Structural properties and binding mechanism of DNA aptamers sensing saliva melatonin for diagnosis and monitoring of circadian clock and sleep disorders.

Circadian desynchrony with the external light-dark cycle influences the rhythmic secretion of melatonin which is among the first signs of circadian rhythm sleep disorders. An accurate dim light melatonin onset (established indicator of circadian rhythm sleep disorders) measurement requires lengthy assays, and antibody affinities alterations, especially in patients with circadian rhythm disorders whose melatonin salivary levels vary significantly, making antibodies detection mostly inadequate. In contrast, aptamers with their numerous advantages (e.g., target selectivity, structural flexibility in tuning binding affinities, small size, etc.) can become preferable biorecognition molecules for salivary melatonin detection with high sensitivity and specificity. This study thoroughly characterizes the structural property and binding mechanism of a single-stranded DNA aptamer full sequence (MLT-C-1) and its truncated versions (MLT-A-2, MLT-A-4) to decipher its optimal characteristics for saliva melatonin detection. We use circular dichroism spectroscopy to determine aptamers' conformational changes under different ionic strengths and showed that aptamers display a hairpin loop structure where few base pairs in the stem play a significant role in melatonin binding and formation of aptamer stabilized structure. Through microscale thermophoresis, aptamers demonstrated a high binding affinity in saliva samples (MLT-C-1F Kd = 12.5 ± 1.7 nM; MLT-A-4F Kd = 11.2 ± 1.6 nM; MLT-A-2F Kd = 2.4 ± 2.8 nM; limit-of-detection achieved in pM, highest sensitivity attained for MLT-A-2F aptamer with the lowest detection limit of 1.35 pM). Our data suggest that aptamers are promising as biorecognition molecules and provide the baseline parameters for the development of an aptamer-based point-of-care diagnostic system for melatonin detection and accurate profiling of its fluctuations in saliva.

Evaluation of Correlation between Sleep and Psychiatric Disorders in a Population of Night Shift Workers: A Pilot Study.

BACKGROUND: Insomnia is the perception of inadequate, insufficient or non-restorative sleep. Of all sleep-related disorders, insomnia is the most common. It is important to remember that the sleep-wake cycle also plays a central role in the genesis of anxiety and depression. The aim of our study is to evaluate the association between sleep disturbances and anxiety and depression in a group of workers of both sexes who perform night shift work. METHODS: Information on sleep disorders was collected by administering the Insomnia Severity Index (ISI) questionnaire. Statistical analysis was conducted using the Chi-square test to assess whether there were any differences between sex for those who were healthy or who were diagnosed with psychiatric disorders. RESULTS: The results showed that there was a good percentage of subjects with insomnia problems, impairing normal daily activities and promoting the onset of fatigue, daytime sleepiness, cognitive performance deficits and mood disorders. CONCLUSION: We highlighted how anxious and depressive anxiety disorders are more pronounced in people who suffer from altered sleep-wake rhythms. Further research in this direction could prove to be fundamental for understanding the genesis of the onset of other disorders as well.

Alterations in Pregnenolone and Testosterone Levels in Male Shift Workers.

Steroid hormone levels are closely related to the endogenous circadian rhythm induced by sleep-wake and dark-light cycles. Shift work that disrupts the circadian rhythm may influence the levels of steroid hormones. The association between shift work and alterations in female sex steroid hormone levels has been studied, but little is known about testosterone and its precursor pregnenolone levels in male shift workers. The present study investigated serum pregnenolone and testosterone levels in a group of shift and daytime male workers. All participants were sampled at the beginning of the morning shift. Lower levels of serum pregnenolone and total testosterone were found in the shift workers compared to the daytime workers. Variations in pregnenolone levels may have consequences for well-being, and they might produce consequences for the levels of hormones downstream of the steroid hormone cascade, such as testosterone. The low levels of testosterone found in shift workers demonstrate the perturbative effect of shift work on testosterone serum levels, which may be independent and/or related to pregnenolone synthesis.

Circadian rhythm sleep disorders and time-of-day-dependent memory deficiency in Presenilin1/2 conditional knockout mice with long noncoding RNA expression profiling changes.

