC, AdeIJK, and AdeFGH was not always related to the presence of mutations in their corresponding regulatory genes. This study highlights (i) the universal presence of the RND-type efflux pumps with variable levels of expression level among the A. baumannii in this collection and (ii) the complexity of their regulation of expression. PMID:27332787

  19. Expression optimization and synthetic gene networks in cell-free systems

    PubMed Central

    Karig, David K.; Iyer, Sukanya; Simpson, Michael L.; Doktycz, Mitchel J.

    2012-01-01

    Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion. PMID:22180537

  20. Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum

    PubMed Central

    Prata, Rhana Berto da Silva; Barbosa, Mayara Garcia de Mattos; Mendes, Mayara Abud; Brandão, Sheila Santos; Amadeu, Thaís Porto; Rodrigues, Luciana Silva; Ferreira, Helen; Costa, Fabrício da Mota Ramalho; dos Santos, Jessica Brandão; Pacheco, Fabiana dos Santos; Machado, Alice de Miranda; Nery, José Augusto da Costa; Hacker, Mariana de Andrea; Sales, Anna Maria; Pinheiro, Roberta Olmo; Sarno, Euzenir Nunes

    2016-01-01

    Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates—a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably

  1. Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum.

    PubMed

    Schmitz, Veronica; Prata, Rhana Berto da Silva; Barbosa, Mayara Garcia de Mattos; Mendes, Mayara Abud; Brandão, Sheila Santos; Amadeu, Thaís Porto; Rodrigues, Luciana Silva; Ferreira, Helen; Costa, Fabrício da Mota Ramalho; Dos Santos, Jessica Brandão; Pacheco, Fabiana Dos Santos; Machado, Alice de Miranda; Nery, José Augusto da Costa; Hacker, Mariana de Andrea; Sales, Anna Maria; Pinheiro, Roberta Olmo; Sarno, Euzenir Nunes

    2016-08-01

    Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates-a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably

  2. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

    PubMed

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær; Rank, Julie; Hansen, Bjarne Gram; Andersen, Mikael Rørdam; Mortensen, Uffe Hasbro

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors. PMID:24879460

  3. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    PubMed Central

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær; Rank, Julie; Hansen, Bjarne Gram; Andersen, Mikael Rørdam; Mortensen, Uffe Hasbro

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors. PMID:24879460

  4. Peri- and Postnatal Effects of Prenatal Adenoviral VEGF Gene Therapy in Growth-Restricted Sheep.

    PubMed

    Carr, David J; Wallace, Jacqueline M; Aitken, Raymond P; Milne, John S; Martin, John F; Zachary, Ian C; Peebles, Donald M; David, Anna L

    2016-06-01

    Uterine artery (UtA) adenovirus (Ad) vector-mediated overexpression of vascular endothelial growth factor (VEGF) enhances uterine blood flow in normal sheep pregnancy and increases fetal growth in the overnourished adolescent sheep model of fetal growth restriction (FGR). Herein, we examined its impact on gestation length, neonatal survival, early postnatal growth and metabolism. Singleton-bearing ewes were evenly allocated to receive Ad.VEGF-A165 (5 × 10(10) particles/ml, 10 ml, n = 17) or saline (10 ml, n = 16) injected into each UtA at laparotomy (0.6 gestation). Fetal growth was serially monitored (blind) by ultrasound until delivery. Lambs were weighed and blood was sampled weekly and a glucose tolerance test performed (68-day postnatal age). Hepatic DNA/RNA was extracted at necropsy (83-day postnatal age) to examine methylation status of eight somatotropic axis genes. IGF1 mRNA and protein expression were measured by RT-PCR and radioimmunoassay, respectively. All pregnancies remained viable following Ad.VEGF-A165 treatment. Fetal abdominal circumference and renal volume were greater in the Ad.VEGF-A165 group compared with the saline group at 21/28 days (P ≤ 0.04) postinjection. At delivery, gestation length (P = 0.07), lamb birthweight (P = 0.08), umbilical girth (P = 0.06), and plasma glucose (P = 0.09) tended to be greater in Ad.VEGF-A165-treated lambs. Levels of neonatal intervention required to ensure survival was equivalent between groups. Absolute postnatal growth rate (P = 0.02), insulin area under the curve (P = 0.04) and carcass weight at necropsy (P = 0.04) were increased by Ad.VEGF-A165 treatment. There was no impact on markers of insulin sensitivity or methylation/expression of key genes involved in somatic growth. Ad.VEGF-A165 gene therapy increased fetal growth in a sheep FGR model, and lambs continued to thrive during the neonatal and early postnatal period. PMID:27103444

  5. Comparison of two expression systems using COS7 cells and yeast cells for expression of heart/muscle-type carnitine palmitoyltransferase 1.

