Taking biological rhythms into account: From study design to results reporting.

Numerous physiological and behavioral processes in living organisms exhibit strong rhythmicity and are regulated within a 24-hour cycle. These include locomotor activity and sleep patterns, feeding-fasting cycles, hormone synthesis, body temperature, and even mood and cognitive abilities, all of which are segregated into different phases throughout the day. These processes are governed by the internal timing system, a hierarchical multi-oscillator structure conserved across all organisms, from bacteria to humans. Circadian rhythms have been seen across multiple taxonomic kingdoms. In mammals, a hierarchical internal timing system is comprised of so-called central and periphereal clocks. Although these rhythms are intrinsic, they are under environmental influences, such as seasonal temperature changes, photoperiod variations, and day-night cycles. Recognizing the existence of biological rhythms and their primary external influences is crucial when designing and reporting experiments. Neglecting these physiological variations may result in inconsistent findings and misinterpretations. Thus, here we propose to incorporate biological rhythms into all stages of human and animal research, including experiment design, analysis, and reporting of findings. We also provide a flowchart to support decision-making during the design process, considering biological rhythmicity, along with a checklist outlining key factors that should be considered and documented throughout the study. This comprehensive approach not only benefits the field of chronobiology but also holds value for various other research disciplines. The insights gained from this study have the potential to enhance the validity, reproducibility, and overall quality of scientific investigations, providing valuable guidance for planning, developing, and communicating scientific studies.

Human social isolation and stress: a systematic review of different contexts and recommendations for future studies

Abstract Objectives The emergence of the coronavirus disease 2019 (COVID-19) pandemic and subsequent lockdowns and social distancing measures adopted worldwide raised questions about the possible health effects of human social isolation. Methods We conducted a systematic review on PubMed, Scopus, and Embase electronic databases using terms related to human social isolation - defined as the isolation of an individual from regular routines and usual social contact - and psychological stress, searching for simulated or naturalistic isolation environments. We present the main results, as well as the validity and limitations of each model. PROSPERO registry number: CRD42021241880. Results Despite the diversity of contexts reviewed, some outcomes almost ubiquitously relate to psychological stress, i.e., longer periods, expectation of a longer period, confinement, lack of social interaction, and support. Based on the results, and considering that most studies were not designed for the purpose of understanding isolation itself, we propose a group of recommendations for future experimental or naturalistic research on the topic. Conclusion Evidence on the impact of different situations in which individuals are subjected to social isolation can assist in development of directed preventive strategies to support people under similar circumstances. Such strategies might increase the general public's compliance with social distancing as a non-pharmacological intervention for emerging infectious diseases.

Understanding the Diurnal Oscillation of the Gut Microbiota Using Microbial Culture.

The composition of the gut microbiota oscillates according to the light-dark cycle. However, the existing literature demonstrates these oscillations only by molecular methods. Microbial cultures are an interesting method for studying metabolically active microorganisms. In this work, we aimed to understand the diurnal oscillation of the intestinal microbiota in Wistar male rats through microbial culture analysis. Over a 24 h period, three animals were euthanized every 6 h. Intestinal segments were dissected immediately after euthanasia and diluted in phosphate-buffered saline (PBS) for plating in different culture media. The CFU/mL counts in feces samples cultured in the Brucella medium were significantly higher at ZT0, followed by ZT6, ZT18, and ZT12 (p = 0.0156), which demonstrated the diurnal oscillation of metabolically active anaerobic bacteria every 6 h using microbial culture. In addition, quantitative differences were demonstrated in anaerobic bacteria and fungi in different gastrointestinal tract tissues.

Human Social Isolation and Stress: A Systematic Review of Different Contexts and Recommendations for Future Studies.

