Hemodialysis-induced chronodisruption and chronotype distribution in patients with chronic kidney disease.

Changes in circadian rhythms have been observed in patients with chronic kidney disease (CKD), and evidence suggests that these changes can have a negative impact on health. This study aimed to investigate the existence of hemodialysis-induced chronodisruption, the chronotype distribution, and their association with sleep quality and quality of life (QoL). This was a cross-sectional study that enrolled 165 patients (mean age: 51.1 ± 12.5 y, 60.6% male) undergoing hemodialysis from three local units. The following instruments were used: the Morning-Eveningness Questionnaire (MEQ); a modified version of the Munich Chronotype Questionnaire (MCQT) to estimate hemodialysis-induced chronodisruption (HIC); the Kidney Disease QoL Short Form (KDQOL-SF); the Epworth Sleepiness Scale (ESS); the Pittsburgh Sleep Quality Index (PSQI) and the 10-Cognitive Screener (10-CS). HIC was present in 40.6% of CKD patients. Morning chronotype was prevalent in CKD patients (69%) compared to evening-type (17.1%) and significantly different from a paired sample from the general population (p < 0.001). HIC and chronotype were associated with different domains of QoL but not with sleep quality. This study suggests that there is a HIC and that morning chronotype is associated with CKD patients undergoing hemodialysis, with implications for QoL.

Layered double hydroxides (LDHs) as efficient and safe carriers for miRNA inhibitors: In vitro and in vivo assessment of biocompatibility.

Layered double hydroxides (LDHs) have been employed as nano-sized carriers for therapeutic/bio-active molecules, including small interfering RNAs (siRNAs). However, the potential of LDHs nanoparticles for an efficient and safe antisense oligonucleotide (AMO) delivery still requires studies. In this research, we have tested the suitability of a Mg-Al-LDH-based nanocarrier loaded with a miRNA-196b-5p inhibitor. LDHs (and LDH-Oligo complex) were synthesized by the coprecipitation method followed by physicochemical characterization as hydrodynamic size, surface charge, crystallinity, and chemical groups. Thymic endothelial cell line (tEnd.1) were transfected with LDH-Oligo and were evaluated for i. cell viability by MTT, trypan blue, and propidium iodide assays; ii. transfection efficiency by flow cytometry, and iii. depletion of miRNA-196b-5p by RT-qPCR. In addition, Drosophila melanogaster larvae were fed LDHs and evaluated for: i. larval motility; ii. pupation rate; iii. larval-pupal transition; iv. lethality, and v. emergence rate. We demonstrated that LDHs nanoparticles are stable in aqueous solutions and exhibit a regular hexagonal shape. The LDH-AMO complex showed a transfection efficiency of 93.95 ± 2.15 % and induced a significant depletion of miRNA-196b-5p 48h after transfection. No cytotoxic effects were detected in tEnd.1 cells at concentrations up to 50 µg/ml, as well as in Drosophila exposed up to 500 µg of LDH. In conclusion, our data suggest that LDHs are biocompatible and efficient carriers for miRNA inhibitors and can be used as a viable and effective tool in functional miRNA inhibition assays.

Associations between chronotype, sleep quality, maternal mental health, and child development in mother-infant dyads.

OBJECTIVE/BACKGROUND: Studies on circadian rhythms throughout development and their physiological and behavioral impacts at early stages are still scarce. Previous studies have shown that mother-infant interactions are important for both sleep and child development. In this cross-sectional study we investigated whether infants' chronotype, sleep and development were associated with their respective mothers' chronotype, sleep, mental health and socioeconomic status. PATIENTS/METHODS: the following were used to evaluate mothers: the Morningness-Eveningness Questionnaire (MEQ), Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS) and Self-Reporting Questionnaire 20 (SRQ-20). To assess the infants' characteristics, the following were used: the 19th question from the Morningness-Eveningness Questionnaire (MEQ), infant nocturnal midpoint of sleep (iMSF), Brief Infant Sleep Questionnaire (BISQ) and Ages and Stages Questionnaire-3 (ASQ3). Socioeconomic aspects were assessed using the Brazilian Economic Class Criterion of the Brazilian Association of Research Companies (ABEP). RESULTS: A hundred and eight mother-infant dyads participated in the study. Sleep disorders were observed in 38 (35%) infants and atypical development (ASQ3) in 35 (32%). The infants' sleep phases were partially explained by the mother's chronotype. Infants' sleep duration was negatively correlated with sleep latency, which was higher in the group with atypical development. Mothers of infants with sleep disorders or discordant chronotypes (32%) had higher Pittsburgh scores (worse sleep quality) and higher SRQ-20 scores (screen for Common Mental Disorders). CONCLUSIONS: We found evidence for the contribution of sleep quality and chronotypes to mothers' mental health and infant development. However, further studies are needed to confirm the influence of sleep and circadian phenotypes in the early stages.