    PubMed

    Hada, Takuya; Kato, Yumiko; Obana, Eriko; Yamamoto, Atsushi; Yamazaki, Naoshi; Hashimoto, Mitsuru; Yamamoto, Takenori; Shinohara, Yasuo

    2012-03-01

    Carnitine palmitoyltransferase 1 (CPT1), catalyzing the transfer of the acyl group from acyl-CoA to carnitine to form acylcarnitine, is located at the outer mitochondrial membrane. Because it is easily inactivated by solubilization, expression systems using living cells are essential for its functional characterization. COS7 cells or yeast cells are often utilized for this purpose; however, the advantages/disadvantages of the use of these cells or the question as to how the CPT1 enzyme expressed by these cells differs are still uncertain. In this study, we characterized the heart/muscle-type isozyme of rat CPT1 (CPT1b) expressed by these two cellular expression systems. The mitochondrial fraction prepared from yeast cells expressing CPT1b showed 25% higher CPT1 activity than that obtained from COS7 cells. However, the expression level of CPT1b in the former was 3.8 times lower than that in the latter; and thus, under the present experimental conditions, the specific activity of CPT1b expressed in yeast cells was estimated to be approximately five times higher than that expressed in COS7 cells. Possible reasons for this difference are discussed. PMID:22266133

  6. The expression pattern of Follistatin-like 1 in mouse central nervous system development.

    PubMed

    Yang, Yang; Liu, Junhua; Mao, Huihua; Hu, Yu-An; Yan, Yan; Zhao, Chunjie

    2009-10-01

    Follistatin-like 1 (Fstl1), also named TSC-36 (TGF-beta-stimulated clone 36), was first cloned from the mouse osteoblastic MC3T3-E1 cell line and can be up-regulated by TGF-beta. To better study the function of Fstl1 during the development of the mouse central nervous system (CNS), we examined Fstl1 expression in the developing mouse CNS, in detail, by in situ hybridization. Our results show that Fstl1 is strongly expressed in the telencephalon, diencephalon, brainstem, limbic system and spinal cord. In the telencephalon, Fstl1 positive cells are mainly located in the ventricular zone (VZ) and the subventricular zone (SVZ); a relatively weak signal was observed in layers II and III of the neocortex at postnatal stages. Fstl1 expression is robust in the developing hippocampus and persists to P20. In the developing diencephalon and hindbrain, abundant Fstl1 signals were also detected in nuclei including the medial habenular nucleus, the medial dorsal nucleus, the cochlear nuclei and so on. In addition, a strong expression of Fstl1 was detected in the thalamencephalic signal center, as well as in the olfactory cortex from E14.5 to P0. Meanwhile, Fstl1 was expressed in the septal area and the cingulate gyrus of the limbic system after birth. A high level of expression was also observed in the ventral horn of the spinal cord. These results indicate that Fstl1 may play an important role during CNS development in the mouse. PMID:19595790

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

    PubMed Central

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

    2015-01-01

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

  8. A novel bidirectional expression system for simultaneous expression of both the protein-coding genes and short hairpin RNAs in mammalian cells

    SciTech Connect

    Hung, C.-F.; Cheng, T.-L.; Wu, R.-H.; Teng, C.-F.; Chang, W.-T. . E-mail: wtchang@mail.ncku.edu.tw

    2006-01-27

    RNA interference (RNAi) is an extremely powerful and widely used gene silencing approach for reverse functional genomics and molecular therapeutics. In mammals, the conserved poly(ADP-ribose) polymerase 2 (PARP-2)/RNase P bidirectional control promoter simultaneously expresses both the PARP-2 protein and RNase P RNA by RNA polymerase II- and III-dependent mechanisms, respectively. To explore this unique bidirectional control system in RNAi-mediated gene silencing strategy, we have constructed two novel bidirectional expression vectors, pbiHsH1 and pbiMmH1, which contained the PARP-2/RNase P bidirectional control promoters from human and mouse, for simultaneous expression of both the protein-coding genes and short hairpin RNAs. Analyses of the dual transcriptional activities indicated that these two bidirectional expression vectors could not only express enhanced green fluorescent protein as a functional reporter but also simultaneously transcribe shLuc for inhibiting the firefly luciferase expression. In addition, to extend its utility for the establishment of inherited stable clones, we have also reconstructed this bidirectional expression system with the blasticidin S deaminase gene, an effective dominant drug resistance selectable marker, and examined both the selection and inhibition efficiencies in drug resistance and gene expression. Moreover, we have further demonstrated that this bidirectional expression system could efficiently co-regulate the functionally important genes, such as overexpression of tumor suppressor protein p53 and inhibition of anti-apoptotic protein Bcl-2 at the same time. In summary, the bidirectional expression vectors, pbiHsH1 and pbiMmH1, should provide a simple, convenient, and efficient novel tool for manipulating the gene function in mammalian cells.

  9. Novel and tightly regulated resorcinol and cumate-inducible expression systems for Streptomyces and other actinobacteria.

