Figure 4c: WT NeRh50 maximum amplitudes and decay rate constants in response to changes in lipid-protein ratio (LPR)

Manuscript figure 4c shows WT maximum amplitude and decay rate constant variation over LPR5, LPR10, and LPR50 for the WT NeRh50 with NH4+ (figure 4c).

The Dataset

The data used for the figure are shown in Table 1

Show the code 
DT::datatable(dfms$dfm_4c, options=list(pageLength=5))
Table 1: Maximum amplitude and decay rate constant at three LPR values, for WT Rh50 (figure 4c).

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The Models

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

Show the code 
# Fit the models
model4c_amp_lm <- lm(amplitude ~ LPR, data=dfms$dfm_4c)
model4c_k_lm <- lm(decayrate ~ LPR, data=dfms$dfm_4c)

# Check model fit
performance::check_model(model4c_amp_lm)
performance::check_model(model4c_k_lm)
(a) amplitude ~ LPR
(b) decayrate ~ LPR
Figure 1: Diagnostic plots for linear model fits of amplitude ~ LPR and decayrate ~ LPR. The splines for linearity/heteroscadicity are odd, but the datapoints are not concerning. There is some evidence of non-normality of residuals in the decay rate data, but nothing especially concerning.

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

Show the code 
EMM <- emmeans::emmeans(model4c_amp_lm, ~ LPR)
confint(emmeans::contrast(EMM, "pairwise", adjust="sidak")) |>
  kableExtra::kbl(digits=3) |>
  kableExtra::kable_styling(
    bootstrap_options = c("hover", "responsive")
  )
EMM <- emmeans::emmeans(model4c_k_lm, ~ LPR)
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. No contrast includes zero in the 95% confidence interval estimate.
(a) Maximum amplitude
contrast estimate SE df lower.CL upper.CL
LPR5 - LPR10 0.914 0.075 21 0.720 1.108
LPR5 - LPR50 1.467 0.084 21 1.250 1.684
LPR10 - LPR50 0.553 0.084 21 0.336 0.770
(b) Decay rate
contrast estimate SE df lower.CL upper.CL
LPR5 - LPR10 11.312 1.314 13 7.716 14.908
LPR5 - LPR50 20.582 1.630 13 16.122 25.042
LPR10 - LPR50 9.270 1.670 13 4.700 13.840

As no contrast includes zero in the 95% confidence interval, we conclude that there are differences in amplitude and decay rate constant for each pairwise comparison between LPR condition.

The Figures

We plot the differences, 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(model4c_amp_lm, ~ LPR),
                               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(model4c_k_lm, ~ LPR),
                               adjust="sidak")) |>
  tidyr::separate(contrast, into=c("group1", "group2"), sep=" - ") |>
  dplyr::mutate(y.position=85) |>
  rstatix::add_significance()

p4c_amp <- ggplot2::ggplot(dfms$dfm_4c, 
                           ggplot2::aes(x=LPR, y=amplitude, color=LPR)) +
  ggplot2::geom_boxplot(outlier.shape = NA) +
  ggplot2::geom_jitter() +
  ggplot2::labs(y="Maximum amplitude (nA)") +
  ggplot2::ylim(0, 3.5) +
  ggplot2::theme_minimal() +
  ggpubr::stat_pvalue_manual(EMM_amp,
                             label="p = {scales::pvalue(p.value)}",
                             size=3, step.increase=0.1)

p4c_k <- ggplot2::ggplot(dfms$dfm_4c, 
                         ggplot2::aes(x=LPR, y=decayrate, color=LPR)) +
  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=0.1)

p4c_amp
p4c_k

# Write figures to file (commented out for webpage)
#ggplot2::ggsave("assets/images/fig4c_WT_maximum_amplitude.pdf", p4c_amp)
#ggplot2::ggsave("assets/images/fig4c_WT_maximum_amplitude.png", p4c_amp)
#ggplot2::ggsave("assets/images/fig4c_WT_decay_rate_constant.pdf", p4c_k)
#ggplot2::ggsave("assets/images/fig4c_WT_decay_rate_constant.png", p4c_k)
(a) Figure 4c, maximum amplitude
(b) Figure 4c, decay rate
Figure 2: WT measurements across three LPRs, replicating figure 4c from the manuscript.

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