Evidence indicates that many university students have poor adherence to a healthy diet accompanied by unhealthy lifestyle behaviors. Chrono-nutrition is an emerging field of research that examines the pattern of optimum daily activity in relation to the human's dietary patterns, and their reflections of variable health indicators such as sleep quality. However, there is a scarcity of research that examines the relationship between adherence to the healthy eating pattern, like the Mediterranean diet (MD), with sleep quality and chronotype among university students.A cross-sectional study was conducted, and convenience sampling was used. Participants were assessed for adherence to the MD using the Mediterranean Diet Quality Index (KIDMED), for sleep quality using the Pittsburgh Sleep Quality Index (PSQI), and for chronotype using the Morningness-Eveningness questionnaire (MEQ).P < 0.001) even after adjustment for age and sex . The regression analysis also showed that those with good adherence to the MD had a significant association with better subjective sleep quality, less sleep latency, sleep disturbance, and daytime dysfunction even after adjustment for age and sex. Those with morningness chronotype had about a six-fold higher chance to have good adherence to the MD .The study included 503 university students, most of them (81.5%) were females. Only 15.1 and 16.9% reported morningness chronotype and good sleep quality, respectively. About half of the students showed medium and high adherence to the KIMED. In-depth analysis revealed that students with good adherence to the MD were more likely to have a good sleep quality (OR = 0.35; 95%CI: 0.21–0.59; Good adherence to the healthy diet presented in the MD among university students is associated with morningness chronotype and with improved overall sleep quality and sleep components. Long-term, controlled intervention research works are warranted for more elaboration on the impact of chronotype and dietary habits on sleep quality and other important aspects such as mental health and academic achievement. The Mediterranean diet (MD), native to countries surrounding the Mediterranean basin, has been consistently associated with improved individual health , 2. The Sleep, an important dimension, and determinant of lifestyle is a complex homeostatic process that involves multiple processes, including sleep-wake homeostasis, circadian cycle, and human dietary and lifestyle behaviors . AdequatRelated to sleep is the chronotype, defined as the expression of an individual's diurnal preferences in experiencing various psychological and biological traits including the wakefulness and timing of sleep . IndividA growing body of evidence signifies the relationship between diet and sleep, with several foods and food components that have been found to improve sleep duration and quality. Among these, tryptophan, and foods containing tryptophan, melatonin, and phytonutrients were looked at as sleep-promoting foods linked to improved sleep outcomes , 26, witWhile the MD and adequate sleep quality are important determinants of health throughout the lifecycle, they are of utmost importance among youth and adolescents. Youth and young adulthood is a period between 15 and 25 years of age, characterized by dramatic psychological and physiological changes, such as increased levels of autonomy in decision-making and increased self-development and identity . People To achieve the study objectives, a cross-sectional survey was conducted among students at the University of Sharjah (UoS), Sharjah, UAE. The UoS is a national university that has a diverse study body, coming from various ethnicities and backgrounds. Data collection was carried out between January and May 2021, using a convenience sampling approach. An email inviting students to take part in the study was sent by the central administration to all students. Students, both males, and females, aged 18 years and above and registered at the university during the time of data collection were eligible to participate in this study. The protocol of this study was designed and implemented in line with the guidelines specified by the Declaration of Helsinki. The survey's protocol and data collection instrument were reviewed and approved by the Research Ethics Committee at the UoS (REC-21-03-03-09-S). Before the start of data collection, participants were presented with a brief introduction about the study, its objectives, and procedure, and were asked to provide a signed electronic informed consent, in case they agreed to participate. Participants were informed that their participation is anonymous and completely voluntary in that they can choose or not to answer any particular question and that they are free to withdraw from the study at any time. Participation was completely voluntary, with no monetary or non-monetary incentives were given to the study participants.Sample size calculations showed that 479 students are needed to estimate a 13.3% prevalence of good adherence to the MD with a power of 80% and an error margin of 3% . The 13.For the data collection, a multicomponent, self-administrated online questionnaire was designed using Google Forms in both the English and Arabic languages. The link for the questionnaire was included within the body of the emails distributed to the participants. A panel of experts, consisting of a clinical nutritionist, a nutrition epidemiologist, and a public health scientist, developed the questionnaire choosing the scales for each of the concepts under study and ensuring its content validity. The questionnaire was first developed in the English language, later translated into Arabic, and back-translated into English. The original and back-translated versions of the questionnaire were compared for consistency and to ensure parallel-form reliability. Following the development of the questionnaire, a pilot test was conducted on 15 students to ensure cultural and context adaptability. The results of the pilot test were not included in the data for this study.The questionnaire consisted of four main sections that addressed (1) sociodemographic characteristics, 2) adherence to the Mediterranean diet, (3) sleep quality, and (4) sleep chronotype. Except for the first section, the questionnaire referred to the month prior to the participation in the study. The average duration for the completion of the questionnaire was 10 min.2.Section Introduction included questions to provide the following information about the participants: age (in years), sex , nationality , marital status , income , smoking status  years spent at the university , cumulative grade point average (CGPA) , and major of studies at the university . In addition, participants were asked to report their weight (kg) and height (cm). Accordingly, the participant's body mass index (BMI) was calculated as weight (kg)/[height (m)]r = 0.60 among college students  was used. A systematic review of tools used showed that the KIDMED is the most widely used index to assess adherence to the MD among youth . The relstudents . This instudents , addressstudents .The third section of the questionnaire addressed the sleep quality among study participants . The AraThe last section of the questionnaire examined the participants' sleep chronotype  using the Morningness-Eveningness Questionnaire (MEQ), first developed by Horne and Östberg . A reviet-test for continuous variable and Chi-square tests for categorical variables. In order to investigate the effect of adherence to the MD on sleep quality, simple and multiple logistic regressions analyses were carried out, with the adherence to the MD (high vs. low) as the independent variable and sleep quality indicators as dependent variables. Sleep quality indicators consisted of the overall PSQI and its seven components. The effect of sleep chronotype on adherence to the MD was also examined using simple and multiple logistic regression analyses. For these analyses, the three classes of adherence to the MD were re-categorized into two categories  and considered as dependent variables, while the five chronotypes were re-categorized into three categories  and entered as the independent variables. For all analyses, variables that showed significant associations with adherence to the MD in the univariate analyses were included in the multiple regression models. Results of the logistic regression analyses were presented as odds ratio (OR) and their corresponding 95% confidence interval (CI). A p-value <0.05 was considered statistically significant.Data analyses for this study were carried out using the Statistical Package for Social Sciences (SPSS) for Mac HD version 23 . Participants' characteristics were described as mean (standard deviation), and frequency and proportions, for continuous and categorical variables, respectively. The characteristics were presented for the overall sample and by adherence to the MD. For the latter, participants were grouped, as per the KIDMED index, into “low”, “medium”, and “high” adherence. Comparisons of participants' characteristics between the poor and high adherence to the KIDMED were carried out using independent student's 2 and about one third were overweight/obese (32.6%). The majority were GCC nationals (88.3%) and single (87.7%), whereas about half of them had low income (51.5%) and 52.7% were in their first-third year of study. More than half of the students (56.5%) had a CGPA of >3/4. The vast majority were non-smokers (92.8%) and from non-medical and health sciences colleges (70.4%). Only the minority had morningness chronotype and a good PSQI . About 54% (272/503) of the students showed medium and high adherence to the KIMED  with a mean age of 22.11(4.2) years and a BMI of 23.97(2.09) kg/mhe KIMED .P = 0.056).P < 0.001). Similarly, those with good PSQI (PSQI score ≤ 5) were about 3 times significantly (P < 0.001) more adherent to the KIDMED (74.1%) compared to those with poor PSQI (50%). In-depth analysis of the association of the KIDMED adherence with the PSQI components showed that those with good subjective sleep quality (60.2%), no sleep latency (60.5%), no sleep disturbance (57.4%), and no daytime dysfunction (60.5%) were significantly more adherent to medium and high KIDMED.P < 0.001).P < 0.001), sleep latency , sleep disturbance , and daytime dysfunction  even after adjustment for age and sex.P <0.001 and adjusted OR = 5.67; 95%CI: 2.86–11.26; P <0.001, respectively).The current study is the first to examine the relationship between adherence to MD, subjectively measured sleep quality using PSQI, and the chronotype among university students. Findings of the current work showed that greater adherence to the MD is associated with better overall sleep quality and sleep components.Sleep quality is strongly affected by food quantity and quality, with dietary inflammatory potential has been proposed as one of the inflecting factors that affect sleep quality and duration . ProvidiAnother underlying mechanism that explains the relationship between MD and improved sleep quality is the ability of the MD to increase melatonin secretion. Many fruits and vegetables are good sources of melatonin, and experimental studies reveal that consumption of these phytochemical-rich foods can improve sleep quality parameters and alleviate sleep disturbances , 57.Further, the relationship between poor sleep quality and food intake has been consistently examined. Poor sleep quality and short sleep duration are associated with greater food intake and lower-quality diet, which are increasingly recognized as risk factors for obesity , 59. It From a health perspective, the morning chronotype has been the preferred chronotype over the evening one, with the former having less association with cardiometabolic risk factors and other health problems, including psychological, neurological, and gastrointestinal morbidities, and lower mortality rates compared to evening chronotypes . These hThe relatively low prevalence of good sleep quality (about 17%) among our university students is also consistent with and mirrors for other reports in the UAE and other parts of the globe. However, this number is lower than previous estimates among students in the UAE, whereby the prevalence rates of good sleep, using similar assessment tools were 34% in 2020 and 44.8% in 2021, respectively , 76. SucHaving three-times higher reported good sleep quality among those with morningness chronotype