scholarly journals Coronaviruses Maintain Viability despite Dramatic Rearrangements of the Strictly Conserved Genome Organization

2002 ◽  
Vol 76 (24) ◽  
pp. 12491-12502 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Haukeline Volders ◽  
Cheri A. Koetzner ◽  
Paul S. Masters ◽  
Peter J. M. Rottier
Keyword(s):  
Gene Order ◽  
High Frequency ◽  
Genome Organization ◽  
Growth Characteristics ◽  
Rna Recombination ◽  
Viral Genes ◽  
Targeted Recombination ◽  
Reduced Risk

ABSTRACT Despite their high frequency of RNA recombination, the plus-strand coronaviruses have a characteristic, strictly conserved genome organization with the essential genes occurring in the order 5′-polymerase (pol)-S-E-M-N-3′. We have investigated the significance of this remarkable conservation by rearrangement of the murine coronavirus genome through targeted recombination. Thus, viruses were prepared with the following gene order: 5′-pol-S-M-E-N-3′, 5′-pol-S-N-E-M-3′, 5′-pol-M-S-E-N-3′, and 5′-pol-E-M-S-N-3′. All of these viruses were surprisingly viable, and most viruses replicated in cell culture with growth characteristics similar to those of the parental virus. The recombinant virus with the gene order 5′-pol-E-M-S-N-3′ was also tested for the ability to replicate in the natural host, the mouse. The results indicate that the canonical coronavirus genome organization is not essential for replication in vitro and in vivo. Deliberate rearrangement of the viral genes may be useful in the generation of attenuated coronaviruses, which due to their reduced risk of generating viable viruses by recombination with circulating field viruses, would make safer vaccines.

scholarly journals Selective elimination of immunosuppressive T cells in patients with multiple myeloma

Leukemia ◽  
2021 ◽  
Author(s):  
Mohamed H. S. Awwad ◽  
Abdelrahman Mahmoud ◽  
Heiko Bruns ◽  
Hakim Echchannaoui ◽  
Katharina Kriegsmann ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Immune Responses ◽  
High Frequency ◽  
Surface Markers ◽  
Selective Elimination ◽  
Related Transcription Factor ◽  
Cell Membrane Protein

AbstractElimination of suppressive T cells may enable and enhance cancer immunotherapy. Here, we demonstrate that the cell membrane protein SLAMF7 was highly expressed on immunosuppressive CD8+CD28-CD57+ Tregs in multiple myeloma (MM). SLAMF7 expression associated with T cell exhaustion surface markers and exhaustion-related transcription factor signatures. T cells from patients with a high frequency of SLAMF7+CD8+ T cells exhibited decreased immunoreactivity towards the MART-1aa26–35*A27L antigen. A monoclonal anti-SLAMF7 antibody (elotuzumab) specifically depleted SLAMF7+CD8+ T cells in vitro and in vivo via macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). Anti-SLAMF7 treatment of MM patients depleted suppressive T cells in peripheral blood. These data highlight SLAMF7 as a marker for suppressive CD8+ Treg and suggest that anti-SLAMF7 antibodies can be used to boost anti-tumoral immune responses in cancer patients.

Effect of Common Anesthetics on Dendritic Properties in Layer 5 Neocortical Pyramidal Neurons

2008 ◽  
Vol 99 (3) ◽  
pp. 1394-1407 ◽  
Author(s):  
Sarah Potez ◽  
Matthew E. Larkum
Keyword(s):  
High Frequency ◽  
Pyramidal Neurons ◽  
Animal Experiments ◽  
Apical Dendrite ◽  
Network Activity ◽  
Calcium Spikes ◽  
Firing Properties ◽  
The Impact

Understanding the impact of active dendritic properties on network activity in vivo has so far been restricted to studies in anesthetized animals. However, to date no study has been made to determine the direct effect of the anesthetics themselves on dendritic properties. Here, we investigated the effects of three types of anesthetics commonly used for animal experiments (urethane, pentobarbital and ketamine/xylazine). We investigated the generation of calcium spikes, the propagation of action potentials (APs) along the apical dendrite and the somatic firing properties in the presence of anesthetics in vitro using dual somatodendritic whole cell recordings. Calcium spikes were evoked with dendritic current injection and high-frequency trains of APs at the soma. Surprisingly, we found that the direct actions of anesthetics on calcium spikes were very different. Two anesthetics (urethane and pentobarbital) suppressed dendritic calcium spikes in vitro, whereas a mixture of ketamine and xylazine enhanced them. Propagation of spikes along the dendrite was not significantly affected by any of the anesthetics but there were various changes in somatic firing properties that were highly dependent on the anesthetic. Last, we examined the effects of anesthetics on calcium spike initiation and duration in vivo using high-frequency trains of APs generated at the cell body. We found the same anesthetic-dependent direct effects in addition to an overall reduction in dendritic excitability in anesthetized rats with all three anesthetics compared with the slice preparation.

