scholarly journals Aluminum particles generated during millisecond electric pulse application enhance adenovirus-mediated gene transfer in L929 cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Angela Tesse ◽  
Franck M. André ◽  
Thierry Ragot
Keyword(s):  
Low Voltage ◽  
Electric Pulse ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
Pulse Number ◽  
Viral Gene ◽  
Aluminum Particles ◽  
L929 Cells ◽  
Electric Pulses ◽  
Direct Cell

AbstractGene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy.

scholarly journals The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

mSphere ◽  
2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Frédéric Dallaire ◽  
Sabrina Schreiner ◽  
G. Eric Blair ◽  
Thomas Dobner ◽  
Philip E. Branton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Viral Gene Expression ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
Viral Gene ◽  
E2f Transcription Factors

ABSTRACT During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication.

scholarly journals Adenovirus Late-Phase Infection Is Controlled by a Novel L4 Promoter

2010 ◽  
Vol 84 (14) ◽  
pp. 7096-7104 ◽  
Author(s):  
Susan J. Morris ◽  
Gillian E. Scott ◽  
Keith N. Leppard
Keyword(s):  
Gene Expression ◽  
Intermediate Phase ◽  
Expression Patterns ◽  
Late Phase ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
Late Gene ◽  
Genome Replication ◽  
Late Gene Expression ◽  
Viral Genome Replication

ABSTRACT During human adenovirus 5 infection, a temporal cascade of gene expression leads ultimately to the production of large amounts of the proteins needed to construct progeny virions. However, the mechanism for the activation of the major late gene that encodes these viral structural proteins has not been well understood. We show here that two key positive regulators of the major late gene, L4-22K and L4-33K, previously thought to be expressed under the control of the major late promoter itself, initially are expressed from a novel promoter that is embedded within the major late gene and dedicated to their expression. This L4 promoter is required for late gene expression and is activated by a combination of viral protein activators produced during the infection, including E1A, E4 Orf3, and the intermediate-phase protein IVa2, and also by viral genome replication. This new understanding redraws the long-established view of how adenoviral gene expression patterns are controlled and offers new ways to manipulate that gene expression cascade for adenovirus vector applications.

scholarly journals COVID-19: Coronavirus Vaccine Development Updates

2020 ◽  
Vol 11 ◽  
Author(s):  
Jing Zhao ◽  
Shan Zhao ◽  
Junxian Ou ◽  
Jing Zhang ◽  
Wendong Lan ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Viral Vector ◽  
Peptide Vaccine ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
Research Progress ◽  
Nucleic Acid Vaccines ◽  
Inactivated Vaccines ◽  
The Common ◽  
Common Genus

Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a newly emerged coronavirus, and has been pandemic since March 2020 and led to many fatalities. Vaccines represent the most efficient means to control and stop the pandemic of COVID-19. However, currently there is no effective COVID-19 vaccine approved to use worldwide except for two human adenovirus vector vaccines, three inactivated vaccines, and one peptide vaccine for early or limited use in China and Russia. Safe and effective vaccines against COVID-19 are in urgent need. Researchers around the world are developing 213 COVID-19 candidate vaccines, among which 44 are in human trials. In this review, we summarize and analyze vaccine progress against SARS-CoV, Middle-East respiratory syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, including inactivated vaccines, live attenuated vaccines, subunit vaccines, virus like particles, nucleic acid vaccines, and viral vector vaccines. As SARS-CoV-2, SARS-CoV, and MERS-CoV share the common genus, Betacoronavirus, this review of the major research progress will provide a reference and new insights into the COVID-19 vaccine design and development.

Calcium influx determines the muscular response to electrotransfer

2012 ◽  
Vol 302 (4) ◽  
pp. R446-R453 ◽  
Author(s):  
Pernille Hojman ◽  
Camilla Brolin ◽  
Hanne Gissel
Keyword(s):  
Cell Membrane ◽  
High Voltage ◽  
Voltage Pulse ◽  
Low Voltage ◽  
Calcium Influx ◽  
High Voltage Pulse ◽  
Electric Pulses ◽  
Tnf Α ◽  
Cell Membrane Permeabilization ◽  
The Times

Cell membrane permeabilization by electric pulses (electropermeabilization), results in free exchange of ions across the cell membrane. The role of electrotransfer-mediated Ca2+-influx on muscle signaling pathways involved in degeneration (β-actin and MurF), inflammation (IL-6 and TNF-α), and regeneration (MyoD1, myogenin, and Myf5) was investigated, using pulse parameters of both electrochemotherapy (8 HV) and DNA delivery (HVLV). Three pulsing conditions were used: 8 high-voltage pulses (8 HV), resulting in large permeabilization and ion flux, and a combination of one high-voltage pulse and one low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca2+ was assessed using 45Ca as a tracer. Using gene expression analyses and histology, we showed a clear association between Ca2+ influx and muscular response. Moderate Ca2+ influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA reducing Ca2+ influx. Larger Ca2+ influx as induced by 8-HV pulses leads to muscle damage and muscle fiber regeneration through recruitment of satellite cells. The extent of Ca2+ influx determines the muscular response to electrotransfer and, thus, the success of a given application. In the case of electrochemotherapy, in which the objective is cell death, a large influx of Ca2+ may be beneficial, whereas for DNA electrotransfer, muscle recovery should occur without myofiber loss to ensure preservation of plasmid DNA.