Depression and anxiety symptoms correlate with diurnal preference, sleep habits, and Per3 VNTR polymorphism (rs57875989) in a non-clinical sample.

BACKGROUND: Evidences suggest that alterations in circadian rhythms trigger the development of mental disorders. Eveningness, sleep behavior, and circadian genes polymorphisms have been associated with depression and anxiety symptomatology. However, the mechanism underlying these interactions is not well understood. We investigated the contribution of diurnal preference, sleep habits, and PER3 VNTR polymorphism (rs57875989) to depression and anxiety symptoms in a Northeast sample from the Brazilian population. METHODS: Eight hundred and four young adults completed the Morningness-Eveningness (MEQ), Munich Chronotype (MCTQ), Center for Epidemiologic Studies - Depression (CES-D), and Beck Anxiety Inventory (BAI) questionnaires. All participants were genotyped and linear regression was performed to test the interactions between the genetic /behavioral variants and depression/ anxiety symptoms. RESULTS: Eveningness and sleep behaviors (bedtime, wake-up time, sleep duration, and midpoint of sleep) were correlated with depression symptomatology, specifically in somatic factors of the CES-D questionnaire. No correlation was found between diurnal preference/sleep habits with anxiety symptoms for both BAI total score and its factors. However, women with PER34/4 genotype showed less interpesonal affect in depression symptomatology and more anxiety symptoms in four factors of the BAI questionnaire. LIMITATIONS: Mainly because this study was based on self-report questionnaires and was limited to undergraduate students aging 18 to 30 years old. CONCLUSION: These results reinforce a role for sleep and diurnal preference in depression, and PER3 VNTR polymorphism in anxiety symptomatology, particularly in women.

Environmental temperature as a mediator on the association between photoperiod at birth and chronotype.

The association between chronotypes and season of birth (SOB) remains an inconclusive issue due, in some extension, to the lack of investigations of mediation mechanisms. We evaluated the association of photoperiod at birth (PAB) with chronotypes and sleep duration in Brazil (n = 810), and the mediating effect of meteorological factors, sex, age and rs4753426 polymorphism in the melatonin receptor MTNR1B. Longer PAB was associated with a delayed mid-sleep phase with a suppressive effect of maximum environmental temperature. No significant interactions were identified for the other variables. These findings suggest that photoperiod and environmental temperature modulate chronotype development at early stages.

Chronobiology of limbic seizures: Potential mechanisms and prospects of chronotherapy for mesial temporal lobe epilepsy.

Mesial Temporal Lobe Epilepsy (mTLE) characterized by progressive development of complex partial seizures originating from the hippocampus is the most prevalent and refractory type of epilepsy. One of the remarkable features of mTLE is the rhythmic pattern of occurrence of spontaneous seizures, implying a dependence on the endogenous clock system for seizure threshold. Conversely, circadian rhythms are affected by epilepsy too. Comprehending how the circadian system and seizures interact with each other is essential for understanding the pathophysiology of epilepsy as well as for developing innovative therapies that are efficacious for better seizure control. In this review, we confer how the temporal dysregulation of the circadian clock in the hippocampus combined with multiple uncoupled oscillators could lead to periodic seizure occurrences and comorbidities. Unraveling these associations with additional research would help in developing chronotherapy for mTLE, based on the chronobiology of spontaneous seizures. Notably, differential dosing of antiepileptic drugs over the circadian period and/or strategies that resynchronize biological rhythms may substantially improve the management of seizures in mTLE patients.