than those with the evening one (30.3% vs. 10.6%), found in this study, is consistent with other reports among university students throughout the world, such as in Brazil , 78, IndAge and sex are factors that are variably associated with the adherence to the MD , with coDespite the well-known health-improving effects and disease-preventing impact of MD against chronic diseases, a low adherence rate to the MD has been repeatedly reported, especially in the Mediterranean countries themselves , 65, witHaving fewer fruits than recommended, skipping breakfast, and frequently visiting fast-food restaurants reported among university students in our work is consistent with the repeatedly observed unhealthy behaviors among the university students in the UAE and other parts of the world , 68. UniThe current work entails some limitations that should be considered when interpreting the current findings. First, diet quantity was not examined in the current work, which may also affect sleep quality and may interact with different components of sleep. The inherent limitations of the cross-sectional design cannot be overridden in this context, where causality cannot be inferred in such an observational design. Other limitations related to the self-reported questionnaires such as recall bias, and response bias are worth to be kept in mind. Of particular importance is the self-reported nature of the assessment of sleep quality, which relies heavily on the participants' subjective assessment. Future studies could include objective measures of sleep quality, using polysomnography . In addiTo conclude, good adherence to the MD among university students is more associated with morningness chronotype and improved overall sleep quality and sleep components. Improving the knowledge and attitude of the university students toward their dietary and lifestyle behaviors, and the significance of chronotype in determining their future disease risk factors are of pivotal importance. Further long-term, controlled intervention research works are warranted for more elaboration on the impact of chronotype and dietary habits on sleep quality.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Sharjah, REC approval no. (REC-21-03-03-09-S). The patients/participants provided their written informed consent to participate in this study.MF: conceptualization, project administration, and funding acquisition. MF and FN: methodology, validation, supervision, and writing—original draft. HH: software. HH and FN: formal analysis and visualization. MF, FN, SK, MB, HA-M, and AA: investigation and data curation. MF, FN, and HH: resources and writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Benzodiazepines have been widely used in clinical practice for over four decades and continue to be one of the most consumed and highly prescribed class of drugs available in the treatment of anxiety, depression, and insomnia. The literature indicates that Benzodiazepine users at a significantly increased risk of Motor Vehicle accidents compared to non-users but the impact on injuries at workplace is not well-defined. We aimed to investigate whether use of benzodiazepine is associated with increased risk of occupational injuries (OI).Methods: PubMed, Embase, and Scopus databases were searched. A meta-analysis was performed to calculate odds ratio (OR) and 95% confidence interval (CI) among case controls, cross-sectional studies, either questionnaire or laboratory exams based.Results: A total of 13 studies met inclusion criteria, involving 324,168 OI from seven different countries, with an estimated occurrence of benzodiazepine positivity of 2.71% (95% CI 1.45–4.98). A total of 14 estimates were retrieved. Of them, 10 were based on laboratory analyses, three on institutional databases, while one study was based on questionnaires. Regarding the occupational groups, three estimates focused on commercial drivers , that exhibited a reduced risk ratio for benzodiazepine positivity compared to other occupational groups . Eventually, no increased risk for benzodiazepine positivity was identified, either from case control studies , or cross sectional studies, when only laboratory based estimates were taken in account .Conclusions: Even though benzodiazepines have the potential to increase injury rates among casual and chronic users, available evidence are insufficient to sustain this hypothesis, particularly when focusing on laboratory-based studies . Benzodiazepines (BDZ) are commonly prescribed central nervous system (CNS) drugs that cause a selective allosteric potentiation of the action of γ-aminobutyric acid (GABA) at GABAA receptors, with eventual inhibition of neural activity  guidelines. Two different databases  were inquired for relevant studies published from their inception to April 3, 2021. The search strategy was a combination of the following keywords [free text and Medical Subject Heading (MeSH) terms]: (benzodiazepine* OR ≪psychotropic drug*≫ OR sedative* OR hypnotic* OR ≪sleeping pill*≫ OR ≪sleeping tablet*≫) AND (accident* OR injur*) AND . Further studies were retrieved from reference lists of relevant articles and consultation with experts in the field. Records we handled using a references management software Endnote X7 software  and duplicates were removed.This systematic review has been conducted according to the PRISMA . To be included, a study should also report the share of injured subjects positive for BDZ use: post-accident analysis of laboratory specimens, questionnaire-based researches, or retrospective assessment of institutional databases were included in the analyses and separately analyzed. Proxies for use of BDZ, i.e., sedatives, sleeping tabs/pills, anxiolytics, were similarly included in the analyses, while more generic case definition  were excluded. In doubtful cases, i.e., the actual shares of positivity for BDZ or their proxies were not overtly reported, corresponding author of the paper was contacted requesting for clarifications. We further explored the reference lists of recent topic-specific reviews to find additional eligible papers. Only articles in English, French, German and Italian were included.Identified studies were independently reviewed for eligibility by two authors (FB and GG) in a two-step-based process: a first screen was performed based on titles and abstracts while full texts were retrieved for the second screen. At both stages, disagreements by reviewers were resolved by consensus.At the second screen level, data were extracted by one author (GG) supervised by a second author (MR) using a standardized data extraction spreadsheet. The data extraction spreadsheet was piloted on 3 randomly selected papers, and modified accordingly with. Data extraction included: study information , study design , characteristics of the study group(s), assessment of BDZ positivity status , total number of participants, population size, and reported estimates  or the information needed to calculate an estimate.Quality of the studies included in meta-analysis was assessed through the Guide methodology for systematic review. Two authors  independently assessed the following domains: recruitment strategy, blinding, exposure assessment, outcome assessment, confounding, incomplete outcome data, selective reporting, and conflict of interest. The quality of individual studies was rated based on fixed and unequivocal criteria in which the end result is one of the following possible statements about the risk of bias: “low,” “probably low,” “probably high,” “high.” Disagreements among raters were resolved through discussion so that a consensus was obtained.The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.p < 0.05.We first performed a descriptive analysis to report the characteristics of the included studies. The prevalence, OR and corresponding 95% Confidential Intervals (CI) were used as the primary measures to assess the frequency and risk of a positive status for the use of BDZ (or their proxies) in occupational injuries. The results were considered statistically significant when I2 statistic was calculated to quantify the amount of inconsistency between studies; it estimates the percentage of total variation across studies that is due to heterogeneity rather than chance. I2 values ranging from 0 to 25% were considered to represent low heterogeneity, from 26 to 50% as moderate heterogeneity and above 50% as substantial heterogeneity, being pooled using a random-effects model.To investigate publication bias, contour-enhanced funnel plots representing Egger test for quantitative publication bias analysis (at a 5% of significance level) were generated. In case of asymmetry at the funnel plots, outliers were excluded irrespective of the results of Egger's test. In fact, Egger's test may yield false positive results if fewer than 10 studied were included. Radial plots were then calculated and visually inspected to rule out small study bias.meta” and “metafor” packages with R (version 4.0.3) and RStudio (version 1.1.463) software.All calculations and illustrations of funnel plots, were performed by means of “The search strategy yielded a total of 2,649 records . After rThe pooled characteristics of the included studies are reported in the The final sample included a data on a total of 408,176 subjects, with 324,168 occupational injuries. Of them, 9,205 cases (2.8%) were positive for the use of, resulting from laboratory analyses , a retrospective analysis of institutional databases , with a further study based on a questionnaire assessment (7.1%).The quality of the included studies is summarized in In the majority of studies, blind status of researchers may have been high or very high, but because of the retrospective design its eventual effect was presumptively not significant. Most of studies had a probably low risk of bias in the outcome assessment, as employed institutional or administrative data, that are assumed to have a high degree of completeness. Confounding was identified at high risk of bias in the majority of studies, because multiple important potential confounders were not evaluated. Three studies had a high or probably high risk of bias in incomplete outcome data as there was insufficient evidence that such data were adequately addressed. We assigned a low or probably low risk of bias in selective reporting to the majority of studies, while it was significantly higher in non-externally validated questionnaire studies , a random effect model was applied in further analyses.As shown in p < 0.01; I2 = 76%, p < 0.01; I2 = 55%, p = 0.06)   with substantial heterogeneity  , but the = 0.04) .p = 0.5802)  , but alsI2 95%, see I2 estimates >55%) precludes drawing more definite conclusions.A growing base of evidence links pre-injury BDZ use with both road accidents and falls of inpatients , and higher in questionnaire-based studies , but some explanations may be tentatively suggested. Firstly, laboratory-based studies assess the potential effect of BDZ at the time of the event, hinting toward the actual effects of the drugs on the CNS, while retrospective ones  more appropriately evaluate chronic consumption of the drugs. In other words, not only retrospective analyses are more likely to report on baseline neuropsychological disorders of the workers than on their status immediately before the accident. Moreover, our analysis was able to retrieve only one questionnaire-based study  may be explained through the actual requirements for BDZ detection. While alcohol, and even some illicit drugs such as tetrahydrocannabinoids and opioids may be detected on site, either through breathalyzer or urine testing, BDZ detection still requires analysis by a toxicology lab, which involves time and expense as well as sophisticated instruments operated by highly qualified staff , or through the analysis of institutional databases for previous receipts, or even questionnaires (Drummer et al., Even though such variables as the posology and the settings of drug uptake, in acute and/or chronic form, strictly model the impact of BDZ on the cognitive side and on the psychophysical performances, only two studies have taken in account such factors in their analyses (Orriols et al., An interesting remark of our study, is the surprisingly low risk for occupational injuries in professional drivers. This is particularly interesting as the use of BDZ has been reported to increase crash risk up to 100 times (Movig et al., In conclusion, the