OBJECTIVES: The emergence of COVID-19 pandemic and subsequent lockdowns and social distancing measures adopted worldwide raised questions about the possible health effects of human social isolation. METHODS: We conducted a systematic review on PubMed, Scopus and Embase electronic databases using terms related to human social isolation - defined as the isolation of an individual from regular routines and usual social contact - and psychological stress, searching for simulated or naturalistic isolation environments. We present the main results, as well as the validity and limitations of each model. PROSPERO registry number: CRD42021241880. RESULTS: Despite the diversity of contexts reviewed, some outcomes almost ubiquitously relate to psychological stress, i.e. longer periods, expectation of a longer period, confinement, lack of social interaction and support. Based on the results, considering that most studies were not designed for the purpose of understanding isolation itself, we propose a group of recommendations for future experimental or naturalistic research on the topic. CONCLUSION: Evidence on the impact of different situations in which individuals are subjected to social isolation can assist in the development of directed preventive strategies to support people under similar circumstances. Such strategies might increase the compliance of the general public to social distancing as a non-pharmacological intervention for emerging infectious diseases.

Sleep disturbances, circadian activity, and nocturnal light exposure characterize high risk for and current depression in adolescence.

STUDY OBJECTIVES: Major depressive disorder (MDD) in adolescence is associated with irregularities in circadian rhythms and sleep. The characterization of such impairment may be critical to design effective interventions to prevent development of depression among adolescents. This study aimed to examine self-reported and actimetry-based circadian rhythms and sleep-wake behavior associated with current MDD and high risk (HR) for MDD among adolescents. METHODS: Ninety-six adolescents who took part in the IDEA-RiSCo study were recruited using an empirically developed depression-risk stratification method: 26 classified as low risk (LR), 31 as HR, and 39 as a current depressive episode (MDD). We collected self-report data on insomnia, chronotype, sleep schedule, sleep hygiene as well as objective data on sleep, rest-activity, and light exposure rhythms using actimetry for 10 days. RESULTS: Adolescents with MDD exhibited more severe insomnia, shorter sleep duration, higher social jetlag (SJL), lower relative amplitude (RA) of activity, and higher exposure to artificial light at night (ALAN) compared with the other groups. They also presented poorer sleep hygiene compared with the LR group. The HR group also showed higher insomnia, lower RA, higher exposure to ALAN, and higher SJL compared with the LR group. CONCLUSIONS: HR adolescents shared sleep and rhythm alterations with the MDD group, which may constitute early signs of depression, suggesting that preventive strategies targeting sleep should be examined in future studies. Furthermore, we highlight that actimetry-based parameters of motor activity (particularly RA) and light exposure are promising constructs to be explored as tools for assessment of depression in adolescence.

Development and testing of methods for detecting off-wrist in actimetry recordings.

STUDY OBJECTIVES: In field studies using wrist-actimetry, not identifying/handling off-wrist intervals may result in their misclassification as immobility/sleep and biased estimations of rhythmic patterns. By comparing different solutions for detecting off-wrist, our goal was to ascertain how accurately they detect nonwear in different contexts and identify variables that are useful in the process. METHODS: We developed algorithms using heuristic (HA) and machine learning (ML) approaches. Both were tested using data from a protocol followed by 10 subjects, which was devised to mimic contexts of actimeter wear/nonwear in real-life. Self-reported data on usage according to the protocol were considered the gold standard. Additionally, the performance of our algorithms was compared to that of visual inspection (by 2 experienced investigators) and Choi algorithm. Data previously collected in field studies were used for proof-of-concept analyses. RESULTS: All methods showed similarly good performances. Accuracy was marginally higher for one of the raters (visual inspection) than for heuristically developed algorithms (HA, Choi). Short intervals (especially < 2 h) were either not or only poorly identified. Consecutive stretches of zeros in activity were considered important indicators of off-wrist (for both HA and ML). It took hours for raters to complete the task as opposed to the seconds or few minutes taken by the automated methods. CONCLUSIONS: Automated strategies of off-wrist detection are similarly effective to visual inspection, but have the important advantage of being faster, less costly, and independent of raters' attention/experience. In our study, detecting short intervals was a limitation across methods.