scholarly journals Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo

2003 ◽  
Vol 77 (2) ◽  
pp. 1382-1391 ◽  
Author(s):  
Michiko Tanaka ◽  
Hiroyuki Kagawa ◽  
Yuji Yamanashi ◽  
Tetsutaro Sata ◽  
Yasushi Kawaguchi
Keyword(s):  
Vero Cells ◽  
Infectious Molecular Clone ◽  
Wild Type ◽  
Molecular Clone ◽  
Viral Genes ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT In recent years, several laboratories have reported on the cloning of herpes simplex virus type 1 (HSV-1) genomes as bacterial artificial chromosomes (BACs) in Escherichia coli and on procedures to manipulate these genomes by using the bacterial recombination machinery. However, the HSV-BACs reported so far are either replication incompetent or infectious, with a deletion of one or more viral genes due to the BAC vector insertion. For use as a multipurpose clone in research on HSV-1, we attempted to generate infectious HSV-BACs containing the full genome of HSV-1 without any loss of viral genes. Our results were as follows. (i) E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAC flanked by loxP sites was inserted into the intergenic region between UL3 and UL4 was constructed. (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii) The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by coinfection of Vero cells with YK304 and a recombinant adenovirus, AxCANCre, expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus, the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulence in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics, and a recombinant virus carrying amino acid substitutions in both copies of the α0 gene was generated. pYEbac102 will have multiple applications to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.

scholarly journals High Frequency Plant Regeneration of Musa paradisiaca cv. Karibale Monthan

2015 ◽  
Vol 3 (2) ◽  
pp. 202-209 ◽  
Author(s):  
R. Shashi Kumar ◽  
V. Krishna ◽  
. Venkatesh
Keyword(s):  
Plant Regeneration ◽  
High Frequency ◽  
Synergetic Effect ◽  
Multiple Shoot ◽  
Coconut Water ◽  
Morphological Variations ◽  
Musa Paradisiaca ◽  
Shoot Initiation

High frequency plant regeneration protocol has been standardized from banana cultivar Musa paradisiaca cv. Karibale Monthan, an endemic cultivar of Malnad region of Karnataka. The fruits are used as glomerular protective to solve kidney problems. To minimize the microbial contamination and to promote healthy growth, explants were treated with 70 % absolute alcohol for 6 min, 0.1 % Mercuric chloride for 10 min and 0.2 % for 10 min, 1 % Sodium hypochlorite for 15 min, 0.1 % Cefotaxime for 5 min and 0.05 % Gentamicin for 5 min. The high frequency shoot initiation (93.33 %) was recorded at 5 mg/l BAP. The synergetic effect of BAP (4 to 6 mg/l), TDZ (0.1 to 1.2 mg/l) and coconut water (0.1 to 0.9 ml/l) induced multiple shoot buds and it was optimized at the concentration of 5 mg/l BAP, 0.5 mg/l TDZ and 0.5 ml/l coconut water with 15.90 ± 1.66 frequency of shoots per propagule. Supplementation of 1.0 mg/l IBA induced 5.33 ± 1.21 numbers of roots with a mean root length of 7.50 ± 1.87 roots. The 99% of plantlets with distinct roots and shoots were successfully acclimatized in the green house and transferred to the field to evaluate the agro-morphological variations. The weight of the bunch (kg), number of hands in a bunch, number of fingers in a hand, length of the finger (cm), girth of the finger (cm) and girth of the pseudostem (cm) exhibited by in vitro plants were higher than the in vivo plants.Int J Appl Sci Biotechnol, Vol 3(2): 202-209 DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12536 

Theophylline minimally alters contractile properties of canine diaphragm in vitro

1990 ◽  
Vol 69 (4) ◽  
pp. 1390-1396 ◽  
Author(s):  
E. Derom ◽  
S. Janssens ◽  
V. De Bock ◽  
M. Decramer
Keyword(s):  
High Frequency ◽  
Force Production ◽  
Contractile Properties ◽  
Diaphragm Muscle ◽  
Muscle Bundle ◽  
Time To Peak ◽  
Diaphragm In Vitro ◽  
Tetanic Tension