effect of the short or long-term use of BDZs on psychomotor performance and cognitive aspects is still unclear as the data are still discordant due to the lack of rigor in the collection of information on drug intake in the studies. It would be relevant to have information on the type of BDZ taken, dosage, time of intake, indication of the prescription of BDZ and concomitant use of other drugs, temporal correlation with the possible injury.Certainly the long-term use of BDZs, especially if they have a long half-life, have deleterious effects on performance and cognitive aspects with increased risk of injuries, the effect size for the use of short-acting BDZ being smaller than for long-acting BDZ.The use of short-acting BDZ for limited periods, as is currently the optimum prescribing practice, may minimize the adverse impact on an individual's cognitive impairment and in depressive patients it could improve the information processing speed after acute treatment (Duan et al., It is imperative that greater efforts are made to curtail inappropriate prescribing, keeping BDZ for selected circumstances only preferring short-acting BDZ for limited periods. This is important not only for the long-term prevention of cognitive impairment or dementia and dependence in a vulnerable population but also to decrease the risk of incidence and injury in workplace. Since BDZs are among the most used drugs in the world with a high risk of creating addiction and data in the literature suggest that the use of hypnotics represent an avoidable risk factor with respect to road accidents, more work is needed to better define the association between use of BDZ and occupational injury risk and subsequent morbidity.The original contributions presented in the study are included in the article/SG and MR: conceptualization. MR, NB, SG, and PL: methodology. MR, NB, and GG: formal analysis. SG and PL: data analysis. SG, PL, MR, NB, and GG: data curation, writing—review and editing, and writing—original draft preparation. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lactobacillus. These findings suggest that Goji berries could be used to produce innovative feeds for rabbits, although further studies are necessary to evaluate their impact on productive performance, gut immune system maturation, as well as resistance to gastrointestinal disorders.The microbial community that inhabits specific areas of the body, developing a symbiotic relationship with the host, is termed the microbiota. The intestinal microbiota plays a pivotal role in different physiological processes and is influenced by many factors, including nutrition. Goji berries are a popular nutraceutical product that have been proposed as a dietary supplement in some livestock species, including rabbits, but their effects on the composition of the microbiota have never been investigated. This study evaluated the effects of Goji berry supplementation on the microbiota of different digestive tracts  of the rabbit, using a modern method of analysis. Our results suggest that Goji berries could modulate the microbiota of the rabbit’s digestive tract increasing the growth of beneficial bacteria, such as Ruminococcaceae, Lachnospiraceae, Lactobacillaceae, and particularly, the genus n = 14) or the same diet supplemented with 3% of Goji berries , from weaning (35 days old) until slaughter (90 days old). At slaughter, samples from the content of the gastrointestinal tracts were collected and analyzed by Next Generation 16S rRNA Gene Sequencing to evaluate the microbial composition. Ammonia and lactic acid were also quantified in caecum. Results showed differences in microbiota composition between the groups for two phyla (Cyanobacteria and Euryarchaeota), two classes (Methanobacteria and Bacilli), five orders, fourteen families, and forty-five genera. Ruminococcaceae (p < 0.05) and Lachnospiraceae (p < 0.01) were more abundant in G than in C group. Lactobacillaceae also showed differences between the two groups, with Lactobacillus as the predominant genus (p = 0.002). Finally, Goji berry supplementation stimulated lactic acid fermentation (p < 0.05). Thus, Goji berry supplementation could modulate gastrointestinal microbiota composition and caecal fermentation.Goji berries show health benefits, although the possible mechanisms of action, including compositional changes in the gut microbiome, are still not fully understood. The aim of this study was to evaluate the effect of Goji berry supplementation on microbiota composition and metabolites in the digestive tracts of rabbits. Twenty-eight New Zealand White rabbits were fed with a commercial feed (control group, C;  Lycium barbarum plant, are often used in traditional Chinese medicine for their nutritional and therapeutic properties, and are also widespread as supplementation in Western diets [Goji berries, the fruits of the rn diets ,2. Theirrn diets , althougrn diets ,5, rats rn diets  and humarn diets . Recentlrn diets ,9,10. Inrn diets  and prodrn diets  energy mrn diets , and mearn diets ,15 in a The microbiota represents a complex ecosystem of microorganisms which inhabits specific niches of the body and plays important roles in physiological processes developing symbiotic relationships with the host . The intLycium barbarum fruit, to favor the growth of a beneficial gut microbiota. Understanding the effects of Goji berry supplementation on the intestinal microbiota can therefore have important implications for the health of rabbits. The rabbit could be also considered as an animal model for diet-induced changes in gut microbiota, as it has already been used for studies exploring the effect of nutrition on productive [The rabbit is a very interesting species because it can be a pet, livestock or animal model. Both in pet and farmed rabbits, the digestive system is a common site of diseases that are often associated with changes in intestinal microbiota ,20. In poductive ,25,26, roductive ,28, and oductive ,30.The aim of this study was to investigate the effect of Goji berry supplementation on the microbiota composition of the different tracts of the digestive system  in the rabbit. For this purpose, the microbiota of all the sections of the digestive apparatus was analyzed using Next Generation 16S rRNA Gene Sequencing. In addition, metabolites from bacterial fermentation in the caecum (lactic acid and ammonia) were also quantified.The experimental trial was conducted in the facilities of the Faculty of Veterinary Medicine of the Agricultural University of Tirana, Tirana, Albania.The rabbits were maintained under the supervision of a responsible veterinarian and in accordance with the Directive 2010/63/EU regarding the protection of animals kept for farming purposes. The lowest number of rabbits necessary to obtain reliable results was used for the trial.n = 14 animals, C), fed with a commercial pellet, and Goji group , fed with the same feed of the C group supplemented with 3% of Goji berries  before pelleting  until slaughter (90 days of age): control group  were collected separately in 15 mL sterile tubes and then stored at −80 °C until examination. Each sample was analyzed individually. The average body weights (±standard error) at weaning were 875 ± 55 g and 893 ± 75, while at slaughter, they were 2310 ± 82 g and 2357 ± 82 g in C and G groups, respectively.Using the commercial QIAamp PowerFecal Pro DNA Kit , the bacterial DNA was extracted from each sample of intestinal contents following the manufacturer’s protocol. DNA quality and quantity were checked using a NanoDrop ND-1000 spectrophotometer  and beta (across-) diversities were used. These indices were estimated starting from the OTU table, after filtering with more than 50 total counts, distributed in at least five samples. Besides the number of observed OTUs directly, within-sample microbial richness, diversity, and evenness were estimated using Chao1 and ACE (abundance-based coverage estimator) for richness, Shannon, Simpson, and Fisher’s alpha for diversity ,34, and https://github.com/filippob/Rare-OTUs-ACE.git, accessed on 15 November 2021) and R  scripts were used to estimate the ACE index and sample-based rarefaction. The figures were generated with the ggplot2 R package [The QIIME 1.9 pipeline  was util package . The R e package  was usedFor the analysis of bacterial metabolites (lactic acid and ammonia), 1 g of caecal content was diluted in 1 mL of 1 M perchloric acid and 8 mL of distilled water. After homogenization, tubes were centrifuged for 10 min at 5000 rpm, and the supernatant was transferred to 2 mL Eppendorf tube and frozen at −20 °C until metabolite quantification. The spectrophotometric method for biological fluids was used for lactic acid determination in accordance with Pryce et al. . Ammoniaijkt is the alpha diversity index value for record i from rabbit j with treatment k and anatomic region t, µ is the intercept, rabbitj is the systematic effect of the individual rabbits, treatmentk is the treatment effect (Goji vs. control), anatomic regiontk(j) is the effect of the anatomic region of the gastrointestinal tract nested within rabbitj, and eijkt is the residual.ij between records within rabbits in the off-diagonal block elements, I is the identity matrix, and σe2 is the residual variance.Differences in alpha diversity indexes between treatments at various taxonomic levels along the rabbit’s gastrointestinal tract were tested with a linear model that took into account the hierarchical structure of within-subject nested data . The model had the following form:A simplified version of Model (1) was used to evaluate differences between Goji and control samples; in particular, where the anatomic region effect was dropped and data from all gastrointestinal sections were analyzed jointly to evaluate the effect of Goji supplementation on the overall rabbit gut microbiota.For Bray–Curtis dissimilarities (beta diversity), differences along the digestive tract were tested non-parametrically using the permutational analysis of variance approach .The microbiota structure of the gastrointestinal tract of C and G groups was characterized by a total of 6,122,359 and 7,156,769 high quality reads (after filtering), respectively, with a mean of 75,584 ± 38,864 reads for C and 90,592 ± 33,296 reads for G group. The evaluation of the sample-based and sequence-based rarefaction curves suggested that the depth of coverage was sufficient to describe the biological diversity within the samples .p = 0.034 and p = 0.004, respectively) between the experimental groups, with higher relative abundances in the upper part of the gastrointestinal tract in G group.Phylum relative abundances distribution along the gastrointestinal tract of C and G groups are summarized in Moreover, Clostridia represented the major class in all anatomic regions, while Ruminococcaceae and Lachnospiraceae were the most abundant families in the Goji group .p = 0.0032) in the G group, and Bacillus was the major genus in the stomach (p = 0.0036). Ruminococcaceae UCG-005, Lachnospiraceae NK4B4 group, and Christensenellaceae R-7 group were genera detected in all the digestive tracts with statistically significant different results between the groups. As reported in p = 0.0018) between the groups with Lactobacillus as the predominant genus in G group compared to C group.p = 0.0176) and jejunum (p = 0.000049). This was confirmed by bootstrapping  ratio followed a clear pattern along the rabbit’s digestive tract starting at around 10 in the stomach, increasing clearly in the duodenum and jejunum, and finally decreasing again in the caecum and colon. The F:B ratio appeared to be significantly lower in the G group , in the p < 0.01) from permutational multivariate analysis of variance . This difference appeared to vary along the gastrointestinal tract, with jejunum, caecum, and colon showing the clearest differences, while the two groups mostly overlapped in the stomach, duodenum, and ileum (p = 0.033). No significant differences in ammonia concentration were detected between the two groups .Regarding lactic acid quantification, G group showed a higher concentration than C group, suggesting higher bacterial activity , and was slightly predominant in the G group. This phylum, not significantly different between the two groups and along the digestive tracts, is known for its role in the stimulation of gut-associated lymphoid tissue ,48. The Methanobrevibacter [Regarding other