Effective recommendations towards healthy routines to preserve mental health during the COVID-19 pandemic

Objective: To assess the adherence to a set of evidence-based recommendations to support mental health during the coronavirus disease 2019 (COVID-19) pandemic and its association with depressive and anxiety symptoms. Methods: A team of health workers and researchers prepared the recommendations, formatted into three volumes (1: COVID-19 prevention; 2: Healthy habits; 3: Biological clock and sleep). Participants were randomized to receive only Volume 1 (control), Volumes 1 and 2, Volumes 1 and 3, or all volumes. We used a convenience sample of Portuguese-speaking participants over age 18 years. An online survey consisting of sociodemographic and behavioral questionnaires and mental health instruments (Patient Health Questionnaire-9 [PHQ-9] and Generalized Anxiety Disorder-7 [GAD-7]) was administered. At 14 and 28 days later, participants were invited to complete follow-up surveys, which also included questions regarding adherence to the recommendations. A total of 409 participants completed the study - mostly young adult women holding university degrees. Results: The set of recommendations contained in Volumes 2 and 3 was effective in protecting mental health, as suggested by significant associations of adherence with PHQ-9 and GAD-7 scores (reflecting anxiety and depression symptoms, respectively). Conclusion: The recommendations developed in this study could be useful to prevent negative mental health effects in the context of the pandemic and beyond.

Effective recommendations towards healthy routines to preserve mental health during the COVID-19 pandemic.

OBJECTIVE: To assess the adherence to a set of evidence-based recommendations to support mental health during the coronavirus disease 2019 (COVID-19) pandemic and its association with depressive and anxiety symptoms. METHODS: A team of health workers and researchers prepared the recommendations, formatted into three volumes (1: COVID-19 prevention; 2: Healthy habits; 3: Biological clock and sleep). Participants were randomized to receive only Volume 1 (control), Volumes 1 and 2, Volumes 1 and 3, or all volumes. We used a convenience sample of Portuguese-speaking participants over age 18 years. An online survey consisting of sociodemographic and behavioral questionnaires and mental health instruments (Patient Health Questionnaire-9 [PHQ-9] and Generalized Anxiety Disorder-7 [GAD-7]) was administered. At 14 and 28 days later, participants were invited to complete follow-up surveys, which also included questions regarding adherence to the recommendations. A total of 409 participants completed the study - mostly young adult women holding university degrees. RESULTS: The set of recommendations contained in Volumes 2 and 3 was effective in protecting mental health, as suggested by significant associations of adherence with PHQ-9 and GAD-7 scores (reflecting anxiety and depression symptoms, respectively). CONCLUSION: The recommendations developed in this study could be useful to prevent negative mental health effects in the context of the pandemic and beyond.

Handling missing data in rest-activity time series measured by actimetry.

A handling procedure of off-wrist episodes in actimetry time series of motor activity is presented using two records (regular vs. irregular sleep-wake cycle and daytime activity) of 14 consecutive days sampled in 1-minute epochs. We generated single missing value (NA) intervals of 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h as well as random NA episodes following probabilistic rules to simulate real-life off-wrist episodes. Then, we replaced these episodes with "zeroes" (i.e., the default of immobility records), mean or median of the remaining 13 days corresponding to the missing bins. Single missing episodes of up to 12 h resulted in less than 5% variation from the original values. The irregular series showed higher variability in acrophase, MESOR, L5, M10 and RA compared to the regular series. Random missing allocation simulating real-life off-wrist episodes resulted in significant changes in most parameters, and the imputation of zeroes significantly increased the variance; however, replacing NA with mean or median resulted in patterns similar to those of NA. We recommend replacing 'zeroes' with NA whenever possible, given the risk of inflating invariance using zeroes. If the parameters cannot be computed in the presence of NA, we recommend using the weekly mean of corresponding timepoints.

Effects of lighting patterns in pubertal development and metabolism of female wistar rats.

