Figure 6d: WT NeRh50 maximum amplitudes and decay rate constants for representative SSME transient currents recorded at pH5-pH8

Manuscript figure 6d shows WT maximum amplitude and decay rate constant variation for representative SSME transient currents recorded at pH5-pH8 (figure 6d).

The Dataset

The data used for the figure are shown in ?@tbl-data-6d

Show the code 
DT::datatable(dfms$dfm_6d, options=list(pageLength=5))
Table 1: Maximum amplitude and decay rate constant for representative SSME transient currents recorded at pH5-pH8 (figure 6d).

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The Models

We use linear models for amplitude and decay rate as dependent variables, with pH as the independent factor, checking fit in Figure 1.

Show the code 
# Fit the models
model6d_amp_lm <- lm(amplitude ~ pH, data=dfms$dfm_6d)
model6d_k_lm <- lm(decayrate ~ pH, data=dfms$dfm_6d)

# Check model fit
performance::check_model(model6d_amp_lm)
performance::check_model(model6d_k_lm)
(a) amplitude ~ pH
(b) decayrate ~ pH
Figure 1: Diagnostic plots for linear model fits of amplitude ~ group and decayrate ~ group. The heteroscadicity spline is odd, but the datapoints are not concerning. There is no strong evidence of non-normality of residuals.

As the model fit is acceptable, we estimate contrasts in Table 2.

Show the code 
EMM <- emmeans::emmeans(model6d_amp_lm, ~ pH)
confint(emmeans::contrast(EMM, "pairwise", adjust="sidak")) |>
  kableExtra::kbl(digits=3) |>
  kableExtra::kable_styling(
    bootstrap_options = c("hover", "responsive")
  )
EMM <- emmeans::emmeans(model6d_k_lm, ~ pH)
confint(emmeans::contrast(EMM, "pairwise", adjust="sidak")) |>
  kableExtra::kbl(digits=3) |>
  kableExtra::kable_styling(
    bootstrap_options = c("hover", "responsive")
  )
Table 2: Contrasts for model fits, with Sidak adjustment. Not all contrasts include zero in the 95% confidence interval estimate.
(a) Maximum amplitude
contrast estimate SE df lower.CL upper.CL
pH5 - pH6 -0.036 0.138 26 -0.428 0.355
pH5 - pH7 -0.192 0.118 26 -0.529 0.146
pH5 - pH8 -1.219 0.118 26 -1.556 -0.882
pH6 - pH7 -0.155 0.131 26 -0.529 0.218
pH6 - pH8 -1.182 0.131 26 -1.556 -0.809
pH7 - pH8 -1.027 0.111 26 -1.343 -0.712
(b) Decay rate
contrast estimate SE df lower.CL upper.CL
pH5 - pH6 5.368 5.451 20 -10.536 21.271
pH5 - pH7 -3.140 4.832 20 -17.236 10.957
pH5 - pH8 -4.824 4.632 20 -18.339 8.691
pH6 - pH7 -8.507 4.832 20 -22.604 5.590
pH6 - pH8 -10.191 4.632 20 -23.706 3.324
pH7 - pH8 -1.684 3.885 20 -13.018 9.650

For decay rate constant, all contrasts include zero in the 95% confidence interval, so we conclude that there are no differences in decay rate constant for each pH condition. There are contrasts that do not include zero for the maximum amplitude data, but only for those contrasts involving pH8.

The Figures

We plot the data, indicating P-values for the contrasts in Figure 2.

Show the code 
# Get p-values, accounting for heteroscedasticity and multiple testing
# Also generate group1 and group2 columns
EMM_amp <- as.data.frame(pairs(emmeans::emmeans(model6d_amp_lm, ~ pH),
                               adjust="sidak")) |>
  tidyr::separate(contrast, into=c("group1", "group2"), sep=" - ") |>
  dplyr::mutate(y.position=2.5) |>
  rstatix::add_significance()

EMM_k <- as.data.frame(pairs(emmeans::emmeans(model6d_k_lm, ~ pH),
                               adjust="sidak")) |>
  tidyr::separate(contrast, into=c("group1", "group2"), sep=" - ") |>
  dplyr::mutate(y.position=70) |>
  rstatix::add_significance()

p6d_amp <- ggplot2::ggplot(dfms$dfm_6d, 
                           ggplot2::aes(x=pH, y=amplitude, color=pH)) +
  ggplot2::geom_boxplot(outlier.shape = NA) +
  ggplot2::geom_jitter() +
  ggplot2::labs(y="Maximum amplitude (nA)") +
  ggplot2::ylim(0, 4.5) +
  ggplot2::theme_minimal() +
  ggpubr::stat_pvalue_manual(EMM_amp,
                             label="p = {scales::pvalue(p.value)}",
                             size=3, step.increase=0.1)

p6d_k <- ggplot2::ggplot(dfms$dfm_6d, 
                         ggplot2::aes(x=pH, y=decayrate, color=pH)) +
  ggplot2::geom_boxplot(outlier.shape = NA) +
  ggplot2::geom_jitter() +
  ggplot2::labs(y="Decay rate constant k (/s)") +
  ggplot2::ylim(0, 105) +
  ggplot2::theme_minimal() +
  ggpubr::stat_pvalue_manual(EMM_k, 
                             label="p = {scales::pvalue(p.value)}", 
                             size=3, step.increase=0.15)

p6d_amp
p6d_k

# Write figures to file (commented out for webpage)
#ggplot2::ggsave("assets/images/fig6d_WT_maximum_amplitude.pdf", p6d_amp)
#ggplot2::ggsave("assets/images/fig6d_WT_maximum_amplitude.png", p6d_amp)
#ggplot2::ggsave("assets/images/fig6d_WT_decay_rate_constant.png", p6d_k)
#ggplot2::ggsave("assets/images/fig6d_WT_decay_rate_constant.pdf", p6d_k)
(a) Figure 6d, maximum amplitude
(b) Figure 6d, decay rate
Figure 2: Maximum amplitude and decay rate constant for four pH regimes.

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