phyla, Verrucomicrobia were found in all sections, while Actinobacteria and Proteobacteria were found in the stomach and small intestine. Although with low levels in the core microbiome, the Euryarchaeota phylum, belonging to the kingdom Archea, was statistically different between the two groups, with higher levels in the G group in all the digestive tracts. All species of this phylum were taxonomically assigned to the methanogenic genus vibacter . Though vibacter , it is ovibacter .Clostridia, anaerobic Gram-positive bacteria present in the intestinal microbiota of human, mouse, chicken, and pig, represented the major class in all anatomic regions, in accordance with Velasco-Galilea and co-workers ; they arThe families of Ruminococcaceae and Lachnospiraceae were present in all anatomic parts, and both were higher in the G group. Ruminococcaceae are usually prevalent in healthy rabbits , while LLactobacillus was the predominant genus. Lactobacilli are rare in the rabbit intestine, occupying less than 1% of the total intestinal bacteria [Lactobacillus abundance in rabbits supplemented with Goji could play a protective role against inflammatory diseases. Components of commensal bacteria can alleviate intestinal inflammation by regulating the expression of both pro-inflammatory and anti-inflammatory factors. Kawashima et al. [Lactobacillus bacteria, showed a regulatory function by triggering anti-inflammatory factor IFN-β production and inhibiting pro-inflammatory factors production.Lactobacillaceae was another family that showed significant differences between the two groups, although present in small quantities. Within this family, bacteria , and thebacteria , probabla et al.  reportedThe F:B ratio was at around 10 in the stomach, then increased in the duodenum and jejunum, and subsequently progressively decreased from the ileum to the caecum and colon. The F:B ratio appeared to be lower in the G group, significantly so in the duodenum and jejunum, less so in the ileum, caecum and colon, as also shown by the bootstrapping analysis. Studies in human microbiota and in animal models, have reported that the F:B ratio was directly related to body weight modifications and in particular to obesity . In obesLactobacillus spp. These conditions could guarantee greater resilience toward dysbiosis in the gut microbiota, which is necessary to maintain homeostasis and, in turn, the healthy status of the gastrointestinal system [The alpha diversity results revealed higher microbial richness and diversity in bacterial composition independently from the treatments in the large intestine. That was an expected result because, as already demonstrated in other livestock species, the microbial densities  along the gastrointestinal tract is maximal in the fermenting compartments . Indeed,l system . The bet2, CH4, CO2) and compounds that are incorporated into microbial cells [w/w of Goji berries in the rabbit diet was able to increase the Lactobacilli population in rabbit meat.The differences in microbiota composition are due to the environmental conditions, such as pH modifications, along the gastrointestinal tract. In adult rabbits, the principal substrates for caecal microorganisms are polysaccharides and protein. Caecal microorganisms ferment available nutrients, converting them to metabolites  could be affected by changes in microbial community composition. Moreover, evaluation of digestive efficiency by performing an in vivo digestibility trial could integrate the study of the microbiota composition of the rabbit. Finally, it could be interesting to evaluate the impact of microbiota modification on the maturation and activity of the immune system, as well as on resistance to infectious diseases, animal welfare and the productive performance of the rabbit.Lycium barbarum fruit increased the growth of the phylum Bacteroidetes as well as of Ruminococcaceae, Lachnospiraceae, and Lactobacillus in the caecum and colon, and as a consequence, lactic acid production. The mechanism of absorption and integration of the bioactive molecules contained in the fruit, and their influence on the microbiota population, should be investigated to appropriately use Goji berries’ probiotic properties. The use of this natural compound needs to be further studied for its implications for both commercial performance and animal resistance to infection, as its supplementation could reduce the incidence of health problems in livestock and consequent antibiotic treatments.The present study demonstrated that Goji berry supplementation can modulate gastrointestinal microbiota composition and caecal fermentations of the rabbit. In particular,


Background and Objectives: Parkinson’s disease (PD) is a prevalent neurodegenerative condition responsible for progressive motor and non-motor symptoms. Currently, no prophylactic or disease-modifying interventions are available. Uric acid (UA) is a potent endogenous antioxidant, resulting from purine metabolism. It is responsible for about half of the antioxidant capacity of the plasma. Increasing evidence suggests that lower serum UA levels are associated with an increased risk of developing PD and with faster disease progression. Materials and Methods: We conducted an electronic medical record database study to investigate the associations between UA levels and different characteristics of PD. Results: Out of 274 datasets from distinct patients with PD, 49 complied with the predefined inclusion and exclusion criteria. Lower UA levels were significantly associated with the severity of parkinsonism according to the Hoehn and Yahr stage , with the motor complications of long-term dopaminergic treatment , and with the presence of neurocognitive impairment . Conclusions: Oxidative stress is considered a key player in the etiopathogenesis of PD, therefore the involvement of lower UA levels in the development and progression of PD is plausible. Data on the potential therapeutic roles of elevating serum UA  are scarce, but considering the accumulating epidemiological evidence, the topic warrants further research. Parkinson’s disease (PD) is a neurodegenerative condition affecting over 6 million people worldwide . ParkinsMost cases of PD are multifactorial and sporadic, but monogenic forms, familial or de novo, with onset at younger ages, also exist . The etiSerum UA levels above the upper reference range  are associated with gout , arteriaHere, we report the results of a small cross-sectional observational study on the associations between serum UA levels and the characteristics of sporadic PD, performed on an electronic medical record database from a tertiary referral center in Romania. This work adds to the evidence that UA may play a role in the progression of motor and nonmotor symptoms in PD. The potential therapeutic role of UA in preventing PD or delaying its progression are also discussed.We conducted a cross-sectional study using the electronic medical record database at Colentina Clinical Hospital, a tertiary referral center for PD in Bucharest, Romania. Our study invested the potential associations between serum UA levels and demographic and clinical characteristics of patients with PD. The study was performed in agreement with the Declaration of Helsinki and was approved by the local Ethics Committee (15/ 7 June 2021). All personal data were anonymized, according to the General Data Protection Regulation (2016/679). The study was conducted as part of the doctoral research of the first author (supervised by B.O.P.).We screened all the available electronic medical records , using the Hippocrate Electronic Medical Record software, which is the electronic medical record database introduced in 2018 at Colentina Clinical Hospital. Study enrollment was based on predefined inclusion and exclusion criteria as follows: we included patients with a diagnosis of sporadic PD at discharge , made by a neurologist working at Colentina Clinical Hospital; in order to be included, the patients must have had the onset of motor manifestations over the age of 50 years, no family history suggesting monogenic PD, and available results of a blood workup, including serum uric acid levels. We excluded patients with concurrent infectious diseases (including COVID-19), systemic inflammatory syndrome, haematological or oncological diseases, and patients with concurrent medication that may alter serum UA levels. We also excluded patients whose datasets were insufficient. If a patient had several datasets/hospital admissions complying with the enrolment criteria, we used the data from the initial evaluation .We collected the following data: sex, age, living environment , diagnosis of PD and of other diseases, medication, PD characteristics , and the serum UA levels .® Statistical Package for Social Sciences® (SPSS®) Statistics Subscription software. For the continuous variables, we calculated the means and standard deviations (SD). The statistical analysis included the Spearman correlation coefficient (rs), for ordinal and nominal categorical variables, and the Pearson coefficient with point-biserial correlation, for binary/dichotomous variables (r). We also performed logistic regression with odds ratio (OR) and 95% confidence interval (95% CI), using the serum UA level as the independent variable, and the motor complications and cognitive impairment as dependent variables. We considered the statistical significance threshold at p < 0.05. Normal distribution of data was assessed using graphical and numerical methods in SPSS®, including the Shapiro–Wilk test and the Kolmogorov–Smirnov test.For statistical analysis, we used the IBMn = 17), followed by stage 3 in 26.5% of the patients (n = 13). Motor complications were present in 33.3% of the patients (n = 15), neurocognitive impairment was found in 32.7% (n = 16), and cerebrovascular disease in 26.5% (n = 13). For more details on the demographic and clinical characteristics of the study population, please see The inclusion process, detailed in n = 6), and above the upper reference range in 6.1% of them (n = 3). We found significant associations between lower serum UA levels and the presence of motor complications , the presence of neurocognitive impairment , and the severity of the parkinsonism, assessed through the Hoehn and Yahr scale —see p = 0.008). We found no association between sex and the Hoehn and Yahr stage, motor complications, or neurocognitive impairment. Logistic regression found significant associations for motor complications  and neurocognitive impairment . The estimated power of the study was 100% for the Hoehn and Yahr stage, 70% for the motor complications, and 50% for the neurocognitive impairment. No significant associations were found with disease duration, cerebrovascular disease, comorbidities, or concomitant medication .The mean serum UA levels of the PD patients was 4.99 mg/dL ± 0.35 (SD 1.27), reference range 3.4–7 mg/dL. Serum UA levels were below the lower reference range in 12.2% of the PD patients . Moreover, the cross-sectional design means that data on the evolution of the disease are only in small part based on previous medical records, most being subjected to recollection bias. Additionally, our study did not include data from patients with probable monogenic PD (as genetic testing was not performed in most patients). Interestingly, higher levels of serum UA are associated with a higher risk of developing PD in carriers of pathologic variants of the leucine-rich repeat kinase 2 (LRRK2) gene, the most common cause of familial PD , and theDespite the above limitations, our findings are important because they add to the evidence that serum UA levels are associated with motor and neurocognitive impairment in patients with PD. This means that serum UA and U deserve further research, both as biomarkers for PD progression, and as plausible therapeutic targets for disease-modyfing interventions. In this respect, serum UA and U levels can be influenced by means of diet; for instance, a purine rich diet  would increase their serum levels . IntraveThe serum UA level is a potential marker for the risk of PD development and progression and might provide the opportunity for potential therapeutic interventions for prodromal PD. Considering the current lack of prophylactic or disease-modyfing therapies in PD, and the increasing epidemiological and experimental evidence in support of a prospective role of UA and U in PD, further reasearch is warranted.