    PubMed

    Horbal, Liliya; Fedorenko, Victor; Luzhetskyy, Andriy

    2014-10-01

    Inducible expression is a versatile genetic tool for controlling gene transcription, determining gene functions and other uses. Herein, we describe our attempts to create several inducible systems based on a cumate or a resorcinol switch, a hammerhead ribozyme, the LacI repressor, and isopropyl β-d-thiogalactopyranoside (IPTG). We successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in actinobacteria that is based on the CymR regulator, the operator sequence (cmt) from the Pseudomonas putida cumate degradation operon and P21 synthetic promoter. Resorcinol-inducible expression system is also functional and is composed of the RolR regulator and the PA3 promoter fused with the operator (rolO) from the Corynebacterium glutamicum resorcinol catabolic operon. Using the gusA (β-glucuronidase) gene as a reporter, we showed that the newly generated expression systems are tightly regulated and hyper-inducible. The activity of the uninduced promoters is negligible in both cases. Whereas the induction factor reaches 45 for Streptomyces albus in the case of cumate switch and 33 in the case of resorcinol toggle. The systems are also dose-dependent, which allows the modulation of gene expression even from a single promoter. In addition, the cumate system is versatile, given that it is functional in different actinomycetes. Finally, these systems are nontoxic and inexpensive, as these are characteristics of cumate and resorcinol, and they are easy to use because inducers are water-soluble and easily penetrate cells. Therefore, the P21-cmt-CymR and PA3-rolO-RolR systems are powerful tools for engineering actinobacteria. PMID:25012786

  10. High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

    PubMed

    Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

    2016-03-01

    An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases. PMID:26666439

  11. Plasmid-borne prokaryotic gene expression: Sources of variability and quantitative system characterization

    NASA Astrophysics Data System (ADS)

    Bagh, Sangram; Mazumder, Mostafizur; Velauthapillai, Tharsan; Sardana, Vandit; Dong, Guang Qiang; Movva, Ashok B.; Lim, Len H.; McMillen, David R.

    2008-02-01

    One aim of synthetic biology is to exert systematic control over cellular behavior, either for medical purposes or to “program” microorganisms. An engineering approach to the design of biological controllers demands a quantitative understanding of the dynamics of both the system to be controlled and the controllers themselves. Here we focus on a widely used method of exerting control in bacterial cells: plasmid vectors bearing gene-promoter pairs. We study two variants of the simplest such element, an unregulated promoter constitutively expressing its gene, against the varying genomic background of four Escherichia coli cell strains. Absolute protein numbers and rates of expression vary with both cell strain and plasmid type, as does the variability of expression across the population. Total variability is most strongly coupled to the cell division process, and after cell size is scaled away, plasmid copy number regulation emerges as a significant effect. We present simple models that capture the main features of the system behavior. Our results confirm that complex interactions between plasmids and their hosts can have significant effects on both expression and variability, even in deliberately simplified systems.

  12. Genome-wide analysis of immune system genes by expressed sequence Tag profiling.

    PubMed

    Giallourakis, Cosmas C; Benita, Yair; Molinie, Benoit; Cao, Zhifang; Despo, Orion; Pratt, Henry E; Zukerberg, Lawrence R; Daly, Mark J; Rioux, John D; Xavier, Ramnik J

    2013-06-01

    Profiling studies of mRNA and microRNA, particularly microarray-based studies, have been extensively used to create compendia of genes that are preferentially expressed in the immune system. In some instances, functional studies have been subsequently pursued. Recent efforts such as the Encyclopedia of DNA Elements have demonstrated the benefit of coupling RNA sequencing analysis with information from expressed sequence tags (ESTs) for transcriptomic analysis. However, the full characterization and identification of transcripts that function as modulators of human immune responses remains incomplete. In this study, we demonstrate that an integrated analysis of human ESTs provides a robust platform to identify the immune transcriptome. Beyond recovering a reference set of immune-enriched genes and providing large-scale cross-validation of previous microarray studies, we discovered hundreds of novel genes preferentially expressed in the immune system, including noncoding RNAs. As a result, we have established the Immunogene database, representing an integrated EST road map of gene expression in human immune cells, which can be used to further investigate the function of coding and noncoding genes in the immune system. Using this approach, we have uncovered a unique metabolic gene signature of human macrophages and identified PRDM15 as a novel overexpressed gene in human lymphomas. Thus, we demonstrate the utility of EST profiling as a basis for further deconstruction of physiologic and pathologic immune processes. PMID:23616578

  13. Tandem-yeast expression system for engineering and producing unspecific peroxygenase.

    PubMed

    Molina-Espeja, Patricia; Ma, Su; Mate, Diana M; Ludwig, Roland; Alcalde, Miguel

    2015-06-01

    Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (∼8mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production. PMID:26002501

  14. PCI express bus design of large format array IRFPA high-speed acquisition system

    NASA Astrophysics Data System (ADS)

