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To test whether the predicted adaptive and neutral mutations have an effect on the thermostability of our model protein, we generated two mutant ctArx1 proteins with either five predicted adaptive or five predicted neutral positions in ctArx1 changed to the respective ancestral residues.
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As a result, the name of the organisation was changed to The Scout Association and major changes were made to the sections and their respective programmes.
Next, the source code files changed by such commits are used to identify the classes changed to fix the respective bugs.
The next day, cells were synchronized by using 10 n M ICI 182780 (Tocris, St . Louis MO, USA) for 24 hours; the following day, cells were washed once with PBS, and the medium was changed to include the respective treatments (10 n M E2 or 10 n M ICI).
Moreover, we thank the referee pointing out the typo regarding P2X channels; we have made some clarifying changes to the respective sentence: "purinergic P2X channels are functional in mouse (Navarro et al., 2011), but not in human sperm (Brenker et al., 2012)".
The mutagenic primers were designed in such a way to change the respective amino acids and to create a Type II restriction enzyme site.
All weights were normalized to tibia length and expressed as percent-wise change compared to the respective sham group.
The mRNA expression values were represented as the fold change relative to the respective control.
Mutations causing a >1.3-fold change compared to the respective parent mutant are considered non-neutral.
Mutations causing a >1.3-fold change compared to the respective parent mutant (dotted line) are considered non-neutral.
The experiment was repeated and the average change relative to the respective parent variant and the standard deviation were determined (Supplementary file 2).
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CEO of Professional Science Editing for Scientists @ prosciediting.com