We examined the effects of theophylline on contractile properties and high-frequency fatigue of canine diaphragm in vitro. Eighteen diaphragm muscle bundles were obtained from 10 anesthetized dogs and equilibrated in oxygenated Krebs solution to 100, 200, or 300 mg/l theophylline. These bundles were compared with 18 matched control bundles from the contralateral hemidiaphragm. No statistically significant differences in twitch tension, tetanic tension, twitch-to-tetanus ratio, time to peak tension, or half-relaxation time were observed. Concentrations of 300 mg/l theophylline, however, significantly (P less than 0.05) increased force production at 10 Hz by 32%. A similar tendency was present at lower concentrations and exhibited a clear dose-response behavior. High-frequency fatigue was similar in control and theophylline-treated bundles. We conclude that supratherapeutic in vitro concentrations of theophylline do not increase maximal tetanic tension and do not protect against muscle fatigue but potentiate relative force production at low stimulation frequencies. This relatively small effect cannot be explained by poor diffusion of the drug in the muscle bundle, because theophylline concentrations in the muscle bath and in the muscle bundle were virtually identical. Moreover, it remains unclear whether this potentially beneficial effect can be achieved at in vivo attainable serum concentrations.

Hydrogen Evolution Catalyzed by Hydrogenase in Cultures of Cyanobacteria

1981 ◽  
Vol 36 (1-2) ◽  
pp. 87-92 ◽  
Author(s):  
Patrick C. Hallenbeck ◽  
Leon V. Kochian ◽  
John R. Benemann
Keyword(s):  
Hydrogen Production ◽  
Hydrogen Evolution ◽  
High Frequency ◽  
Nitrogenase Activity ◽  
Inhibitory Effect ◽  
Hydrogenase Activity ◽  
Oxygen Inhibition ◽  
Oxygen Sensitive

Abstract Cultures of Anabaena cylindrica, grown on media containing 5 mᴍ NH4Cl (which represses heterocyst formation), evolved hydrogen after a period of dark incubation under an argon atmosphere. This hydrogen production was not due to nitrogenase activity, which was nearly undetectable, but was due to a hydrogenase. Cultures grown on media with tungsten substituted for molybdenum had a high frequency of heterocysts (15%) and inactive nitrogenase after nitrogen starvation. The hydrogenase activity of these cultures was three-fold greater than the activity of non-heterocystous cultures. The effects of oxygen inhibition on hydrogen evolution by hetero-cystous cultures suggest that two pools of hydrogenase activity exist - an oxygen sensitive hydrogen evolution in vegetative cells and a relatively oxygen-resistent hydrogen evolution in heterocysts. In either case, inhibition by oxygen was reversible. Light had an inhibitory effect on net hydrogen evolution. Hydrogen production in vitro was much higher than in vivo, indicating that in vivo hydrogenase activity is limited by endogenous reductant supply.

Hepatitis a virus: Growth characteristics of in vivo and in vitro propagated wild and attenuated virus strains

1984 ◽  
Vol 14 (4) ◽  
pp. 373-386 ◽  
Author(s):  
Daniel W. Bradley ◽  
Charles A. Schable ◽  
Karen A. McCaustland ◽  
E. H. Cook ◽  
Bert L. Murphy ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Growth Characteristics ◽  
Virus Growth ◽  
Attenuated Virus ◽  
Virus Strains

In vitro and in vivo high‐frequency ultrasound response of microbubbles

2006 ◽  
Vol 119 (5) ◽  
pp. 3438-3438
Author(s):  
Orlando Aristizábal ◽  
Daniel H. Turnbull ◽  
Jeffrey A. Ketterling
Keyword(s):  
High Frequency ◽  
High Frequency Ultrasound ◽  
Frequency Ultrasound

scholarly journals In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A, or E1/E4 Deleted

1998 ◽  
Vol 72 (3) ◽  
pp. 2022-2032 ◽  
Author(s):  
M. Lusky ◽  
M. Christ ◽  
K. Rittner ◽  
A. Dieterle ◽  
D. Dreyer ◽  
...  
Keyword(s):  
Recombinant Adenovirus ◽  
Virus Genome ◽  
Minor Role ◽  
Immunodeficient Mice ◽  
Viral Genomes ◽  
Viral Genes ◽  
Adenovirus Vectors ◽  
A Minor

ABSTRACT Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

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