Alzheimer's disease (AD) patients exhibit sleep and circadian disturbances prior to the onset of cognitive decline, and these disruptions worsen with disease severity. However, the molecular mechanisms behind sleep and circadian disruptions in AD patients are poorly understood. In this study, we investigated sleep pattern and circadian rhythms in Presenilin-1/2 conditional knockout (DKO) mice. Assessment of EEG and EMG recordings showed that DKO mice displayed increased NREM sleep time but not REM sleep during the dark phase compared to WT mice at the age of two months; at the age of six months, the DKO mice showed increased wakefulness periods and decreased total time spent in both NREM and REM sleep. WT exhibited time-of-day dependent modulation of contextual and cued memory. Compared with WT mice, 4-month-old DKO mice exhibited the deficiency regardless trained and tested in the same light/night phase or not. Particularly interesting was that DKO showed circadian modulation deficiency when trained in the resting period but not in the active period. Long noncoding RNAs (lncRNAs) are typically defined as transcripts longer than 200 nucleotides, and they have rhythmic expression in mammals. To date no study has investigated rhythmic lncRNA expression in Alzheimer's disease. We applied RNA-seq technology to profile hippocampus expression of lncRNAs in DKO mice during the light (/resting) and dark (/active) phases and performed gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses of the cis lncRNA targets. Expression alteration of lncRNAs associated with immune response and metallodipeptidase activity may contribute to the circadian disruptions of DKO mice. Especially we identified some LncRNAs which expression change oppositely between day and light in DKO mice compared to WT mice and are worthy to be studied further. Our results exhibited the circadian rhythm sleep disorders and a noteworthy time-of-day-dependent memory deficiency in AD model mice and provide a useful resource for studying the expression and function of lncRNAs during circadian disruptions in Alzheimer's disease.

Circadian Interventions as Adjunctive Therapies to Cognitive-Behavioral Therapy for Insomnia.

The circadian system plays a key role in the sleep-wake cycle. A mismatch between the behavioral timing of sleep and the circadian timing of sleepiness/alertness can contribute to insomnia. Patients who report primarily difficulty falling asleep or early morning awakenings may benefit from circadian interventions administered adjunctively to cognitive-behavioral therapy for insomnia. Specific circadian interventions that clinicians may consider include bright light therapy, scheduled dim light, blue-blocking glasses, and melatonin. Implementation of these interventions differs depending on the patient's insomnia subtype. Further, careful attention must be paid to the timing of these interventions to ensure they are administered correctly.

Dysfunction of Prkcaa Links Social Behavior Defects with Disturbed Circadian Rhythm in Zebrafish.

Protein kinase Cα (PKCα/PRKCA) is a crucial regulator of circadian rhythm and is associated with human mental illnesses such as autism spectrum disorders and schizophrenia. However, the roles of PRKCA in modulating animal social behavior and the underlying mechanisms remain to be explored. Here we report the generation and characterization of prkcaa-deficient zebrafish (Danio rerio). The results of behavioral tests indicate that a deficiency in Prkcaa led to anxiety-like behavior and impaired social preference in zebrafish. RNA-sequencing analyses revealed the significant effects of the prkcaa mutation on the expression of the morning-preferring circadian genes. The representatives are the immediate early genes, including egr2a, egr4, fosaa, fosab and npas4a. The downregulation of these genes at night was attenuated by Prkcaa dysfunction. Consistently, the mutants demonstrated reversed day-night locomotor rhythm, which are more active at night than in the morning. Our data show the roles of PRKCA in regulating animal social interactions and link the social behavior defects with a disturbed circadian rhythm.

Current Insights into the Risks of Using Melatonin as a Treatment for Sleep Disorders in Older Adults.

Exogenous melatonin is commonly used for sleep disorders in older adults, and its use is increasing over time. It appears to have modest efficacy in treating insomnia and circadian rhythm sleep-wake disorders. Melatonin is commonly perceived to be a safe alternative to other hypnotics and is available without prescription in some jurisdictions. New evidence suggests that endogenous melatonin has pleomorphic effects on multiple organ systems, many of which are poorly understood. This narrative review summarizes the current evidence regarding the safety of melatonin in older adults (defined by age over 65 years). Melatonin appears to have a favorable safety profile in this population, however there is a dearth of evidence regarding the safety of prolonged use. There are several factors which increase the risk of adverse effects of melatonin in older adults, and these should be taken into consideration when prescribing to this population.

Exercise, diet, and sleep habits of nurses working full-time during the COVID-19 pandemic: An observational study.

