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In this work, we confirmed stability changes in the variant proteins using GdnHCl-induced equilibrium unfolding experiments at various temperatures (Figure 3A).
Some methods analyze changes in the variant allele frequencies (VAFs) of single-nucleotide mutations; i.e. the fraction of tumor cells that contain each mutation (Miller et al., 2014; Roth et al., 2014).
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Therefore, the variation in characteristics over time is due to the variation of the characteristics of the representative variant (and not due to a change in the variant chosen).
The FASTA format sequence of AKR1C2 was taken from UniProt[11] (accession number: P52895) and edited to reflect the amino acid change in the variant.
The mechanism behind this was a change in the variant structure of the virus population, plus changes in virus morphology.
Multiple passage of one isolate resulted in increased pathogenicity to a resistant codling moth colony and was accompanied by a change in the variant structure of the virus population (Berling et al. 2009).
As a first hint of allosteric networks throughout these proteases, the binding of an activated peptidomimetic inhibitor caused a structural change in the variant protease that reverted it to a wild type conformation.
Due to limitation of PCR reaction in co-amplification, we further performed RT-qPCR of individual splicing variants from each of the selected gene, and found that there are reciprocal changes in the splicing variant levels (Supplementary Fig. 17).
During its career the Phantom underwent many changes in the form of numerous variants developed.
Regarding that the limitation of co-amplification PCR may not fully reflect alternative splicing scenario, we further conducted qRT-PCR assay using isoform-specific primers to confirm there are indeed reciprocal changes in the splicing variant (Supplementary Fig. 17).
Instead, we found that 7 genes (Spcs3, Wdr17, Npy1r, Psd3, Gdf1, Fcho1, and Eps15l1) had base substitutions causing amino acid changes in the circadian variant.
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