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Indeed, it is likely that the recombinants observed represent a small fraction of amount of homologous repair, since the sibling genomes will presumably be the more available partners for repair but will not contribute restriction resistant genomes.
The survivors of restriction enzyme selection are homoplasmic reflecting a strong selection against the starting genomes that acts to completion in a single generation with little opportunity for a slow amplification of the resistant genomes.
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Replication of the DEB025res genome proved, unlike replication of the WT genome, to be efficient in CypA KD cells, indicating that the drug resistant genome is less dependent on CypA (and thus likely on the cis-trans isomerase activity) for efficient replication.
Furthermore, preliminary data suggests that rescue of fertility by having ∼2.5% of resistant genome upon cutting, is inefficient, meaning that many recombinant genomes are required for in each stem cell to produce progeny.
To detect endonuclease-resistant genomes in crude viral preps (CVPs) or Optiprep fractions, only benzonase-treated CVPs were utilized so that all non-encapsidated genomes were degraded.
Thus, for a capsid inhibitor, trans-encapsidation of newly arising drug-resistant genomes by the intracellular excess of drug-susceptible capsids should provide an additional mechanism to reduce the reservoir of drug-resistant genomes packaged in drug-resistant capsids.
Thus, failure to select V-073-resistant genomes at high MOIs could be overcome by preventing the entry of the drug-susceptible genomes.
Therefore, targeting the viral NTPase with guanidine allows the growth of drug-resistant genomes even in the presence of an excess of drug-susceptible ones.
The virus stock used was deliberately prepared from cells infected at low MOI to ensure that drug-resistant genomes were packaged in drug-resistant capsids.
Late treatment may be directed at a population that contains 0.2% drug-resistant genomes, but many of these will have been trans-encapsidated by the other viral variants present in their cells of origin.
Although both the dORF replicon cells and the previously reported full-genome replicons secreted RNase-resistant genomes, there was a striking difference between these two full-genome replicons when NP-40 treatment was carried out on their supernatants.
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