BACKGROUND: Healthy diet, exercise, and sleep practices may mitigate stress and prevent illness. However, lifestyle behaviors of acute care nurses working during stressful COVID-19 surges are unclear. PURPOSE: To quantify sleep, diet, and exercise practices of 12-hour acute care nurses working day or night shift during COVID-19-related surges. METHODS: Nurses across 10 hospitals in the United States wore wrist actigraphs and pedometers to quantify sleep and steps and completed electronic diaries documenting diet over 7-days. FINDINGS: Participant average sleep quantity did not meet national recommendations; night shift nurses (n = 23) slept significantly less before on-duty days when compared to day shift nurses (n = 34). Proportionally more night shift nurses did not meet daily step recommendations. Diet quality was low on average among participants. DISCUSSION: Nurses, especially those on night shift, may require resources to support healthy sleep hygiene, physical activity practices, and diet quality to mitigate stressful work environments.

Impacto de los turnos de trabajo en la calidad del sueño del personal de enfermería en dos hospitales de tercer nivel de Canarias / Impact of shift work on the sleep quality of nursing staff attending two tertiary hospitals in the Canary Island

Fundamentos: Se sabe que las características de la actividad laboral influyen en la calidad del sueño de los trabajadores. Un elemento de la actividad laboral es la turnicidad. El objetivo de este estudio fue analizar la calidad del sueño del personal de enfermería de dos hospitales de tercer nivel en Canarias y su correlación con el turno de trabajo. Métodos: Estudio observacional, analítico y transversal, de una muestra no aleatoria de 275 enfermeros y técnicos en cuidados auxiliares de enfermería (TCAEs). Se midió calidad de sueño mediante el test de Pittsburgh Sleep Quality Index (PSQI) y características sociodemográficas y laborales con un cuestionario creado ad hoc. Resultados: El 65,8% del personal de enfermería tiene una mala calidad del sueño. Se observó que la turnicidad laboral afecta a la calidad del sueño. Los trabajadores con turno antiestrés (D/N) tienen mejor calidad de sueño (M=7,31; ±DE=4,0) comparado con el turno rotatorio (M/T/N) (M= 9,06; ± DE=4,3); p=0,01. Conclusiones: Las alteraciones del sueño son frecuentes entre el personal de enfermería, siendo el turno antiestrés (D/N) el que menos afecta a la calidad del sueño (AU)

Attention-Deficit/Hyperactivity Disorder and Delayed Sleep Phase Syndrome in Adults: A Randomized Clinical Trial on the Effects of Chronotherapy on Sleep.

Delayed sleep phase syndrome (DSPS) is the most common sleep disturbance in adults with attention-deficit/hyperactivity disorder (ADHD). We previously showed that chronotherapy with melatonin effectively advanced the dim-light melatonin onset (DLMO), a biomarker for the internal circadian rhythm, by 1.5 h and reduced ADHD symptoms by 14%. Melatonin combined with bright light therapy (BLT) advanced the DLMO by 2 h, but did not affect ADHD symptoms. This article explores whether sleep times advanced along with DLMO, leading to longer sleep duration and better sleep in general, which might explain the working mechanism behind the reduction in ADHD symptoms after treatment with melatonin. This article presents exploratory secondary analysis on objective and self-reported sleep characteristics from a three-armed double-blind randomized placebo-controlled clinical trial (RCT), which included 49 adults (18-55 years) with ADHD and DSPS. Participants were randomized to receive sleep education and 3 weeks of (1) 0.5 mg/day placebo, (2) 0.5 mg/day melatonin, or (3) 0.5 mg/day melatonin plus 30 min of bright light therapy (BLT) between 0700 and 0800 h. Sleep was assessed at baseline, directly after treatment, and 2 weeks after the end of treatment. Objective measures were obtained by actigraphy, self-reported measures by various sleep questionnaires and a sleep diary. Melatonin with or without BLT did not advance sleep times, improve sleep in general, or strengthen wake-activity rhythms. So even though the DLMO had advanced, sleep timing did not follow. Adding extensive behavioral coaching to chronotherapy is necessary for advancing sleep times along with DLMO and to further alleviate ADHD symptoms.

Drugs Used in Circadian Sleep-Wake Rhythm Disturbances.