scholarly journals Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector

Vaccine ◽  
2007 ◽  
Vol 25 (15) ◽  
pp. 2886-2891 ◽  
Author(s):  
Haroldo Toro ◽  
De-chu C. Tang ◽  
David L. Suarez ◽  
Matt J. Sylte ◽  
Jennifer Pfeiffer ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
In Ovo

Transient Electroluminescence from PPV under Strong Voltage Pulses

1997 ◽  
Vol 488 ◽  
Author(s):  
V. Savvate'ev ◽  
R. Pogreb ◽  
D. Davidov ◽  
H. Chayet ◽  
R. Neumann ◽  
...  
Keyword(s):  
Peak Power ◽  
Electric Pulse ◽  
Metal Electrode ◽  
Emission Pulse ◽  
Light Emitting ◽  
Electric Pulses ◽  
Pulsed Mode ◽  
Polymer Metal ◽  
High Fields ◽  
Transient Electroluminescence

AbstractWe have studied the transient electroluminescence (EL) from poly(phenylenevinylene), PPV, as a function of electric field under strong electric pulses up to fields of E≈ 109 V/m with emphasis on (a) the time delay, τ, between the electric pulse and the onset of the EL emission pulse and (b) the EL intensity as a function of the field. A monotonic decrease of τ with increasing E is explained by an increase of the carriers mobility according to Frenkel- Poole model. The EL intensity at high fields is proportional to E3 suggesting that the contacts at the polymer-metal electrode interfaces are practically ohmic. We demonstrate significantly improved brightness, peak power and lifetimes for polymer-based light emitting diodes working under such a pulsed mode.

scholarly journals Phylogenetic Analysis and Structural Predictions of Human Adenovirus Penton Proteins as a Basis for Tissue-Specific Adenovirus Vector Design

2007 ◽  
Vol 81 (15) ◽  
pp. 8270-8281 ◽  
Author(s):  
Ijad Madisch ◽  
Soeren Hofmayer ◽  
Christian Moritz ◽  
Alexander Grintzalis ◽  
Jens Hainmueller ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Secondary Structure ◽  
Rational Design ◽  
Immune Escape ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
Site Directed Mutagenesis ◽  
Penton Base ◽  
Base Function ◽  
Tissue Specific

ABSTRACT The penton base is a major capsid protein of human adenoviruses (HAdV) which forms the vertices of the capsid and interacts with hexon and fiber protein. Two hypervariable loops of the penton are exposed on the capsid surface. Sequences of these and 300 adjacent amino acid residues of all 51 HAdV and closely related simian adenoviruses were studied. Adjacent sequences and predicted overall secondary structure were conserved. Phylogenetic analysis revealed clustering corresponding to the HAdV species and recombination events in the origin of HAdV prototypes. All HAdV except serotypes 40 and 41 of species F exhibited an integrin binding RGD motif in the second loop. The lengths of the loops (HVR1 and RGD loops) varied significantly between HAdV species with the longest RGD loop observed in species C and the longest HVR1 in species B. Long loops may permit the insertion of motifs that modify tissue tropism. Genetic analysis of HAdV prime strain p17′H30, a neutralization variant of HAdV-D17, indicated the significance of nonhexon neutralization epitopes for HAdV immune escape. Fourteen highly conserved motifs of the penton base were analyzed by site-directed mutagenesis of HAdV-D8 and tested for sustained induction of early cytopathic effects. Thus, three new motifs essential for penton base function were identified additionally to the RGD site, which interacts with a secondary cellular receptor responsible for internalization. Therefore, our penton primary structure data and secondary structure modeling in combination with the recently published fiber knob sequences may permit the rational design of tissue-specific adenoviral vectors.

scholarly journals 43IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS WITH 6-DMAP FOLLOWING FUSION

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
G.-S. IM ◽  
L. Lai ◽  
Z. Liu ◽  
Y. Hao ◽  
C.M. Murphy ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Developmental Rate ◽  
Cell Number ◽  
Blastocyst Stage ◽  
In Vitro Development ◽  
Electric Pulses ◽  
Developmental Rates ◽  
Vitro Development

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P<0.05) developmental rates to the blastocyst stage. In Experiment 2, developmental rates to the blastocyst stage for C, D0 or D20 were 10.0, 12.3, and 19.9%, respectively. Developmental rate to the blastocyst stage was higher (P<0.05) in D20. Fragmentation rates were 19.9, 10.8, and 9.0%, respectively. Regardless of Ca2+ concentration in fusion medium, continued treatments with chemicals following electric pulses supported more development of porcine activated oocytes. Treating NT embryos with 6-DMAP alone after fusion was completed by using 1.0mM Ca2+ in fusion medium improved the developmental rates to the blastocyst stage and prevented fragmentation accompanied by electric fusion. This study was supported by NIH NCRR 13438 and Food for the 21st Century.

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