Charcot-Marie-Tooth type 1A (CMT1A) comprises ~50% of all CMT cases. CMT1A is a slowly progressive motor and sensory neuropathy that leads to significant disability. We aimed to investigate the quality of life (QoL) in Serbian patients with CMT1A and to assess sociodemographic and clinical features associated with their QoL.Forty-five genetically confirmed patients with CMT1A were included −60% women . SF-36, Medical Research Council (MRC) Sum Score, CMT Examination Score (CMTES), Overall Neuropathy Limitation Scale (ONLS), Beck Depression Inventory (BDI), and Krupp's Fatigue Severity Scale (FSS) were used in the study.p < 0.05), longer disease duration , more pronounced muscle weakness measured by MRC-SS , presence of tremor (p < 0.05), worse CMTES , more severe disability in upper  and lower limbs  measured by ONLS scores, use of walking aids (p < 0.01), and with depression (p < 0.01) and fatigue (p < 0.01). Worse scores on CMTES , BDI , and FSS  were significant independent predictors of worse QoL in patients with CMT1A .Regarding SF-36, Mental Health and Social Functioning were the scales with the best achievements, whereas Role Physical was the worst domain. Worse QoL in patients with CMT1A was associated with elder age (rho = −0.34, Besides impairment made directly by CMT1A itself, QoL in these patients was also strongly affected by the presence of depression and fatigue. Since CMT1A is still not a curable disease, it is of interest to identify factors associated with QoL that are amenable to treatment. PMP22 gene (Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disease, with a prevalence of up to 82.3 per 1,00,000 inhabitants as registered in Norway . Previous studies reported reduced QoL in patients with CMT –15. MostMost previous studies examined QoL of a heterogeneous group of patients with CMT, including also patients diagnosed solely based on clinical features without a confirmed gene mutation. It is known that the CMT is a heterogeneous disease, presented differently depending on the underlying mutation and disease subtype. Because of the above-mentioned heterogeneity, we included only genetically confirmed patients with CMT1A, which is an important advantage of our study. Furthermore, our study is one of the few that examined the QoL of these patients in developing countries, which is of great importance considering the well-known association of Human Development Index (HDI) and QoL in the general population .We aimed to investigate QoL in Serbian patients with CMT1A and to assess sociodemographic and clinical features associated with their QoL, especially ones that are potentially amenable to treatment or prevention.PMP22 gene was carried out in the Genetic Laboratory of the Neurology Clinic by the real-time quantification PCR (RT-PCR) using the TaqMan assay on the ABI7500Fast apparatus . The HSA gene was used as an endogenous control. The analysis was carried out in separate reactions, for each sample in triplicate, and the ΔΔCt model was applied in data processing  project, which was established to collect data on patients with CMT who have been diagnosed and/or followed at the Neurology Clinic, University Clinical Center of Serbia as the largest tertiary neuromuscular center in the country. Determination of the number of copies of the ocessing . In 10 yAll patients were tested during 2018 and 2019. By reviewing medical records and surveying patients, the following sociodemographic data and clinical features were obtained: gender, age at disease onset, age at the time of referral to the genetic analysis, disease duration, and family history. We differed between juvenile disease onset (if symptoms started before the age of 20) and adult onset of the disease (if the first symptom was noticed after the age of 20). Evaluation of the limb muscle strength was done using the Medical Research Council (MRC) 0–5 point scale . Total MRC sum score (MRC-SS) was obtained by adding individual assessments of the following muscle groups on both sides: shoulder abductors, elbow flexors, wrist extensors, hip flexors, knee extensors, and foot dorsiflexors . CharcotSerbian version of the SF-36 questionnaire was used to measure health-related QoL . It is a2 test, Fisher test, Mann–Whitney U test, and Student's t-test were used as appropriate. Correlations were estimated using Spearman's rho. To obtain independent predictors of worse QoL, all variables that were associated with total SF-36 score (p < 0.01) in univariate analysis were included in the multiple linear regression analysis (stepwise method) with the total SF-36 score being a dependent variable. For all statistical tests, significant testing was two-sided, where p < 0.01 was considered highly significant and p < 0.05 significant.The normality of data was evaluated using the Kolmogorov–Smirnov test. For group comparisons, the χp < 0.01).The main sociodemographic and clinical features of Serbian patients with CMT1A are shown in Results on the SF-36 questionnaire are shown in p < 0.01), BDI , and FSS  were significant independent predictors of worse QoL in patients with CMT1A .In the linear regression analysis, the SF-36 total score was a dependent variable, whereas all parameters that correlated with the total SF-36 score were independent variables. Worse scores on CMTES , which was expected considering CMT1A is an untreatable, progressive, chronic disorder . SurprisWe observed the lowest scores in RP, GH, and PF domains of SF-36. Similar findings with the lowest scores in GH, VT, PF, and RP domains of the SF-36 scale were previously observed in CMT1A , 32 and In Serbian patients with CMT1A, decreased QoL in both mental and physical domains (MCS and PCS) was associated with greater muscle weakness, more severe disability, use of walking aids, presence of fatigue, and depression. Three earlier studies showed that disability and limb muscle weakness are the main predictors of worse PCS in a heterogeneous group of patients with CMT , 8, 15. Besides physical issues, we noticed a strong correlation between mental aspects of QoL and physical impairment/disability measured through the MRC-SS scale, CMTES scale, and ONLS. Two previous studies also found lower limb weakness/ability to toe-walk as independent predictors of worse scores on mental health domains of SF-36 in patients with CMT1A  and CMT We observed that worse PCS, but not MCS, in patients with CMT1A was associated with elder age, longer disease duration, and presence of tremor. Several studies also observed an association between age/disease duration and PCS, but not MCS –7. This Besides impairment made by CMT itself , the main predictor of worse QoL in CMT1A was the presence of depression and fatigue. The prevalence of fatigued patients in our CMT1A cohort was 56%, which is in line with previous reports where authors emphasized fatigue as a significant independent predictor of worse QoL in CMT , 12, 36.The prevalence of depression in patients with CMT1A was 28.9 vs. 2.2% in controls. Some previous studies did not find the excess prevalence of depression in patients with CMT –40. On tOur study has several limitations. First, the number of patients is relatively small, but not so small for such a rare disease. However, our study has the advantage that all patients were from the same cultural background. Another limitation of the study is that we did not assess correlations between QoL and nerve conduction study (NCS) results. However, we intentionally decided not to use the NCS part of the CMT Neuropathy Score (CMTNS), but only symptoms and signs scores, so-called CMTES. The reason for this is that previous studies showed that NCS items have a floor effect. For example, the ulnar SAP is absent in almost all patients with CMT, even in mildly affected individuals. Finally, it would be of interest to have prospective, longitudinal data on QoL in CMT1A. Since our patients were tested in the pre-COVID era, we believe that further assessment during the pandemic and in the post-COVID era will be of specific interest based on the previous literature . It is oIn conclusion, our results confirmed a significant reduction of QoL in patients with CMT1A. We identified several factors associated with QoL, some of them being amenable to treatment, which is of crucial importance in developing new strategies for QoL improvement in untreatable, chronic disorders, such as CMT1A.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethical Committee of the Faculty of Medicine, University of Belgrade. The patients/participants provided their written informed consent to participate in this study.VI, BB, VS, and AK contributed to the concept or design of the study. VI, BB, AP, MB, IBa, IBo, and AS contributed to the analysis and interpretation of data. VI and BB produced the first draft of the manuscript. All authors provided input into subsequent drafts and reviewed and approved the final version for submission.This study was supported by the Ministry of Education, Science and Technological Development of Serbia, #175083.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study investigated, through wrist actigraphy, the activity-rest pattern, estimate nocturnal sleep parameters, and quantify the exposure of light (daylight and blue light) during social isolation due to COVID-19.The participants  wore the actigraph in nondominant wrist for 7 days. Derivation of 25 nocturnal sleep parameters was inferred from PIM mode raw data including sleep, wake, activity, and fragmentation statistics. A hierarchical cluster analysis determined the participants profiles. Mann-Whitney and independent Student t tests, linear stepwise regression and Kendalls test were applied. The significant level was a = 0.05.Two clusters were formed, normal sleepers (n = 13) and short sleepers (n = 6). The participants of both clusters went to sleep after midnight, spent approximately 1 h of being awake during time in bed, their latency to persistent sleep was normal, though true sleep minutes was less than 7 h, showed a normal sleep efficiency. Daytime activity was moderate, and a circadian rhythm was irregular. The regressions showed that bedtime and nocturnal activity contributed to the variance of daytime activity and the beginning of it (p< 0.001). The midpoint during the time in bed was the most significant predictor for the start of less period activity at night (p< 0.001).Actigraphy inferred that during social isolation the individuals presented, despite normal sleep latency and efficiency, inconsistent sleep parameters and irregular circadian rhythm. Moreover, decreased exposure to daylight during the morning was observed. The pandemic due to Coronavirus disease (Covid-19) is today, in Brazil, a phenomenon of great magnitude and extension, which has caused such great losses in terms of human lives, economy and quality of life of individuals.2. Circadian rhythms are 24 h daily cycles that can be entrained or phase- shifted not only by our internal clock but also by external factors such as daily schedules, social rhythms, and daylight exposition3. This is important because activity and behavior during wakefulness can infuence the duration and quality of sleep and, conversely, the duration and quality of sleep can affect daytime function4.The 17th of March 2020, the São Paulo Government implemented measures to prevent risks of direct transmission of COVID-19. The Brazilian population has been subjected to a substantial period of social isolation with a restriction of movements, cancellation of all events promoted by the Governments that generate