This article focuses on melatonin and other melatonin receptor agonists and summarizes their circadian phase shifting and sleep-enhancing properties, along with their associated possible safety concerns. The circadian system and circadian rhythm sleep-wake disorders are described, along with the latest American Academy of Sleep Medicine recommendations for the use of exogenous melatonin in treating them. In addition, the practical aspects of using exogenous melatonin obtainable over the counter in the United States, consideration of the effects of concomitant light exposure, and assessing treatment response are discussed.

Self-Reported Sleepiness after 2, 4, and 7 Consecutive Night Shifts and Recovery Days in Danish Police Officers.

BACKGROUND: Night shift work often implies shorter sleep duration and this can lead to sleepiness, which has been associated with an increased risk of accidents and injuries. The aim is to study how the number of consecutive night shifts affects self-reported sleepiness. PARTICIPANTS AND METHODS: The study was a quasi-experimental, within-subject crossover study with 73 police officers. Three work schedules of two, four, and seven consecutive night shifts followed by the same number of recovery days, i.e., days worked or days off, was performed by all participants. Sleepiness was self-reported using the Karolinska sleepiness scale (KSS) every fourth hour on the last night shift and the last recovery day in each sequence. RESULTS: We observed differences in the level of sleepiness between recovery days and night shift days but no differences in the pattern of sleepiness levels on night shift days in the different work schedules. The highest levels of KSS were observed before bedtime (at 07:00 after a night shift and 23:00 on a recovery day). CONCLUSION: The number of consecutive night shifts did not affect the self-reported levels of self-reported sleepiness among Danish police officers.

Circadian rhythms and disorders of the timing of sleep.

The daily alternation between sleep and wakefulness is one of the most dominant features of our lives and is a manifestation of the intrinsic 24 h rhythmicity underlying almost every aspect of our physiology. Circadian rhythms are generated by networks of molecular oscillators in the brain and peripheral tissues that interact with environmental and behavioural cycles to promote the occurrence of sleep during the environmental night. This alignment is often disturbed, however, by contemporary changes to our living environments, work or social schedules, patterns of light exposure, and biological factors, with consequences not only for sleep timing but also for our physical and mental health. Characterised by undesirable or irregular timing of sleep and wakefulness, in this Series paper we critically examine the existing categories of circadian rhythm sleep-wake disorders and the role of the circadian system in their development. We emphasise how not all disruption to daily rhythms is driven solely by an underlying circadian disturbance, and take a broader, dimensional approach to explore how circadian rhythms and sleep homoeostasis interact with behavioural and environmental factors. Very few high-quality epidemiological and intervention studies exist, and wider recognition and treatment of sleep timing disorders are currently hindered by a scarcity of accessible and objective tools for quantifying sleep and circadian physiology and environmental variables. We therefore assess emerging wearable technology, transcriptomics, and mathematical modelling approaches that promise to accelerate the integration of our knowledge in sleep and circadian science into improved human health.

The Effects of the Exogenous Melatonin on Shift Work Sleep Disorder in Health Personnel: A Systematic Review.

(1) Background: To know the medical documentation related to exogenous melatonin in sleep disorders caused by shift work in health personnel; (2) Methods: Systematic and critical review. Data were obtained by looking up the bibliographic data base: MEDLINE (via Pubmed), Embase, Cochrane Library, Scopus, Web of Science, Latin American and Caribbean literature in Health Sciences (LILACS) and Medicine in Spanish (MEDES). The used terms, as descriptors and text in the title and abstract record fields, were "Health Personnel", "Melatonin" and "Sleep Disorders", Circadian Rhythm, by using the following filters: "Humans", "Adult: 19+ years" and "Clinical Trial". The search update was in December 2021. The documentary quality of the articles was assessed using the CONSORT questionnaire. (3) Results: Having applied the inclusion and exclusion criteria, 10 clinical essays were selected out of 98 retrieved references. CONSORT scores ranged from a minimum of 6.0 to a maximum of 13. 7 with a median of 10.2. According to the SIGN criteria, this review presented "1-"evidence with a grade of recommendation B. The intervention dose via administration of exogenous melatonin ranged between 1 and 10 mg. It was not mentioned whether the route of administration was by fast or slow absorption. The outcomes showed decreased daytime sleepiness, lessened sleep onset latency, diminished night-time awakenings, increased total sleep period and improved daytime attention in the melatonin-treated group; (4) Conclusions: Exogenously administered melatonin is effective in shift worker health personnel that are suffering from sleep disorders, and given its low adverse effects and tolerability, it might be recommended. A great disparity was evidenced in terms of dose, follow-up periods and type of melatonin, small participant population, same age ranges and young age. Therefore, new trials would be needed to amend these observations in order to have full evidence that is able to ensure the efficacy of exogenous melatonin in the studied population.