    Huang, Zewu; Zheng, Xing; Zeng, Xingxin; Liu, Ziji

    2012-10-01

    In this paper, a novel solution of PCI Express Bus was designed to improve the data transfer rate for large format array infrared imaging acquisition system. In this structure, an embedded PCI Express hard intellectual property (IP) block of Stratix IV GX FPGA was used, and the protocol stack module is totally compliant with PCI Express base specification Gen 2.0 which includes PHY-MAC, Data Link, and transaction layers. In order to communicate with CPU through computer PCIe root port, a pipeline structure was established with two SSRAMs to carry out the function of real-time data process. The DMA mode was adopted for the high-speed data transmission on the PCI Express Bus. Some other control logic parts such as detector drive signal generator - display controller and PCIe configuration module were also designed and introduced in this paper. According to the evaluation, the data transmission speed was up to 5.6Gbps, which means that this system could meet the qualifications of infrared imaging data acquisition. Compared with traditional infrared imaging data acquisition systems, this solution is more integrated and faster, so it is suitable for larger format and higher frame rate of infrared focal plane image acquisition in nowadays and future.

  15. Klf4 Regulates the Expression of Slurp1, Which Functions as an Immunomodulatory Peptide in the Mouse Cornea

    PubMed Central

    Swamynathan, Sudha; Buela, Kristine-Ann; Kinchington, Paul; Lathrop, Kira L.; Misawa, Hidemi; Hendricks, Robert L.; Swamynathan, Shivalingappa K.

    2012-01-01

    Purpose. The secreted Ly6/uPAR-related protein-1 (Slurp1), associated with the hyperkeratotic disorder mal de Meleda, is abundantly expressed in corneas. Here, we examine its corneal expression and functions. Methods. Gene expression was quantified by quantitative PCR (qPCR), immunoblots, and immunofluorescent staining. Effect of Kruppel-like factor 4 (Klf4) on Slurp1 promoter was evaluated by chromatin immunoprecipitation (ChIP) and transient transfections. Adenoviral vectors were used to express Slurp1 in corneas. Leukocytic infiltration in bacterial lipopolysaccharide (LPS)–, herpes simplex virus type 1 (HSV-1)–, or adenovirus (serotype 5)–treated mouse corneas was characterized by flow cytometry. Results. Corneal expression of Slurp1 increased sharply upon mouse eyelid opening, concurrent with the elevated expression of Klf4. Slurp1 was significantly decreased in Klf4 conditional null (Klf4CN) corneas that displayed elevated expression of cytokines and cytokine receptors, as well as neutrophil influx consistent with a proinflammatory environment. In additional models of corneal inflammation, Slurp1 expression was abrogated within 24 hours of LPS injection or HSV-1 or adenoviral infection, accompanied by a predominantly neutrophilic infiltrate. Neutrophilic infiltration was enhanced in HSV-1-infected Klf4CN corneas lacking Slurp1. SLURP1 promoter activity was stimulated by KLF4, suppressed by IL-4, IL-13, and TNFα, and unperturbed by IFN-γ. Slurp1 downregulation and neutrophil influx were comparable in HSV-1-infected wild-type (WT) and Ifng−/− mouse corneas. Mouse corneas infected with Slurp1-expressing adenoviral vectors displayed reduced signs of inflammation and restricted neutrophilic infiltration compared with those infected with control vectors. Conclusions. Klf4 regulates the expression of Slurp1, a key immunomodulatory peptide that is abundantly expressed in healthy corneas and is downregulated in proinflammatory conditions. PMID:23139280

  16. Semi-analytical expression of stochastic closed curve attractors in nonlinear dynamical systems under weak noise

    NASA Astrophysics Data System (ADS)

    Guo, Kongming; Jiang, Jun; Xu, Yalan

    2016-09-01

    In this paper, a simple but accurate semi-analytical method to approximate probability density function of stochastic closed curve attractors is proposed. The expression of distribution applies to systems with strong nonlinearities, while only weak noise condition is needed. With the understanding that additive noise does not change the longitudinal distribution of the attractors, the high-dimensional probability density distribution is decomposed into two low-dimensional distributions: the longitudinal and the transverse probability density distributions. The longitudinal distribution can be calculated from the deterministic systems, while the probability density in the transverse direction of the curve can be approximated by the stochastic sensitivity function method. The effectiveness of this approach is verified by comparing the expression of distribution with the results of Monte Carlo numerical simulations in several planar systems.

  17. Recombinant Human Butyrylcholinesterase As a New-Age Bioscavenger Drug: Development of the Expression System

    PubMed Central

    Ilyushin, D.G.; Haertley, O.M.; Bobik, T.V.; Shamborant, O.G.; Surina, E.A.; Knorre, V.D.; Masson, P.; Smirnov, I.V.; Gabibov, A.G.; Ponomarenko, N.A.

    2013-01-01

    Butyrylcholinesterase (BChE) is a serine hydrolase (EC 3.1.1.8) which can be found in most animal tissues. This enzyme has a broad spectrum of efficacy against organophosphorus compounds, which makes it a prime candidate for the role of stoichiometric bioscavenger. Development of a new-age DNA-encoded bioscavenger is a vival task. Several transgenic expression systems of human BChE were developed over the past 20 years; however, none of them has been shown to make economic sense or has been approved for administration to humans. In this study, a CHO-based expression system was redesigned, resulting in a significant increase in the production level of functional recombinant human butyrylcholinesterase as compared to the hitherto existing systems. The recombinant enzyme was characterized with Elman and ELISA methods. PMID:23556132

  18. An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles

    PubMed Central

    Santi, Luca; Batchelor, Lance; Huang, Zhong; Hjelm, Brooke; Kilbourne, Jacquelyn; Arntzen, Charles J.; Chen, Qiang; Mason, Hugh S.

