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Simulated Shift Work in Rats Perturbs Multiscale Regulation of Locomotor Activity Wan-Hsin Hsieh1,2*, Carolina Escobar3, Tatiana Yugay1,4, Men-Tzung Lo1,2, Benjamin Pittman-Polletta1,4, Roberto Salgado-Delgado5, Frank A.J.L. Scheer1,4, Steven A. Shea1,4,6, Ruud M. Buijs7 and Kun Hu1,2,4* 1 Medical Biodynamics Program, Division of Sleep Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 2 Center for Dynamical Biomarkers and Translational Medicine, National Central University, Chungli 32001, Taiwan 3 Departamento de Anatomia, Facultad de Medicina, Edificio “B” 4o Piso, Universidad Nacional Autónoma de México, México DF 04510, México 4 Medical Chronobiology Program, Division of Sleep Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 5 Laboratorio de Biología Celular Fisiología, Facultad de Ciencias, Universidad Autónoma de San Luis Potosí, San Luis Potosí 78290, México 6 Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, Portland, OR 97239, United States 7 Departamento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México DF 04510, México 1 0.02 0.02 Week1 Week2 0.015 0.015 0.01 0.01 0.005 0.005 0 24-hour 12-hour 8-hour 6-our 4-hour 0.02 0 * 24-hour 12-hour 8-hour 4-hour 0.02 Week3 0.015 6-our Week4 0.015 * 0.01 0.01 * * 0.005 0 0.005 24-hour 12-hour 8-hour 6-our 4-hour 0 24-hour 12-hour 8-hour 6-our 4-hour Figure S1. Power density of harmonics of 24 hours rhythm during weekends from Week 1 to Week 4.The blue and red bar represent normal and shift workers and P < 0.01 is indicated by “*”. 2 Figure S2. Mean daily activity profiles during weekends for shift work animals (a) and normal work animals (c). Detail activity changes during the light and dark transitions during weekends for shift work animals (b) and normal work animals (d). Shaded areas indicated the dark phase of LD cycles. To obtain the mean daily activity pattern, activity data in each day were first normalized with respect to the associated mean activity level. Then averaged daily patterns were obtained from two days during weekend. The mean activity levels were obtained from five 30-min windows near the light and dark transitions and after scheduled work. “*” indicates a significant dependence on week (P < 0.05). For normal work animals, there are no significant changes in their activity responses to light-dark (P > 0.47) or dark-light transitions (P > 0.32). 3 Figure S3. Actograms of seven rats under normal work schedule for three or four weeks. 4 Figure S4. Actograms of 12 rats under shift work schedule for five weeks. 5 Figure S5. DFA results of surrogate data derived from the activity recording of a shift worker during weekends. We artificially imposed an additional 24-hour rhythm in the activity data of shift workers during weekends such that the power density at 24-hour was equal to the value for normal workers. The fluctuation amplitude calculated from raw data was also provided for comparison. We found that the scaling behavior at time scales < 4 hours (1 = 0.97 ± 0.02 vs. 0.98 ± 0.02 for raw data; P > 0.33) and at 4-12 hours (2 = 0.86 ± 0.02 vs. 0.85 ± 0.02 for raw data, P > 0.05) remained unchanged. 6