Figure 1.
Determination of circadian period in vivo and in vitro.
A. In vivo period length was obtained from salivary melatonin content profiles of normal sighted subjects under a constant routine protocol for three consecutive cycles. Data from one representative subject (Start25) are shown. Open circles, melatonin measurement values. B. From the same subject, fibroblast cultures were obtained from two separate biopsies and infected with a lentiviral bioluminescent circadian reporter. After synchronization with dexamethasone, circadian oscillations in bioluminescence were recorded from eight measures over five days. Three cycles of this oscillation are shown aligned with the physiological data of part A. Period calculations for both panels were conducted by cosinor fitting, and the best-fit curve is shown in color, along with its period length and goodness of fit R2.
Figure 2.
Relationship between physiological period length and fibroblast period length.
A. In vitro period length was obtained from skin fibroblasts from nine sighted human subjects of normal chronotype, and compared to physiological period in the same individuals measured in two constant routine studies (Basel Start and Likri). The solid line shows the best-fit linear regression (1st polynomial order) represented by the data, and the dashed lines represent 95% confidence intervals to the indicated regression line, with goodness-of-fit R2 shown at lower right. The overall p-value (vs. null-hypothesis slope of 0) is shown at upper-left. For this panel and also panels B and C, identical statistical measures are depicted. In addition, the range and average for each group of subjects are shown in Table 1, and data for individual subjects are listed in Methods S1. B. The same comparison was performed for 11 sighted subjects whose physiological period was measured in controlled laboratory conditions in two studies (Novosibirsk). C. A further comparison was performed for 8 totally blind subjects whose physiological period was measured at home (Guildford). Fibroblasts from the asterisked subject showed abnormal clock properties in vitro at different temperatures and were excluded from statistical analysis. D. The results of all five studies are graphed on the same axes: Yellow, Basel; Red, Novosibirsk; Blue, Guildford.
Table 1.
Period lengths obtained from subjects by physiological measurements and from fibroblast molecular analyses.
Figure 3.
Relationship between population averages of human physiological period length and fibroblast period length.
A. The average in vivo period lengths obtained from both protocols with sighted individuals were significantly different from that of blind subjects (p<0.01, 0.05). B. Nevertheless, the fibroblast-derived values were not significantly different among all three populations. (1-way Anova). Data are presented as a standard boxplot, with bars showing the smallest and largest observed values, and box dimensions and midline reflecting lower quartile (Q1), median (Q2), and upper quartile (Q3) values.
Figure 4.
Circadian period and wake timing.
A. Period length measured in fibroblasts is plotted against the circadian phase angle of sleep, calculated as the difference between the measured time of dim-light melatonin onset on the first day of the protocol and the time of subject spontaneous waking that morning. This value could only be determined in one of the five studies presented (Basel constant routine). Linear regression and statistical analyses were as in Figure 2. B. Period length in vivo is plotted versus the same sleep phase angle.