    2009-01-01

    Virus like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNV) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post infection. Oral immunization of CD1 mice with 100 or 250 μg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic. PMID:18325641

  19. Construction and application of a food-grade expression system for Lactococcus lactis.

    PubMed

    Lu, Wenwei; Kong, Jian; Kong, Wentao

    2013-06-01

    A food-grade host/vector expression system for Lactococcus lactis was constructed using alanine racemase gene (alr) as the complementation marker. We obtained an alanine racemase auxotrophic mutant L. lactis NZ9000Δalr by double-crossover recombination using temperature-sensitive integration plasmid pG(+)host9 and a food-grade vector pALR with entirely lactococcal DNA elements, including lactococcal replicon, nisin-inducible promoter PnisA and the alr gene from Lactobacillus casei BL23 as a complementation marker. By using the new food-grade host/vector system, the green fluorescent protein and capsid protein of porcine circovirus type II were successfully overexpressed under the nisin induction. These results indicate that this food-grade host/vector expression system has application potential as an excellent antigen delivery vehicle, and is also suitable for the use in the manufacture of ingredients for the food industry. PMID:22674186

  20. Expression Optimization and Inducible Negative Feedback in Cell-Free Systems

    SciTech Connect

    Karig, David K; Iyer, Sukanya; Simpson, Michael L; Doktycz, Mitchel John

    2012-01-01

    Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells. Cell-free approaches, on the other hand, offer simpler and more flexible contexts, but few synthetic systems based on cell-free protein expression have been constructed. Here, we evaluate cell-free regulatory systems based on T7 promoter driven expression, and we demonstrate negative feedback, an essential motif in many natural and engineered systems. First, we characterize variants of TetR and LacI repressible T7 promoters in a cell-free context and examine sequence elements that determine expression efficiency. Then, we explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in E. coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom up fashion.

  1. AMTEC radioisotope power system design and analysis for Pluto Express Fly-By

    SciTech Connect

    Hendricks, T.J.; Huang, C.; Sievers, R.K.

    1997-12-31

    The Pluto Express Fly-By program requires a Radioisotope Power System (RPS) to supply spacecraft power for various internal functions and mission instruments and experiments. AMTEC (Alkali-Metal Thermal-Electric Conversion) power conversion is the DOE-selected technology for an advanced, high-efficiency RPS to power the Pluto Express Fly-By spacecraft. An AMTEC-based RPS using the General Purpose Heat Source (GPHS) has been conceptually designed to satisfy the Pluto Express power requirements. Integrated AMTEC cell and system thermal/electrical design analyses, structural design analyses, and mass analyses were performed to define an optimum system design. Using fresh radioisotope fuel at beginning of mission, the RPS produces 102 watts of power, has a mass of 8.35 kg (specific power density = 12.2 watts/kg), with a system conversion efficiency of 20.3%. Mass/power scale-up estimates have also been generated, indicating that a 150-watt version of this RPS would weigh approximately 11.3 kg. This paper presents and discusses the key features of this RPS design, the design and analysis methodology, and the numerous system and AMTEC cell tradeoff studies establishing the optimum AMTEC-based RPS.

  2. Unbiased expression mapping identifies a link between the complement and cholinergic systems in the rat central nervous system.

    PubMed

    Lindblom, Rickard P F; Ström, Mikael; Heinig, Matthias; Al Nimer, Faiez; Aeinehband, Shahin; Berg, Alexander; Dominguez, Cecilia A; Vijayaraghavan, Swetha; Zhang, Xing-Mei; Harnesk, Karin; Zelano, Johan; Hübner, Norbert; Cullheim, Staffan; Darreh-Shori, Taher; Diez, Margarita; Piehl, Fredrik

    2014-02-01

    The complement system is activated in a wide spectrum of CNS diseases and is suggested to play a role in degenerative phenomena such as elimination of synaptic terminals. Still, little is known of mechanisms regulating complement activation in the CNS. Loss of synaptic terminals in the spinal cord after an experimental nerve injury is increased in the inbred DA strain compared with the PVG strain and is associated with expression of the upstream complement components C1q and C3, in the absence of membrane attack complex activation and neutrophil infiltration. To further dissect pathways regulating complement expression, we performed genome-wide expression profiling and linkage analysis in a large F2(DA × PVG) intercross, which identified quantitative trait loci regulating expression of C1qa, C1qb, C3, and C9. Unlike C1qa, C1qb, and C9, which all displayed distinct coregulation with different cis-regulated C-type lectins, C3 was regulated in a coexpression network immediately downstream of butyrylcholinesterase. Butyrylcholinesterase hydrolyses acetylcholine, which exerts immunoregulatory effects partly through TNF-α pathways. Accordingly, increased C3, but not C1q, expression was demonstrated in rat and mouse glia following TNF-α stimulation, which was abrogated in a dose-dependent manner by acetylcholine. These findings demonstrate new pathways regulating CNS complement expression using unbiased mapping in an experimental in vivo system. A direct link between cholinergic activity and complement activation is supported by in vitro experiments. The identification of distinct pathways subjected to regulation by naturally occurring genetic variability is of relevance for the understanding of disease mechanisms in neurologic conditions characterized by neuronal injury and complement activation. PMID:24353269