Modern lifestyle is characterized by constant exposure to artificial light, which is associated with alterations in biological rhythms, abnormalities to reproductive cycles and metabolic changes. In this study, we aimed to evaluate the effects of four different lighting patterns on puberty timing and on possible metabolic changes in female Wistar rats. Additionally, we developed a machine learning algorithm to automatically classify the stages of the estrous cycle. Adult Wistar rats mated during a week at a photoperiod station where they were exposed to combined red-green-blue lights (RGB) during the photoperiod that varied its spectral composition (i.e., variable color temperature) during the day (RGB-v; N = 14), RGB during the photoperiod with a fixed light color temperature (RGB-f; N = 13) during the whole photoperiod; constant darkness (DD; N = 13) and constant fixed light (LL; N = 15). Experiments were performed only on female litters from postnatal day (PND) 22 to 50. Body weight, puberty onset, estrous cyclicity and serum metabolic parameters were measured. We also collected pictures of vaginal smears to create a dataset of 15,936 images to construct an automatic classifier based on convolutional neural networks. No significant differences were found in the age of vaginal opening; however, the RGB-v group showed a significantly lower number of complete and consecutives cycles. Also, the RGB-f group showed the first complete estrous cycle significantly earlier than the RGB-v group. Female rats housed in LL condition presented significantly lower mean body weight from PND 33 to PDN 47 compared to the other groups. Furthermore, higher levels of plasma triglycerides were found in the DD group compared to RGB-f and RGB-v. HDL levels were significantly lower in RGB-v compared to RGB-f and LL groups. Total cholesterol was significantly lower in RGB-v compared to all groups. Visceral fat was significantly higher in RGB-f compared to the LL group. These results suggest that both changes in photoperiod and lighting quality affect pubertal development and alter lipid profiles and visceral fat accumulation.

Relationship Between Circadian Strain, Light Exposure, and Body Mass Index in Rural and Urban Quilombola Communities.

Industrialization has greatly changed human lifestyle; work and leisure activities have been moved indoors, and artificial light has been used to illuminate the night. As cyclic environmental cues such as light and feeding become weak and/or irregular, endogenous circadian systems are increasingly being disrupted. These disruptions are associated with metabolic dysfunction, possibly contributing to increased rates of overweight and obesity worldwide. Here, we aimed to investigate how activity-rest rhythms, patterns of light exposure, and levels of urbanization may be associated with body mass index (BMI) in a sample of rural and urban Quilombola communities in southern Brazil. These are characterized as remaining social groups who resisted the slavery regime that prevailed in Brazil. Quilombola communities were classified into five groups according to their stage of urbanization: from rural areas with no access to electricity to highly urbanized communities. We collected anthropometric data to calculate BMI, which was categorized as follows: from ≥ 18.5 kg/m2 to < 25 kg/m2 = normal weight; from ≥ 25 kg/m2 to < 30 kg/m2 = overweight; and ≥ 30 kg/m2 = obese. Subjects were asked about their sleep routines and light exposure on workdays and work-free days using the Munich Chronotype Questionnaire (N = 244 included). In addition, we analyzed actimetry data from 121 participants with seven consecutive days of recordings. Living in more urbanized areas and higher intradaily variability (IV) of activity-rest rhythms were associated with an increased risk of belonging to the overweight or obese group, when controlling for age and sex. These findings are consistent with preclinical data and point to potential strategies in obesity prevention and promotion of healthy metabolic profiles.

Circadian Strain, Light Exposure, and Depressive Symptoms in Rural Communities of Southern Brazil.

Irregular light-dark cycles and circadian/sleep disturbances have been suggested as risk or co-occurring factors in depression. Among a set of metrics developed to quantify strain on the circadian system, social jetlag (SJL) has been put forward as a measure of the discrepancy between biological and social clocks. Here, we approached the question on whether light exposure and SJL would also be associated with depressive symptoms in Quilombola communities in Southern Brazil. These rural communities are void of potential confounders of modern lifestyles and show low levels of SJL. 210 Quilombolas (age range 16-92; 56% women) were asked about their sleep times and light exposure using the Munich ChronoType Questionnaire (MCTQ). The Beck Depression Inventory (BDI) was used to assess depressive symptoms. Additionally, we analyzed 7-day actimetry recordings in 124 subjects. BDI scores higher than 10 (having clinically significant depressive symptoms; controlled for age and sex in the multivariate analysis) were positively associated with SJL >1 h and negatively associated with median light exposure during the day, especially in the morning from 8:00 to 10:00. Our results suggest that low light exposure during the day, and higher levels of SJL are associated with depressive symptoms; longitudinal and experimental studies are needed to understand the underlying mechanisms. Nevertheless, we highlight the potential of treatment strategies aimed at decreasing circadian strain and insufficient light exposure, which are suggested as areas of further research in Psychiatry.