Melatonin receptor 1B -1193T>C polymorphism is associated with diurnal preference and sleep habits.

BACKGROUND: Melatonin modulates the master circadian clock through the activation of G-protein-coupled receptors MT1 and MT2. It is presumed, therefore, that genetic variations in melatonin receptors can affect both sleep and circadian phase. We investigated whether the -1193T > C (rs4753426) polymorphism in the promoter of MT2 receptor gene (MTNR1B) is associated with diurnal preference and sleep habits. This polymorphism was previously associated with sunshine duration, suggesting a role in circadian entrainment. METHODS: A total of 814 subjects who completed the Morningness-Eveningness and the Munich Chronotype questionnaires were genotyped for the selected polymorphism. Linear and multinomial regression were performed to test the interaction between gene variants and diurnal preference/sleep habits. RESULTS: The -1193C variant was associated with the extreme morningness phenotype in a codominant model (C/C vs. T/T), recessive model (C/C + C/T vs. T/T) and alleles (C vs. T). A negative correlation was found between -1193C alleles and social jetlag scores. The frequency of -1193T allele was higher in the group that stay in bed more than 8 h/night compared to the group that stay in bed less than 8 h/night on weekends. CONCLUSION: To the best of our knowledge, these data provide the first insights into the role of MTNR1B gene in the regulation of sleep, biological rhythms, and entrainment in humans.

Green Tobacco Sickness among Brazilian farm workers and genetic polymorphisms.

OBJECTIVE: Green Tobacco Sickness (GTS) is an occupational illness caused by dermal absorption of nicotine from tobacco leaves. It affects thousands of farm workers worldwide. Brazil is the second tobacco producer in the world; despite this, there are few studies on GTS among Brazilian harvesters. This study aimed to determine the prevalence of GTS among a population of tobacco workers from a producing area in northeastern Brazil and investigate whether the occurrence of the disease was influenced by factors such age, gender and smoking status. In addition, it was investigated if there was association between the onset of GTS and genetic polymorphisms in genes that encode some detoxification enzymes. A semi-structured questionnaire was used to collect demographic, behavioral and occupational data from the referred workers. Polymorphisms were tested through the Polymerase Chain Reaction technique. RESULTS: The total prevalence of GTS found was 56.9%, with a significant difference between genders (71.7% for women and 35.3% for men, p < 0.0001). No association was identified between the investigated polymorphisms and GTS. This study confirms the occurrence of GTS among tobacco harvesters in Brazil with high prevalence. The investigation suggests the need to take preventive measures to protect tobacco workers against this disease.

Usual normalization strategies for gene expression studies impair the detection and analysis of circadian patterns.

Recent studies have shown that transcriptomes from different tissues present circadian oscillations. Therefore, the endogenous variation of total RNA should be considered as a potential bias in circadian studies of gene expression. However, normalization strategies generally include the equalization of total RNA concentration between samples prior to cDNA synthesis. Moreover, endogenous housekeeping genes (HKGs) frequently used for data normalization may exhibit circadian variation and distort experimental results if not detected or considered. In this study, we controlled experimental conditions from the amount of initial brain tissue samples through extraction steps, cDNA synthesis, and quantitative real time PCR (qPCR) to demonstrate a circadian oscillation of total RNA concentration. We also identified that the normalization of the RNA's yield affected the rhythmic profiles of different genes, including Per1-2 and Bmal1. Five widely used HKGs (Actb, Eif2a, Gapdh, Hprt1, and B2m) also presented rhythmic variations not detected by geNorm algorithm. In addition, the analysis of exogenous microRNAs (Cel-miR-54 and Cel-miR-39) spiked during RNA extraction suggests that the yield was affected by total RNA concentration, which may impact circadian studies of small RNAs. The results indicate that the approach of tissue normalization without total RNA equalization prior to cDNA synthesis can avoid bias from endogenous broad variations in transcript levels. Also, the circadian analysis of 2-Cycle threshold (Ct) data, without HKGs, may be an alternative for chronobiological studies under controlled experimental conditions.

Association of TNFA (-308G/A), IFNG (+874 A/T) and IL-10 (-819 C/T) polymorphisms with protection and susceptibility to dengue in Brazilian population.