  3. Expression pattern of drought stress marker genes in soybean roots under two water deficit systems

    PubMed Central

    Neves-Borges, Anna Cristina; Guimarães-Dias, Fábia; Cruz, Fernanda; Mesquita, Rosilene Oliveira; Nepomuceno, Alexandre Lima; Romano, Eduardo; Loureiro, Marcelo Ehlers; de Fátima Grossi-de-Sá, Maria; Alves-Ferreira, Márcio

    2012-01-01

    The study of tolerance mechanisms for drought stress in soybean is fundamental to the understanding and development of tolerant varieties. Using in silico analysis, four marker genes involved in the classical ABA-dependent and ABA-independent pathways of drought response were identified in the Glycine max genome in the present work. The expression profiles of the marker genes ERD1-like, GmaxRD20A-like, GmaxRD22-like and GmaxRD29B-like were investigated by qPCR in root samples of drought sensitive and tolerant soybean cultivars (BR 16 and Embrapa 48, respectively), submitted to water deficit conditions in hydroponic and pot-based systems. Among the four putative soybean homologs to Arabidopsis genes investigated herein, only GmaxRD29B-like was not regulated by water deficit stress. Distinct expression profiles and different induction levels were observed among the genes, as well as between the two drought-inducing systems. Our results showed contrasting gene expression responses for the GmaxRD20A-like and GmaxRD22-like genes. GmaxRD20A-like was highly induced by continuous drought acclimating conditions, whereas GmaxRD22-like responses decreased after abrupt water deprivation. GmaxERD1-like showed a different expression profile for the cultivars in each system. Conversely, GmaxRD20A-like and GmaxRD22-like genes exhibited similar expression levels in tolerant plants in both systems. PMID:22802707

  4. Role of plant expression systems in antibody production for passive immunization.

    PubMed

    Virdi, Vikram; Depicker, Ann

    2013-01-01

    Passive immunization is a method to achieve immediate protection against infectious agents by administering pathogen-specific antibodies. It has proven to be lifesaving for many acute infections, and it is now also used for cancer treatment. Passive immunization therapies, however, are extremely expensive because they require large amounts of specific antibodies that are produced predominantly in mammalian expression systems. The cost for manufacturing plant-made antibodies is estimated to be comparatively low since plant production systems require relatively less capital investments. In addition, they are not prone to mammalian pathogens, which also eases downstream processing along with making it a safe expression system. Moreover, some of the recent developments in transient expression have enabled rapid, cGMP (current Good Manufacturing Practices) compliant manufacturing of antibodies. Whether lower production costs will be reflected in a lower market price for purified antibodies will be known when more plant-produced antibodies come to the market. Promisingly, the current molecular techniques in the field of in planta expression have enabled high-level production of a variety of antibodies in different plant organs, like roots/tubers/fruits, leaves and seeds, of a variety of plants, like potato, tobacco, maize, rice, tomato and pea, providing a very wide range of possible plant-based passive immunization therapies. For instance, the production of antibodies in edible tissues would allow for a unique, convenient, needle-less, oral passive immunization at the gastric mucosal surface. The technological advances, together with the innate capacity of plant tissues to assemble complex antibodies, will enable carving a niche in the antibody market. This non-exhaustive review aims to shed light on the role of plants as a flexible expression system for passive immunotherapy, which we envisage to progress alongside the conventional production platforms to manufacture

  5. Expression and one-step purification of the antimicrobial peptide cathelicidin-BF using the intein system in Bacillus subtilis.

    PubMed

    He, Qing; Fu, Ai-yun; Li, Tian-jiao

    2015-04-01

    The intein expression system has been widely applied in Escherichia coli to express various proteins and peptides. However, the removal of endotoxin from the recombinant proteins expressed in E. coli is very difficult and therefore complicates the purification process. In this study, we constructed an intein-based expression vector for an antimicrobial peptide (cathelicidin from Bungarus fasciatus) and expressed the intein fusion peptide in a Bacillus subtilis expression system. The fusion peptide was secreted into the culture medium, identified by Western blot and purified by affinity chromatography and intein self-cleavage in just one step. Approximately, 0.5 mg peptide was obtained from 1 litre of culture medium. The purified peptide showed antimicrobial activity. Our results indicate that the intein expression system may be a safe and efficient method to produce soluble peptides and proteins in B. subtilis. PMID:25578306

  6. A transient three-plasmid expression system for the production of high titer retroviral vectors.

    PubMed

    Soneoka, Y; Cannon, P M; Ramsdale, E E; Griffiths, J C; Romano, G; Kingsman, S M; Kingsman, A J

    1995-02-25

    We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants. PMID:7899083

  7. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.