SHIFTPLAN: a randomized controlled trial investigating the effects of a multimodal shift-work intervention on drivers' fatigue, sleep, health, and performance parameters.

BACKGROUND: Shift work is commonly associated with health problems resulting from circadian misalignment and sleep restriction. About one in three shift workers is affected by insomnia and up to 90% report regular fatigue and/or sleepiness at the workplace. Epidemiological data shows that shift workers are at increased risk of cardiovascular disease, diabetes, obesity, breast cancer, mental-health problems, and shift-work disorder, which conditions typically lead to reduced work performance, processing errors, accidents at work, absenteeism, and reduced quality of life. Given these widespread and debilitating consequences, there is an urgent need for treatments that help improve the sleep, health, and functional performance of the shift-working population. The most common non-pharmacological recommendations are improved scheduling, bright-light exposure, napping, psychoeducation promoting sleep hygiene, and cognitive-behavioral techniques. The objectives of the present study are to investigate the effects of a multimodal shift-work intervention on perceived fatigue, sleepiness, physical and mental health, sleep parameters, and absenteeism. METHODS: A randomized controlled interventional study comparing the two groups each comprising at least 80 drivers of a public transport company, using self-report questionnaires and health checks completed at intake and after 3 and 6 months following the start of the intervention or waiting-list period. The intervention consists of (a) healthy scheduling taking into account shift-rotation direction and speed, chronotype, resting time, and napping; (b) an education program specifically developed for shift workers; and (c) a dedicated information campaign for shift planners. The primary outcome is symptomatic burden in terms of sleepiness, and the key secondary outcome is symptomatic burden in terms of fatigue. Supplementary secondary outcomes are sleep parameters, absenteeism, general and clinical health, changes in mood, and anxiety. DISCUSSION: Expected outcomes are significant improvements on all primary and secondary outcome parameters in the intervention group. To our knowledge, ours is the first randomized controlled study to systematically investigate the effects of a multimodal program on multiple health, sleep, and performance parameters in shift workers. Our research also aims at providing evidence-based practice guidelines for healthy scheduling in general and thus contribute to diminishing the serious health and economic burdens associated with shift work overall. TRIAL REGISTRATION: EDGE registration number: 000339. CLINICALTRIALS: gov NCT05452096.

Sleep Disturbances and Health Consequences Induced by the Specificity of Nurses' Work.

INTRODUCTION: Nursing staff working in a shift or night system are exposed to sleep disorders, which has a direct impact on the emergence of dangerous health consequences for them. Melatonin secretion is abnormal at night and the circadian rhythm is disturbed. The aim of the study was to assess the occurrence of sleep disorders and their consequences for the body in a group of representative nursing staff working in a shift and night system. PARTICIPANTS: The study was conducted among 126 nurses who are generally healthy, employed in health care facilities in the Malopolskie voivodship. METHODS: The Athens Insomnia Scale consisting of 8 test items was used to obtain research material: falling asleep, waking up at night, waking up in the morning, total sleep time, sleep quality, well-being the next day, mental and physical fitness the next day, and sleepiness during the next day. As well as an original questionnaire. RESULTS: The research showed significant negative consequences of shift work on the health of health-care workers. The subjects noticed symptoms related to the nervous system, such as increased nervous tension 53%, lack of patience in 62% of all respondents. As many as 85% pointed to the negative impact of shift work on their family life, 82% of all respondents on social life and 56% of all respondents on sex life. The other variables were not confirmed. CONCLUSIONS: Symptoms of insomnia are common among night-work nurses.

Ultra-low-dose early night ramelteon administration for the treatment of delayed sleep-wake phase disorder: case reports with a pharmacological review.