crowds of people, such as events sporting, artistic, cultural, political, scientific, and commercial. Many people have been in home confinement situations; non-essential services adopted a non-face-to-face regime , and were authorized to operate the services of urgent needs of the community . These changes in lifestyle have negative consequences for well-being, which impact sleep quality that can be related to changes in the sleep/wake cycle and circadian rhythms6, not allowing the clinical evaluation of research participants. Nevertheless, studies are required to show objective data on the rhythmicity of the circadian cycle and sleep in healthy individuals during that time. In this sense, this study hypothesizes that social isolation can alter the sleep–wake pattern, generating irregular and less robust activity–rest patterns. Furthermore, during this period the daily exposure daylight may have decreased because people stayed at home, which may have led to increased exposure to blue light. Thus, this study aimed to investigate, through wrist actigraphy, the activity–rest pattern, estimate nocturnal sleep parameters, and quantify the exposure of light (daylight and blue light) in healthy individuals who were in social isolation during the COVID-19 pandemic.Previous studies have been presented using online questionnaires, as the lifestyle changes during social isolationThis study was approved by the Ethics Committee of of Piracicaba Dental School, University of Campinas (FOP-UNICAMP) . The participants provided their written informed consent to participate.2, to sleep alone , to be in social isolation and living in Piracicaba city, São Paulo state, Brazil. Individuals who were self-reporting sleep disorders and respiratory diseases by a standardized questionnaire, for example, obstructive sleep apnea and asthma, and neurological disorders were excluded. Demographic data, such as age, sex, marital status, and education were obtained through questionnaires. To calculate the BMI, self-reported height and weight were obtained from which BMI was calculated as weight in kilograms (kg) divided by height in square meters (m2).The base population of this study were well-known young adults, specifically students from the high-schools and universities of Piracicaba, S P, Brazil. First, they were considered as potential participants and then were invited to participate via WhatsApp, composing a convenience sample. The inclusion criteria were age 18 to 35 years, body mass index (BMI) of 18.5–24.9 kg/m7. In this study, 24 h periods were recorded for seven consecutive days; however, this study was cross-sectional and descriptive-observational.In the framework of chronobiological study designs, longitudinal sampling corresponds to obtaining data on the same individual as a function of timeth to June 8th, 2020. The degree of social isolation began from March 17th in the State of São Paulo, Brazil, when the acquisition of actigraphy data began. Schools were closed, the home-office was encouraged and activities with many people were restricted. However, the measures adopted of social isolation were not so strict, which gave people relative freedom, i.e., they could decide to stay at home and go out only if necessary or go out to walk/run. Initially, the participants should receive the actigraph in the lab of the Dental School, but due to the closure of educational institutions, the device has just been delivered and removed at the participants’ homes.The data were collected from March 168, showing excellent sensitivity (95.69%), good accuracy (80.24%), predictive value for the sleep of 81.52% and predictive value for wakefulness of 68.93% in individuals with sleep-disordered breathing. All participants were instructed to use the device on the wrist of the non-dominant hand for 7 days  applied to evaluate the circadian rhythms and sleep parameters of the selected sample. This device was previously validated byr 7 days , startinStandardized procedures were performed on the device configuration  before handing it over to the participant, as follows:- The device was programmed to record activity counts in the proportional integral mode (PIM), every 30 s, and added to generate a 1 min epoch; similarly, light was recorded every 30 s.- The calculations of day length were based on the times of sunrise and sunset  computed based on the local latitude, longitude, date, and geopolitical time zone.- The information stored in the actigraph was transferred via a USB adapter  to a computer using the software provided by the manufacturer . All configurated were made on the same computer.9. These participants were not included in the final study.- The examiner (DZRD) was previously trained to handle actigraphy related to data acquisition and analysis. Five participants, aged 24.6 ± 4.16 years, properly instructed, used the device for four consecutive days. After 12 days, the data were acquired again in the same way allowing the determination of intra-examiner reliability using the intraclass correlation coefficient, which was 97-99, showing “good” reliability, according to Koo and Li10 and are included in four statistics, namely, sleep, wake, activity, and fragmentation, providing a detailed analysis method to infer these parameters objectively, allowing a description of events during the periods11. The derivation of each sleep parameter uses only the sleep scores. The complete definition of each parameter can be seen in the supplementary materials, as well as the normal values and ranges, so the studied variables were listed below:The sleep parameters were calculated based on the Cole-Kripke algorithmA) Sleep statisticsi. Time in bed (TIB)ii. Sleep period (SLP)iii. Sleep minutes during TIB (SMIN)iv. True sleep minutes (TSMIN)v. Sleep onset latency (SOL)vi. Latency to persistent sleep (LPS)vii. Percent sleep (PSLP)viii Sleep efficiency (SE)ix. Sleep episodes (SEP)x. Mean sleep episode (MSEP)xi. Long sleep episodes (LSEP)xii. Longest sleep episode (LGSEP)B) Wake statisticsi. Wake minutes during TIB (WMIN)ii. Wake after sleep onset (WASO)iii. Number of awakenings during TIB (NA)iv. Mean wake episode (MWEP)v. Long wake episodes (LWEP)vi. Longest wake episode (LGWEP)C) Activity statisticsi. Mean activity during TIB (AMEAN)ii. Activity standard deviation during TIB (ASD)iii. Activity index (ACTX)D) Fragmentation statisticsi. Sleep fragmentation index (SFX) ii. Brief wake ratio (BWR)Circadian rhythm non-parametric variablesi. Activity counts for the most active 10 h period (M10) and Onset-M10ii. Activity counts for the least active 5 h period (L5) and Onset-L5iii. Inter-daily stability (IS)iv. Intra-daily variability (IV)v. Relative amplitude (RA)vi. Circadian function index (CFI)Complementary measuresi. Chronotype by SLP and TIBii. Exposure to daylight and blue lightFor data statistical analyses, the Statistical Package for Social Sciences, version  was used. Descriptive statistics were performed for all variables and sample characterization expressed as mean, standard deviation, median, and quartile amplitude.12. The hierarchical cluster analysis was performed using the farthest neighbor method for calculating distances between clusters and obtain the dendrogram. The sleep parameters that contributed to the formation of clusters can be seen in Supplementary Table 112. After analyzing the plot dendrogram, it was decided to inform a priori the number of clusters to be performed for identifying clusters of participants with similar sleep parameters. Thus, two clusters were chosen (clusters 1 and 2). The differences between clusters were assessed by the Mann–Whitney test for clustering validation.To study the participants’ profiles, a cluster analysis was performed, which aims to organize a set of cases into homogeneous groups, in such a way that the individuals belonging to a group are similar to each other and different from the restData distribution was assessed using the Shapiro–Wilk test and the quartile–quartile plot (QQ-plot) graphs, and the homogeneity Levene test. The sleep parameters and circadian rhythms  show data not normally distributed, whereas the circadian rhythms  were normally distributed. To compare the differences between clusters and differences between weekdays and weekends, the Mann–Whitney test was applied. For the variables I V, IS, and CFI the differences between clusters, the independent Student t-test was applied.Exploratory analyses were conducted to investigate potential relationships between circadian rhythm variables (M10 and L5) and sleep parameters predictors . The relationships were tested using the linear stepwise regression model. The blue light and sunlight intensity were correlated with Onset SLP and Onset L5, using Kendall’s test.The level of significance was defined as alpha equal to 0.05.The characteristics of the study population are shown in LWEP and SFX all parameters were significantly lower for normal sleepers.Nevertheless, the similar values of NA between clusters were above the normal range.Although the other parameters were similar for both clusters, in the pre-selection, they were considered different as much as possible.Differences between weekdays and weekend was only for SLP, since short sleepers had lower values, suggesting that they slept one hour and thirty minutes less during the weekend when compared with the normal sleepers (p < 0.005) . New sleep behaviors can be assumed during social isolation due to changes in daytime and night-time activities and routines can infuence the parameters mentioned above19. An interesting observation is the similarity between median values of Onset SLP  and the values of Onset L5  mainly in the short sleepers. This finding shows the need for further studies that implement a methodological analysis for the derivation of the sleep parameters presented in this study to test whether the Onset SLP and the Onset L5 match.Bedtime can be considered delayed in the present study, probably due to isolation during the COVID-19 pandemic. This is an important observation, since later sleep timing has been associated with poor health results20 due to a delay in mid-sleep on workdays21. One possibility is that social isolation increased fexibility regarding social schedules, determining the postponed time of awakening on workdays20.Get up was nearly 9 a.m., without difference between weekdays and weekend. Recent studies have found that during social isolation, the sleep-wake time difference between weekdays and weekend days decreased24. Conversely, Korman et al.20, observed a delay in sleep onset during social isolation that did not affected the sleep duration, due to the postponed wake-up time. The respective differences can be attributed to different measurements, since the study of Korman et al.20, used questionnaires in a large sample, whereas the others used actigraphy.The SOL showed normal values in both clusters, despite the late bedtime, and was similar with other studies before Covid-19 pandemic26 but are in contrast with Cellini et al.27 and Haghayegh et al.28, who showed that participants spent adequate TIB, but the sleep quality was not good enough, mainly because of a high amount of WASO, differing from the participants in the present study. Some studies during social isolation due to Covid19 pandemic agreed with Cellini et al.27, showing a decrease in sleep quality in this time21. On the other hand, many people experienced increased fexibility regarding social schedules, leading to improve individual sleep– wake timing and overall more sleep21, that can be occurred in