    PubMed

    Livet, Jean; Weissman, Tamily A; Kang, Hyuno; Draft, Ryan W; Lu, Ju; Bennis, Robyn A; Sanes, Joshua R; Lichtman, Jeff W

    2007-11-01

    Detailed analysis of neuronal network architecture requires the development of new methods. Here we present strategies to visualize synaptic circuits by genetically labelling neurons with multiple, distinct colours. In Brainbow transgenes, Cre/lox recombination is used to create a stochastic choice of expression between three or more fluorescent proteins (XFPs). Integration of tandem Brainbow copies in transgenic mice yielded combinatorial XFP expression, and thus many colours, thereby providing a way to distinguish adjacent neurons and visualize other cellular interactions. As a demonstration, we reconstructed hundreds of neighbouring axons and multiple synaptic contacts in one small volume of a cerebellar lobe exhibiting approximately 90 colours. The expression in some lines also allowed us to map glial territories and follow glial cells and neurons over time in vivo. The ability of the Brainbow system to label uniquely many individual cells within a population may facilitate the analysis of neuronal circuitry on a large scale. PMID:17972876

  8. Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    PubMed Central

    Yang, Liang; Wang, Xu; Deng, Weixian; Mo, Weiliang; Gao, Jie; Liu, Qing; Zhang, Chuanyu; Wang, Qin; Lin, Chentao; Zuo, Zecheng

    2016-01-01

    Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein–protein interactions. PMID:27446167

  9. Heat flux expressions that satisfy the conservation laws in atomistic system involving multibody potentials

    NASA Astrophysics Data System (ADS)

    Fu, Yao; Song, Jeong-Hoon

    2015-08-01

    Heat flux expressions are derived for multibody potential systems by extending the original Hardy's methodology and modifying Admal & Tadmor's formulas. The continuum thermomechanical quantities obtained from these two approaches are easy to compute from molecular dynamics (MD) results, and have been tested for a constant heat flux model in two distinctive systems: crystalline iron and polyethylene (PE) polymer. The convergence criteria and affecting parameters, i.e. spatial and temporal window size, and specific forms of localization function are found to be different between the two systems. The conservation of mass, momentum, and energy are discussed and validated within this atomistic-continuum bridging.

  10. Heat flux expressions that satisfy the conservation laws in atomistic system involving multibody potentials

    SciTech Connect

    Fu, Yao Song, Jeong-Hoon

    2015-08-01

    Heat flux expressions are derived for multibody potential systems by extending the original Hardy's methodology and modifying Admal & Tadmor's formulas. The continuum thermomechanical quantities obtained from these two approaches are easy to compute from molecular dynamics (MD) results, and have been tested for a constant heat flux model in two distinctive systems: crystalline iron and polyethylene (PE) polymer. The convergence criteria and affecting parameters, i.e. spatial and temporal window size, and specific forms of localization function are found to be different between the two systems. The conservation of mass, momentum, and energy are discussed and validated within this atomistic–continuum bridging.

  11. Modular and coordinated expression of immune system regulatory and signaling components in the developing and adult nervous system

    PubMed Central

    Monzón-Sandoval, Jimena; Castillo-Morales, Atahualpa; Crampton, Sean; McKelvey, Laura; Nolan, Aoife; O’Keeffe, Gerard; Gutierrez, Humberto

    2015-01-01

    During development, the nervous system (NS) is assembled and sculpted through a concerted series of neurodevelopmental events orchestrated by a complex genetic programme. While neural-specific gene expression plays a critical part in this process, in recent years, a number of immune-related signaling and regulatory components have also been shown to play key physiological roles in the developing and adult NS. While the involvement of individual immune-related signaling components in neural functions may reflect their ubiquitous character, it may also reflect a much wider, as yet undescribed, genetic network of immune–related molecules acting as an intrinsic component of the neural-specific regulatory machinery that ultimately shapes the NS. In order to gain insights into the scale and wider functional organization of immune-related genetic networks in the NS, we examined the large scale pattern of expression of these genes in the brain. Our results show a highly significant correlated expression and transcriptional clustering among immune-related genes in the developing and adult brain, and this correlation was the highest in the brain when compared to muscle, liver, kidney and endothelial cells. We experimentally tested the regulatory clustering of immune system (IS) genes by using microarray expression profiling in cultures of dissociated neurons stimulated with the pro-inflammatory cytokine TNF-alpha, and found a highly significant enrichment of immune system-related genes among the resulting differentially expressed genes. Our findings strongly suggest a coherent recruitment of entire immune-related genetic regulatory modules by the neural-specific genetic programme that shapes the NS. PMID:26379506