Delayed sleep-wake phase disorder (DSWPD) is a common circadian sleep-wake phase disorders brings serious social impairment of the patients. Melatonin is the main medication option; however, it has not been approved in some countries, and over-the-counter melatonin is under poor quality control. The melatonin receptor agonist ramelteon might be a potential treatment option, but there are few reports regarding its use in DSWPD patients. Existing pharmacological and chronobiological studies suggest that an ultra-low dose of ramelteon in the early night is beneficial for DSWPD. Here, we present our clinical experience together with a pharmacological review and discussion. Twenty-three DSWPD patients, of whom 18 patients had a treatment history of a normal dose of ramelteon, were prescribed low-dose ramelteon (median: 0.571 mg, 1/14 of a tablet) to be taken in the early night (mean: 18:10). After the treatment, the mean sleep schedule was significantly advanced, and clinical symptoms were improved. CITATION: Shimura A, Kanno T, Inoue T. Ultra-low-dose early night ramelteon administration for the treatment of delayed sleep-wake phase disorder: case reports with a pharmacological review. J Clin Sleep Med. 2022;18(12):2861-2865.

[Social jetlag: possibilities of micronutrient support].

The concept of social jetlag refers to asynchronous communication of a person's biological clock with tempo of modern living, which occurs mainly as a result of intensive work. At the core of social jetlag is sleep deprivation or chronic sleep restriction caused by social factors: pervasive use of electronic solutions and networks, intensive round the clock operation, chronic diseases. The aim of the work was to determine vitamins, minerals, and other micronutrients, the availability of which is important for supporting the organism in case of circadian rhythm sleep disorders and sleep restrictions, the so-called social jetlag. Material and methods. The analysis of 78 sources from PubMed and Google Scholar bibliographic bases was carried out with a detailed analysis of data from the published studies. Results. Circadian rhythm sleep disorders and sleep restrictions affects cognitive functions, increasing risk of anxiety and depressive disorders, enhances processes of chronic inflammation, oxidative stress, cardiometabolic disorders. Scientific evidence has been collected that lack of such elements as magnesium, folates, omega-3 polyunsaturated fatty acids and probiotics in diet can worsening effects of social jetlag and increase the risk of chronic diseases. Preventive course intake of this micronutrients is reasonable in people predisposed to social jetlag. Conclusion. In risk groups of people predisposed to social jetlag, along with diverse diet and adequate nutrition, sleep hygiene, it is necessary to provide targeted supplementation with magnesium, folates, omega-3 polyunsaturated fatty acids and probiotic products.

Melatonin supplementation for the treatment of infantile spasms: protocol for a randomised placebo-controlled triple-blind trial.

INTRODUCTION: Infantile spasms (IS) is a type of severe epileptic encephalopathy that occurs in infancy and early childhood. IS is characterised clinically by epileptic spasms, often accompanied by sleep disorder and abnormal circadian rhythm. The endogenous circadian rhythm disorder, in turn, can make spasms worse. Melatonin has also been found to have anticonvulsant and neuroprotective properties by adjusting the circadian rhythm. However, there are lack of relevant studies on controlling IS by using melatonin. This study aims to analyse the therapeutic effect of melatonin supplementation for the treatment of IS. METHODS AND ANALYSIS: This is a triple-blinded (trial participant, outcome assessor and the data analyst), prospective, randomised controlled trial to be conducted in the Department of Paediatrics, The First Medical Center of Chinese PLA General Hospital, Beijing, China from November 2020. Patients (n=70) aged 3 months to 2 years with IS will be recruited in this study after receiving written consent from their parents or guardians. Patients will be randomly divided into two equal groups and treated with a combination of adrenocorticotropic hormone, magnesium sulfate and either melatonin or placebo. Clinical data from the patients in the two groups before and after the treatment will be collected and compared. The primary outcome will be assessed 2 weeks later by seizure diaries and reported as the average reduced rate of spasms frequency. Secondary outcomes include the response rate (the rate of spasms-free), electroencephalogram hypsarrhythmia assessment and the psychomotor development assessment (Denver Developmental Screening Test). Sleep quality and safety will also be assessed. ETHICS AND DISSEMINATION: The protocol for this study was approved by the Ethics Committee of Chinese PLA General Hospital (reference number S2020-337-01) and was reported according to the Standard Protocol Items: Recommendations for Interventional Trials statement. Findings of this research will be disseminated through national and international meetings, conferences and peer-reviewed journals. TRIAL REGISTRATION NUMBER: ChiCTR2000036208.