part with the participants of the present study.Moreover, the sleep parameters, WASO and SE, presented also normal values, suggesting that, although participants slept late and presented light activity during TIB, sleep quality was adequate, that is, greater than 90% in both clusters. These findings agree with previous studies using other devices16; circadian phase or questionnaire to estimate chronotype were not considered. There was significantly difference between clusters, with a later chronotype for the short sleepers, although no differences were found between clusters for sleep-wake-up time, considering TIB and SLP. This was an unexpected result, as late chronotypes prefer to wake up later in the morning and sleep later at night11, but the condition of social isolation during the pandemic could be an infuencing factor. Moreover, variations in chronotype can be associated with variations in the timing of numerous physiological and behavioral variables29.The chronotype was estimated by midpoint of the TIB and SLP, as done also by Wright et al.30. According to Pépin et al.31, the activity pattern did not appear to have a significant clinical impact on people’s activity compared to the pre-pandemic period. M10 was in line with L5 values, indicating that participants presented slight movement intensity during the time of less activity that usually occurs during the sleep period32, agreeing with previous studies30. Differences in circadian rhythm between clusters can be explained because during the social isolation the first one had a little bit to exposure to light during the early hours of the morning than the second one, which can lead to the delay of the biological clock, since sunlight has been considered as an important zeitgeberts34.The activity pattern for M10 in this study did not differ between clusters, suggesting that the participants perform a moderate activity in the wake period during daytime, as seen also by Forner-Cordero et al.35. However, during the COVID-19 pandemic, the participants were kept in their homes, probably reducing the physical activity.The stepwise regression models showed that both sleep parameters, M10 and L5, explained a significant amount of the variance in the level of day/night activity. Of the various factors that may infuence M10, ASD contributed significantly to the variation of daytime activity, demonstrating that activity during the sleep period may have had the effect of decreasing the amount of daytime activity. This is an interesting result since activity at night can stabilize day-to-day habits, thus creating a more regular circadian rhythm35, which was observed in the present sample. WASO was other significant contributor, suggesting that fragmented nights by nocturnal activity can also contribute to Onset M10 delay. Moreover, AMEAN significantly infuenced L5 due to nighttime activity during the least active period of the light–dark cycle. The preference time to sleep and wake up (Midpoint TIB) was the most significant predictor for the variation of the Onset-L5, because the participants of this study preferred to go to sleep late and wake up later. This sleep behavior produces a delay at the beginning of M10 during the light–dark cycle, and consequently, it makes L5 start later.A relevant finding of the study was that the bedtime was a significant and negative contributor to the beginning of daytime activity because a late bedtime overlaps sleep in the middle of the morning, consequently it will lead to a few hours of daytime activity. The lack of activity significantly increases the fragmentation of the activity–rest rhythm20, social restrictions lead to robust shifts in daily behavior and in exposure to daylight that can explain the present findings. Moreover, participants could be exposed to less daylight during social isolation, due to the characteristics of the house, such as small windows and no outside area19. When analyzing the actograms, a degree of variability was evidenced during the 7 days  data acquisition was in a small convenience sample of healthy adults of different ages only during the period of social isolation, determining that the results cannot be extrapolated to other populations and requiring new assessment of the participants in the post-pandemic period. Further, (iii) the fact that most studies use methodologies and devices different from the present study determine that the respective comparisons should be interpreted with caution. Moreover, (iv) no measures of daily sleepiness and nap were collected, as well as data prior to the period of social isolation; therefore, any strong assumptions about sleep disorders cannot be made. Finally, (v) the cross-sectional design of the study does not allow for the determination of a causal effect of circadian rhythm on sleep parameters.The findings should be interpreted considering the limitations of the study that include (i) the lack of PSG data to validate findings (the gold standard of sleep assessment). However, the adopted methodology provided a clear and detailed protocol for scoring sleep. Moreover, Rodrigues and EckelliDespite the aforementioned limitations, it is important to guide individuals in general to maintain a more consistent sleep schedule, with regular sleep time in an adequate and specific environment, use electronic equipment sparingly at appropriate times, avoiding stimulating drinks before bedtime and avoiding light during sleep. These cares and/or habits can contribute to mitigate the sleep disturbances reported in this study.In conclusion, actigraphy inferred that during social isolation the individuals presented, despite normal sleep latency and efficiency, inconsistent sleep parameters and irregular circadian rhythm. Moreover, decreased exposure to daylight during the morning was observed.


Introduction: This study aimed to examine the effectiveness of the national school-based intervention on both obesity and high blood pressure in Chinese children and adolescents aged 6–18 years.Methods: The national school-based cluster non-randomized controlled trial was done in seven provinces from September 2013 to February 2014. A total of 23,175 children and adolescents in the control group and 25,702 in the intervention group were included in this trial with a mean follow-up of 6.7 ± 0.9 months. Mixed-effects regression models were used to evaluate the effect of the interventions on body weight and blood pressure (BP).Results: A significant upward in the body mass index (BMI) levels but downward in systolic BP (SBP), diastolic BP (DBP), BMI Z-scores, SBP Z-scores, and DBP Z-scores were witnessed in the intervention group compared to those in the control group (<0.001). Subgroup analyses presented significant intervention effects in children aged 6–12 years for BMI, SBP, DBP, and their standardized values Z-scores, but no effective results were found in adolescents aged 13–18 years. Stratification analyses based on the dynamic weight changes presented non-differential HBP, SHBP, and DHBP prevalence gaps between the control and intervention groups. Children aged 6–12 years with higher BMI percentiles at baseline presented obvious declines in SBP and DBP standardized values Z-scores.Conclusion: A mean 6-month multi-centered school-based comprehensive obesity intervention in China yields a small to null effect on obesity and hypertension with increasing age; the early age before 12 years may be the key period for interventions, and the younger, the better. Precise and high-intensity interventions targeting the population at different stages of childhood and adolescence are urgently needed to be developed.Clinical Trial Registration:https://www.clinicaltrials.gov/, identifier: NCT02343588 Obesity continues to rise rapidly in many countries . In ChinSchools have been regarded as one promising setting for scalable obesity interventions. Since the 1990s, many local school-based intervention studies for obesity prevention have been conducted among Chinese children and adolescents , 8. MostThe Health Lifestyles Intervention in Chinese Children and Adolescents (HLI-CCA) was a multicenter cluster non-randomized controlled school-based intervention aiming to prevent obesity in children and adolescents. It included elements of education around nutrition and physical activity as well as modifications to school environments and engagement of families . We hypoThis national trial with a school-based cluster non-randomized controlled design took place in seven provinces (centers) including Liaoning, Ningxia, Shanghai, Chongqing, Hunan, Tianjin, and Guangdong . This stn classes from each grade (n depended on the average size of classes and was no <200 students per school). According to the protocol, the schools and the participants were adjusted slightly to meet the matched standards of balanced schools in the same stratification in each center and the equal distribution of participants in the control or the intervention group .A multistage cluster non-randomized sampling was used to obtain the representative sample of children and adolescents in each center, where schools meeting the survey requirements, willing to participate, and fulfilling the inclusion criteria were selected according to the trial protocol. In the end, 12–17 primary and secondary schools were recruited in each center. Altogether, 92 schools were included in our study and were assigned to the control or the intervention group. In each school, all participants selected were extracted from All participants underwent a regular physical examination every year, and those who had one or more of the following conditions were excluded based on the previous medical history and physical examination data: (1) serious organ disease ; (2) abnormal physical development ; (3) physical impairment or deformity ; or (4) acute disease symptoms  during the past month and not yet recovered. Furthermore, all principals of the selected schools were assessed comprehensively through a face-to-face interview to ensure the smooth execution of the project.According to the trial protocol, a planned sample size of 7,000 has 90% power to detect a 10% difference in the obesity intervention group at 5% statistical significance (two-sided) . A samplThe HLI-CCA aimed to deliver a general healthy lifestyle message encouraging a healthy energy balance. In schools assigned to the intervention, the HLI-CCA was delivered to children and adolescents with integrated intervention strategies focused on changing specific behaviors related to energy intake and expenditure, such as decreasing the consumption of sweetened fizzy drinks, increasing the consumption of vegetables, ensuring proper protein intake, reducing sedentary behaviors including screen time, and maintaining at least 1 h of moderate to vigorous physical activity. Integrating the above intervention elements, the comprehensive intervention strategy of the HLI-CCA includes the following four parts during the whole intervention period, which were described in the previously published protocol in detail .As shown in Project members, school managers, class teachers, school doctors, parents, and students themselves were all involved in the intervention project. The investigators arranged to supervise the intervention in schools throughout the program. All schools completing the trial would be offered all intervention materials from the project team to reduce loss of follow-up. In the control schools, no specific intervention strategies or activities were carried out throughout the study period, but only records of