  12. Expression of receptor protein tyrosine phosphatase δ, PTPδ, in mouse central nervous system.

    PubMed

    Shishikura, Maria; Nakamura, Fumio; Yamashita, Naoya; Uetani, Noriko; Iwakura, Yoichiro; Goshima, Yoshio

    2016-07-01

    Protein tyrosine phosphate δ (PTPδ), one of the receptor type IIa protein tyrosine phosphates, is known for its roles in axon guidance, synapse formation, cell adhesion, and tumor suppression. Alternative splicing of this gene generates at least four (A-D) isoforms; however, the major isoform in vivo is yet to be determined. The protein localization has neither been revealed. We have generated anti-mouse PTPδ-specific monoclonal antibody and analyzed the protein expression in wild-type and Ptpδ knockout mice. Immunoblot analysis of various organs revealed that neuronal tissues express both C-and D-isoforms of PTPδ, whereas non-neuronal tissues express only C-isoform. Immunohistochemistry of wild-type or Ptpδ heterozygous sections showed that olfactory bulb, cerebral cortex, hippocampus, cerebellum, and several nuclei in brain stem exhibit moderate to strong positive signals. These signals were absent in Ptpδ knockout specimens. Higher magnification revealed differences between expression patterns of PTPδ mRNA and its protein product. In hippocampus, weak mRNA expression in CA1 stratum pyramidale but strong immunostaining in the stratum lacunosum moleculare was observed, suggesting the axonal expression of PTPδ in the entorhinal cortical afferents. Olfactory mitral cells exhibited mRNA expression in cell bodies and protein localization in their dendritic fields, glomerular and external plexiform layers. Nissl staining showed that the external plexiform layer was reduced in Ptpδ knockout mice. Golgi-impregnation confirmed the poor dendritic growth of homozygous mitral cells. These results suggest that PTPδ may localize in axons as well as in dendrites to regulate their elaboration in the central nervous system. PMID:27026654

  13. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    PubMed

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  14. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

    PubMed Central

    Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  15. A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

    DOE PAGESBeta

    Linger, Jeffrey G.; Taylor, II, Larry E.; Baker, John O.; Vander Wall, Todd; Hobdey, Sarah E.; Podkaminer, Kara; Himmel, Michael E.; Decker, Stephen R.

    2015-03-18

    One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated amore » native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose

  16. Integrative Food-Grade Expression System Based on the Lactose Regulon of Lactobacillus casei

    PubMed Central

    Gosalbes, María José; Esteban, Carlos David; Galán, José Luis; Pérez-Martínez, Gaspar

    2000-01-01

    The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L. casei. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3′ end of lacG and the complete lacF gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of L. casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the E. coli gusA gene and the L. lactis ilvBN genes in L. casei. Following the same expression pattern as that for the lactose genes, β-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in L. casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses. PMID:11055930

  17. Immunohistochemical expression of p53 and its clinicopathological correlation with modified Anneroth's histological grading system

    PubMed Central

    Dave, Kajal V; Chalishazar, Monali; Dave, Vishal R; Panja, Pritam; Singh, Manisha; Modi, Tapan G

    2016-01-01

    Introduction and Objectives: Oral squamous cell carcinoma (OSCC) is an epithelial neoplasm generally beginning as focal overgrowth of altered stem cells near the basement membrane, moving upward and laterally, replacing the normal epithelium. Histopathological grading has been used for many decades in an attempt to predict the clinical behavior of oral squamous cell carcinoma. In the present study, Forty biopsies were studied for histological grading and p53 expression. The p53 expression was studied in relation to clinical parameters such as age, sex of patient and site of tumors. Relation between histological grade of malignancy and p53 protein expression was analysed. All cases were classified according to Anneroth's histological malignancy grading system (1987). Materials and Methods: 40 cases of OSCC were assessed for clinical parameters, Anneroth's histological grading and immunohistochemically stained with p53 protien. Statistical Analysis: The results obtained were analyzed using Spearman's Co-relation. Observations and Results: The positive expression of p53 was found in 62% of carcinomas studied. Positivity of p53 showed correlation with histological grade of malignancy and with individual parameters like degree of keratinization, nuclear polymorphism, number of mitoses and lymphoplasmacytic infiltration while showed a negative correlation with pattern of invasion. Conclusion: Our study showed a significant correlation between parameters of tumor cell population, lymphoplasmacytic infiltration and p53 expression. A significant association between high grade of malignancy and p53 overexpression and insignificant correlation of p53 with age, sex of the patient and site of the tumor was found. PMID:27194859

  18. The adenoviral E1A N-terminal domain represses MYC transcription in human cancer cells by targeting both p300 and TRRAP and inhibiting MYC promoter acetylation of H3K18 and H4K16

    PubMed Central

    Zhao, Ling-Jun; Loewenstein, Paul M.; Green, Maurice

    2016-01-01

    Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells.

  19. Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases

    PubMed Central

    Klinger, Johannes; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host. PMID:26648951