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Finally, DNA# 54 was defined as 12Val-mutated by TheraScreen analysis, but this mutation could not be unequivocally confirmed in the PyroMark assay.
Using reverse genetics, combined mutations of Q253H and A284T could adapt vvIBDV to non-permissive CEF cells (rGx-F9VP2) but any single mutation could not.
Though, we could confirm the causal gene, the causal mutation could not be ascertained as we could not identify any recombinants in the material tested among the five potential causal mutations, owing to small population size.
We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD.
Situation therefore is quite distinct from the case with Pngl, where the equivalent C-to-A mutation could not rescue the developmental defect (Table 2).
It is however possible that some mutations such as 769N have a heavy fitness cost, as parasites harbouring the mutation could not be propagated in vitro under standard culture conditions.
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The five key mutations could not be reversed first, because the receptor would be rendered useless.
Thus a prerequisite of unlinked loci within the whole group is needed and the recombination events between any two mutations could not be considered.
However, these loss-of-function-mutations could not be associated with specific diseases in general.
Therefore, the somatic status of these mutations could not be ascertained.
In this study, we observed only a fairly small number of candidates in the comparison between EAS and CEU, which indicates that the divergence time of the two populations is so short that new mutations could not reach fixation without strong selection.
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