their daily activities according to ordinary monitoring were conducted by project members. Detailed quality control measures and process evaluation were implemented according to the protocol, including the specialized intervention manual, professional trainings for all the intervention school doctors, health education teachers, and physical education teachers, routine supervision by project managers, and appropriate incentives to avoid loss of follow-up .At baseline and follow-up examinations, information on the demographic characteristics, parental education level, and occupation were collected by questionnaire. All the anthropometric measurements were taken in a sensitive manner in private rooms in both the control and intervention schools. All the measurements followed a standardized procedure by professionals who had passed the training course.Participants underwent a complete anthropometric measurement at baseline and outcome including height, weight, waist circumference, hip circumference, and blood pressure. About 5% of students would be rechecked, and if the error exceeds 10%, all the students should be measured again. All the participants wore only light clothing and stood erect, barefoot, and at ease while being measured. Height was measured to the nearest 0.1 cm with a portable stadiometer , and weight was measured to the nearest 0.1 kg with a standardized scale . According to the WHO anthropometric standardization and comparison of different standards, waist circumference and hip circumference were measured to the nearest 0.1 cm, and their measurements were located at 1 cm above the umbilicus and the maximal protrusion of the buttocks, respectively , 14.Blood pressure was measured using an auscultation mercury sphygmomanometer  with an appropriate cuff for children. Three cuff sizes  were selected according to the age and upper arm circumference of the children, which stipulated that the cuff bladder width should cover 50–75% of the mid-arm circumference. The mid-upper arm circumference determined the cuff size. The cuff was placed ~2 cm above the crease of the elbow. The child was seated comfortably for at least 10 min prior to the first reading. The feet of children were placed on a platform during BP measurement. Blood pressure was measured twice, with a 1-min break between each measurement. The participants were asked to remain quiet and to sit still while each reading was being taken. Systolic blood pressure was determined by the onset of the first Korotkoff sound (K1) and diastolic blood pressure determined by the fifth Korotkoff sound (K5). The stadiometers, scales, steel tape, and auscultation mercury sphygmomanometer were calibrated before use, and similar instruments were used in all measurements at the investigated schools.Z-scores] and categorical variables . BMI was calculated as body weight (in kilograms) divided by height (in meters) squared. Overweight and obesity were classified using the sex- and age-specific BMI reference values developed by the International Obesity Task Force (IOTF)  , Stable group (BMI Z-score changes between −0.5 and +0.5), and Increase group (BMI Z-score changes +0.5 or greater).The two primary outcomes were body mass index (BMI) and blood pressure (BP), including continuous variables [BMI, systolic BP (SBP), diastolic BP (DBP), and their standardized values e (IOTF) , 16. BMIhcharts) . BMI valhcharts) . BMI Z-sZ-scores were calculated according to reference in the Fourth Report.Systolic HBP (SHBP) and diastolic HBP (DHBP) were defined as SBP and DBP greater than or equal to the reference age-, sex-, and height-specific 95th centile, respectively, according to the National High Blood Pressure Education Program reference in the Fourth Report. HBP was defined as the SHBP or DHBP of children . The SBPp-values. Similarly, mixed-effects models for binomial responses were used to evaluate the intervention on categorical variables  with odds ratios (ORs), whose values more than 1 meant an increased effect in the variables after intervention, while values <1 meant an effective declined effect with significant p-values. Random intercepts with assumed Gaussian distribution were also used in the mixed-effects regression models.Baseline characteristics were described as the mean (SD) for continuous variables or number (percentage) for categorical variables. The analysis of mixed-effects regression models was used to evaluate the effect of intervention on outcomes after adjusting for age, sex, provinces, urban/rural areas, and the baseline disequilibrium for multiple variables. Two-level  mixed-effects regression models were used in our analyses due to the grouping structure of the data consisting of multiple levels of nested groups. Linear mixed-effects regression models were used to evaluate the intervention on continuous variables  with β coefficients, whose positive values meant an increase in the variables after intervention, while negative values meant an effective decline with significant p-values of 0.05.Subgroup analyses were used in our study by age groups as follows: 6–9, 10–12, 13–15, and 16–18 years, which were used to identify the effective or sensitive subgroups for intervention. Allowing for the different intervention effects between the age groups in this study and the age composition of the stages of primary and high schools in China, the study further evaluated the differences in the intervention effects between the two age groups of 6–12 and 13–18 years based on the analysis of the four age groups above. Stratification analyses based on the dynamic change of BMI during the intervention periods were used to assess whether the body weight changes had mediating effects on the intervention effects by age groups. Stratification analyses based on BMI percentiles at baseline were used to explore which groups with different initial body weights were sensitive to intervention. Due to the blood pressure sustained increment along with age by genders, we used the blood pressure standardized values to assess the highly sensitive population with weight at baseline. In each BMI percentile group, changed values of variables = —. The same analyses of the mixed-effects regression models were conducted for the different BMI percentile groups with β coefficients and ORs to further quantify the intervention effects of BMI on blood pressure. Statistical analyses were conducted using Stata version 14.0 (StataCorp). Statistical significance was defined using a two-sided test with The baseline characteristics of the study participants are shown in 2 compared to 0.11 kg/m2 in the control group. The β coefficient of 0.04 (p <0.001) indicated a greater increase in BMI in the intervention group. Both control and intervention groups had declines in the prevalence of overweight and obesity, but with a non-significant trend for lower levels in the intervention effects  after adjusting for age, sex, provinces, urban/rural areas, and the baseline disequilibrium for multiple variables. As for the BMI Z-scores, the negative significant β coefficient of −0.0035 (p = 0.002) indicated that there was a significant decrease of the BMI Z-scores in the intervention group compared to the control group.After an average 6 months' intervention with participation rates of 94.3% in the intervention group and 94.8% in the control group, the intervention group had an increase of BMI values of 0.14 kg/mZ-scores for children and adolescents aged 6–18 years, respectively (p <0.001). Similar results were found in both sexes, with 0.78 mmHg (SBP) and 0.40 mmHg (DBP) absolute decreases in boys and 0.75 and 0.53 mmHg absolute decreases in girls, respectively. However, overall, no significant difference for the intervention effects existed for HBP, SHBP, and DHBP, except for DHBP in girls, although we saw a larger decline in the prevalence of HBP, SHBP, and DHBP in the intervention group than that in the control group. For example, the prevalence of HBP decreased from 8.6 to 5.8% with −2.8 percentage points, which was higher than that in the control group (−2.6 percentage points) with non-statistical effects of the OR value   and DBP (−0.88 mmHg) levels was found in the intervention compared with the control group (0.33 and −0.41 mmHg). The negative significant β coefficients, after adjusting for age, sex, provinces, urban/rural areas, and the baseline disequilibrium for multiple variables, predicted that the intervention would lead to a 0.77 and a 0.46-mmHg absolute decrease in SBP and DBP, as well as to a 0.047 and a 0.033 decrease in the SBP and DBP = 0.107) .Z-scores, with statistically significant β coefficients, particularly in those aged 6–9 years with relatively better intervention effects. However, there seemed no obvious intervention effects for adolescents aged 13–18 years . Childhood and adolescence are two different but closely linked periods before adulthood. Adolescence is a critical transition period between childhood and adulthood, which brings rapid physical growth and behavioral alterations. However, girls begin reaching puberty with an average age of menarche at about 12 years, although with a 2-year lag in boys . After tlescents . They welescents . Thus, oPrevious studies reported that the risk of HBP would increase for individuals whose body status changed from normal weight to overweight and obesity during a long period, but they failed to examine the effects of weight loss on preventing HBP in adults , 41. WeiZ-scores) and risk  outcomes.The present study is the first and largest multicenter school-based and family-involved obesity intervention and BP effectiveness assessment study in Chinese children and adolescents. Although the selection of schools in each center did not realize the randomization, willingly participating schools will increase the degree of cooperation for intervention and the effectiveness of the implementation program. Thus, the design of the multicenter cluster non-randomized controlled trial provided a strong reference for obesity intervention on HBP prevention in Chinese children and adolescents with a broad age band and a large sample size, especially valuable for the present weak to null results among adolescents. However, the undesirable intervention results produced in those older might suggest that we should not waste resources in a general intervention among all adolescents in the future. The more precise, the more helpful; in other words, adolescents could not obtain more benefits from a general intervention, but more need of a precise intervention because the target population among them is small. In addition, the primary outcomes of the present study were fully assessed using a comprehensive set of variables around the theme of weight and blood pressure in order to capture potential intervention effects on later cardiovascular risks, including weight and blood pressure levels and their standardized . Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.YD conceptualized and designed the study, completed the statistical analyses, drafted the initial manuscript, and reviewed and revised the manuscript. YS, YM, and JM contributed to the conceptualization and design of the study, supervised the data collection, statistical analyses, initial drafting of the manuscript, and reviewed and revised the manuscript. ZZ and HW assisted with the statistical analyses and critically reviewed and revised the manuscript. PH and BD assisted with the data processing, statistical analyses, and the interpretation of the data. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.