OBJECTIVE: To evaluate gene polymorphisms and their association with susceptibility to dengue. METHODS: A retrospective case-control study was performed with 262 subjects, comprising 78 dengue fever (DF) patients, 49 dengue hemorrhagic fever (DHF) patients and 135 healthy controls. Genotypic and allelic profiles were identified using polymerase chain reaction based in real time and amplification-refractory mutation system. RESULTS: We observed a protective association of IL-10 (-819 C/T) C allele (P = 0.028, OR = 0.56, CI = 0.34-0.91) against DHF, while the C/T (P = 0.047, OR = 2.10, CI = 1.01-4.38) and T/T (P = 0.008, OR = 3.82, CI = 1.38-10.59) genotypes were associated with DHF and DF, respectively. The dominant model TNFA -308 GA + AA (P = 0.043, OR = 0.45, CI = 0.20-1.00) genotypes were found to have protective effect against dengue infection. A protective association among the IFNG (+874 A/T) A/T genotype against DF (P = 0.02, OR = 0.46, CI = 0.24-0.89) and DHF (P = 0.034, OR = 0.43, CI = 0.19-0.95) was observed. When the studied single-nucleotide polymorphism was analyzed in combination, the combination GTA (P = 0.022, OR = 2.95, CI = 1.18-7.41) was statistically significantly associated with susceptibility to DF and the combination GCT (P = 0.035, OR = 0.28, CI = 0.08-0.90) with protection against the development of DHF. CONCLUSIONS: This research identifies the association of the IFNG (+874 A/T), TNFA (-308G/A), IL-10 (-819 C/T) genotypes as a factor for protection, susceptibility and severity to dengue.

Do suicide attempts occur more frequently in the spring too? A systematic review and rhythmic analysis.

BACKGROUND: Seasonal variations in suicides have been reported worldwide, however, there may be a different seasonal pattern in suicide attempts. The aim of this study was to perform a systematic review on seasonality of suicide attempts considering potential interfering variables, and a statistical analysis for seasonality with the collected data. METHOD: Observational epidemiological studies about seasonality in suicide attempts were searched in PubMed, Web of Science, LILACS and Cochrane Library databases with terms attempted suicide, attempt and season. Monthly or seasonal data available were evaluated by rhythmic analysis softwares. RESULTS: Twenty-nine articles from 16 different countries were included in the final review. It was observed different patterns of seasonality, however, suicide attempts in spring and summer were the most frequent seasons reported. Eight studies indicated differences in sex and three in the method used for suicide attempts. Three articles did not find a seasonal pattern in suicide attempts. Cosinor analysis identified an overall pattern of seasonal variation with a suggested peak in spring, considering articles individually or grouped and independent of sex and method used. A restricted analysis with self-poisoning in hospital samples demonstrated the same profile. LIMITATIONS: Grouping diverse populations and potential analytical bias due to lack of information are the main limitations. CONCLUSIONS: The identification of a seasonal profile suggests the influence of an important environmental modulator that can reverberate to suicide prevention strategies. Further studies controlling interfering variables and investigating the biological substrate for this phenomenon would be helpful to confirm our conclusion.

Genetic Polymorphisms Might Predict Suicide Attempts in Mental Disorder Patients: A Systematic Review And Meta-Analysis.

The aim of the present study was to analyze if the genetic polymorphisms might predict suicide attempts in mental disorder patients. The literature review and meta-analysis were conducted using the PubMed/Medline, Web of science and Scopus database using the terms: "5-HTT or SLC6A4 or 5-SERT and suicide, suicidal ideation or suicidal behavior or suicidal attempt". Thirty articles were analyzed. We found 17 articles that showed association and 13 articles that showed no association between LPR serotonin transporter polymorphism and suicide. A higher study of suicide identified the serotonin transporter polymorphism in patients with schizophrenia, mental disorder, major depression and bipolar disorder. There is an association between the serotonin-transporter-linked polymorphic region and suicidal behavior. The mental disorders with greater relationship with the suicide were the bipolar disorder, major depression and schizophrenia. The L allele had higher risk for suicide.

Identification of endogenous reference genes for the analysis of microRNA expression in the hippocampus of the pilocarpine-induced model of mesial temporal lobe epilepsy.

Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.

Validation of suitable reference genes for expression studies in different pilocarpine-induced models of mesial temporal lobe epilepsy.

It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

Predicted microRNAs for mammalian circadian rhythms.

There is little evidence for the involvement of microRNAs (miRs) in the regulation of circadian rhythms, despite the potential relevance of these elements in the posttranscriptional regulation of the clock machinery. The present work aimed to identify miRs targeting circadian genes through a predictive analysis of conserved miRs in mammals. Besides 23 miRs previously associated with circadian rhythms, we found a number of interesting candidate genes, equally predicted by the 3 software programs used, including miR-9, miR-24, miR25, miR-26, miR-27, miR-29, miR-93, miR-211, miR-302, and miR-346. Moreover, several miRs are predicted to be regulated by circadian transcription factors, such as CLOCK/BMAL, DEC2, and REV-ERBalpha. Using real-time PCR we demonstrated that the selected candidate miR-27b showed a daily variation in human leukocytes. This study presents predicted feedback loops for mammalian molecular clock and the first description of an miR with in vivo daily variation in humans.

Lack of association between rs211037 of the GABRG2 gene and juvenile myoclonic epilepsy in Brazilian population.

BACKGROUND: Juvenile myoclonic epilepsy (JME) is an idiopathic generalized epilepsy syndrome with genetic basis and accounts for 10% of all forms of epilepsy. Despite the existence of rare mutations responsible for some familial forms inherited in a Mendelian pattern, the genetics of JME is complex and probably involves multiple genes. Because of widespread distribution in the central nervous system (CNS) and their ability to produce postsynaptic inhibition, GABA (A) receptor subunits (GABRs) encoding genes represent high ranking candidates for epilepsy susceptibility. AIM: This case/control study was designed to investigate whether the rs211037 of the GABRG2 gene is a risk factor for JME in the Brazilian population. MATERIALS AND METHODS: The polymorphism was genotyped in 98 patients and 130 controls using polymerase chain reaction-restriction fragment length polymorphism method. Descriptive and statistical analyses were performed using SNP stat software. RESULTS: Genotype proportions and allele frequencies for the rs211037 polymorphism of the GABARG2 gene did not differ significantly between the groups, even when the odds ratio was adjusted for clinical variables. CONCLUSION: These results present no evidence for an association of rs211037 with JME. Further studies are required to investigate the involvement of the GABRG2 gene in the genetic susceptibility to this epileptic syndrome.

Identification of novel candidate genes for globin regulation in erythroid cells containing large deletions of the human beta-globin gene cluster.

The genetic mechanisms underlying the continued expression of the gamma-globin genes during the adult stage in deletional hereditary persistence of fetal hemoglobin (HPFH) and deltabeta-thalassemias are not completely understood. Herein, we investigated the possible involvement of transcription factors, using the suppression subtractive hybridization (SSH) method as an initial screen to identify differentially expressed transcripts in reticulocytes from a normal and a HPFH-2 subject. Some of the detectable transcripts may participate in globin gene regulation. Quantitative real-time PCR (qRT-PCR) experiments confirmed the downregulation of ZHX2, a transcriptional repressor, in two HPFH-2 subjects and in a carrier of the Sicilian deltabeta-thalassemia trait. The chromatin remodeling factors ARID1B and TSPYL1 had a very similar pattern of expression with an incremental increase in HPFH and decreased expression in deltabeta-thalassemia. These differences suggest a mechanism to explain the heterocellular and pancellular distribution of fetal hemoglobin in deltabeta-thalassemia and deletional HPFH, respectively. Interestingly, alpha-globin mRNA levels were decreased, similar to beta-globin in all reticulocyte samples analyzed.

Simple fluorescent PCR method for detection of large deletions in the beta-globin gene cluster.

We developed a semi-automated approach to detect large deletions in the beta-globin gene cluster, based on the quantitative differences in the amplifications of samples by a fluorescent PCR. With this strategy, we were able to detect the presence of HPFH-2 in an African-Brazilian subject, confirmed by sequencing analysis. Differently from other PCR strategies, GAP-PCR for example, it has the potential to identify new deletions.