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

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

Varicella Zoster Virus ORF25 Gene Product: An Essential Hub Protein Linking Encapsidation Proteins and the Nuclear Egress Complex

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.Fil: Vizoso Pinto, María Guadalupe. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Pothineni, Venkata R.. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; AlemaniaFil: Haase, Rudolf. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; AlemaniaFil: Woidy, Mathias. Ludwig Maximilians Universitat; AlemaniaFil: Lotz Havla, Amelie. Ludwig Maximilians Universitat; AlemaniaFil: Gersting, Soren W.. Ludwig Maximilians Universitat; AlemaniaFil: Muntau, Ania C.. Ludwig Maximilians Universitat; AlemaniaFil: Haas, Jurgen. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; AlemaniaFil: Sommer, Marvin. University of Stanford; Estados UnidosFil: Arvin, Ann M.. University of Stanford; Estados UnidosFil: Baiker, Armin. Bavarian Health and Food Safety Authority; Alemani

The interactome of Streptococcus pneumoniae and its bacteriophages show highly specific patterns of interactions among bacteria and their phages

Although an abundance of bacteriophages exists, little is known about interactions between their proteins and those of their bacterial hosts. Here, we experimentally determined the phage-host interactomes of the phages Dp-1 and Cp-1 and their underlying protein interaction network in the host Streptococcus pneumoniae. We compared our results to the interaction patterns of E. coli phages lambda and T7. Dp-1 and Cp-1 target highly connected host proteins, occupy central network positions, and reach many protein clusters through the interactions of their targets. In turn, lambda and T7 targets cluster to conserved and essential proteins in E. coli, while such patterns were largely absent in S. pneumoniae. Furthermore, targets in E. coli were mutually strongly intertwined, while targets of Dp-1 and Cp-1 were strongly connected through essential and orthologous proteins in their immediate network vicinity. In both phage-host systems, the impact of phages on their protein targets appears to extend from their network neighbors, since proteins that interact with phage targets were located in central network positions, have a strong topologically disruptive effect and touch complexes with high functional heterogeneity. Such observations suggest that the phages, biological impact is accomplished through a surprisingly limited topological reach of their targets

Varicella zoster virus infection of malignant glioma cell cultures: a new candidate for oncolytic virotherapy?

BACKGROUND: Glioblastoma multiforme is a highly aggressive tumor with a median survival of 14 months despite all standard therapies. Focusing on alternative treatment strategies, we evaluated the oncolytic potential of varicella zoster virus (VZV) in malignant glioma cell cultures. MATERIALS AND METHODS: Replication of wildtype and mutant VZV was comparatively analyzed in glioma cell lines (U87, U251 and U373) and in primary malignant glioma cells (n=10) in vitro by infectious foci assay, immunofluorescence microscopy and western blot analysis. Additionally, the tumor-targeting potential of VZV-infected human mesenchymal stem cells was evaluated. RESULTS: VZV replicated efficiently in all the glioma cells studied here followed by rapid oncolysis in vitro. The attenuated vaccine VZV mutant rOKA/ORF63rev[T171] exhibited most efficient replication. Human mesenchymal stem cells were found suitable for targeting VZV to sites of tumor growth. CONCLUSION: VZV exhibits an intrinsic oncolytic potential in malignant glioma cell cultures and might be a novel candidate for virotherapy in glioblastoma multiforme

Cell-Free Culture Supernatant of Bifidobacterium breve CNCM I-4035 Decreases Pro-Inflammatory Cytokines in Human Dendritic Cells Challenged with Salmonella typhi through TLR Activation

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro-inflammatory pathways.This study was supported by Hero Spain S. A. through a number 3143 contract signed with the Fundación General Universidad de Granada Empresa and co-sponsored by a CDTI project, a public entity of the Ministry of Economy and Competitiveness of the Spanish Government