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To compare our whole genome mutation frequencies with phenotype-selected mutation frequencies, we used a one-way frequency table with χ analysis.
The mutation frequencies we observed were very low (e.g. between 0.03 to 0.5 events per plant upon VOT infection) and hence screening a large population was necessary.
The mutation frequencies we observed are thus ∼35 150 times higher than reported previously for a case of standard EMS mutagenesis in Arabidopsis.
To determine whether ribavirin and favipiravir treatment resulted in greater mutation frequencies, we carried out sequence analysis of individual molecular clones isolated from populations subjected to 4 passages in the presence of either 200 μM ribavirin or favipiravir.
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To verify that inhibition of c-Abl is the responsible mechanism for the enhanced mutation frequency, we examined MEFs derived from c-Abl−/− mice retransfected with either KD-Abl or wt-Abl.
Here, by assessing evolutionary selective advantage from longitudinal data, which we demonstrated was independent of viral load, rather than cross-sectional mutation frequency we were able to ascertain the direction of causality: viral escape from CTL appears to contribute to clinical decline.
To quantify global changes in mutation frequency, we plotted the mean z-score for each of the twelve possible nucleotide changes as a function of time and drug concentration (e.g. A to C, A to G, A to T, C to A, C to G, C to T, etc).
In addition to the mutation frequency, we examined whether metastasis-derived mutations occur at certain genomic regions.
In line with this somatic mutation frequency we found only one somatic mutation for each of the three patients.
Because of an increased mutation frequency, we assume that DNA damage accumulates in some of these mutants.
We identified the ancestral SP for each SP in the recurrent tumor by taking into account size and shared mutation frequency: We hypothesize that two SPs that share many of their mutations are likely to be closely related clones.
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