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.

Neuroprotective Effect of Exogenous Galectin-1 in Status Epilepticus.

Intrahippocampal pilocarpine microinjection (H-PILO) induces status epilepticus (SE) that can lead to spontaneous recurrent seizures (SRS) and neurodegeneration in rodents. Studies using animal models have indicated that lectins mediate a variety of biological activities with neuronal benefits, especially galectin-1 (GAL-1), which has been identified as an effective neuroprotective compound. GAL-1 is associated with the regulation of cell adhesion, proliferation, programmed cell death, and immune responses, as well as attenuating neuroinflammation. Here, we administrated GAL-1 to Wistar rats and evaluated the severity of the SE, neurodegenerative and inflammatory patterns in the hippocampal formation. Administration of GAL-1 caused a reduction in the number of class 2 and 4 seizures, indicating a decrease in seizure severity. Furthermore, we observed a reduction in inflammation and neurodegeneration 24 h and 15 days after SE. Overall, these results suggest that GAL-1 has a neuroprotective effect in the early stage of epileptogenesis and provides new insights into the roles of exogenous lectins in temporal lobe epilepsy (TLE).

Multi-omic Analysis of the Gut Microbiome in Rats with Lithium-Pilocarpine-Induced Temporal Lobe Epilepsy.

Evidence supports that the gut microbiota and bacteria-dependent metabolites influence the maintenance of epileptic brain activity. However, the alterations in the gut microbiota between epileptic versus healthy individuals are poorly understood. We used a multi-omic approach to evaluate the changes in the composition of gut metagenome as well in the fecal metabolomic profile in rats before and after being submitted to status epilepticus (SE)-induced temporal lobe epilepsy (TLE). The 16S ribosomal RNA (rRNA) sequencing of fecal samples coupled to bioinformatic analysis revealed taxonomic, compositional, and functional shifts in epileptic rats. The species richness (Chao1 index) was significantly lower in the post-TLE group, and the ß-diversity analysis revealed clustering separated from the pre-TLE group. The taxonomic abundance analysis showed a significant increase of phylum Desulfobacterota and a decrease of Patescibacteria in the post-TLE group. The DESEq2 and LEfSe analysis resulted in 18 genera significantly enriched between post-TLE and pre-TLE groups at the genus level. We observed that epileptic rats present a peculiar metabolic phenotype, including a lower concentration of D-glucose and L-lactic acid and a higher concentration of L-glutamic acid and glycine. The microbiota-host metabolic correlation analysis showed that the genera differentially abundant in post-TLE rats are associated with the altered metabolites, especially the proinflammatory Desulfovibrio and Marvinbryantia, which were enriched in epileptic animals and positively correlated with these excitatory neurotransmitters and carbohydrate metabolites. Therefore, our data revealed a correlation between dysbacteriosis in epileptic animals and fecal metabolites that are known to be relevant for maintaining epileptic brain activity by enhancing chronic inflammation, an excitatory-inhibitory imbalance, and/or a metabolic disturbance. These data are promising and suggest that targeting the gut microbiota could provide a novel avenue for preventing and treating acquired epilepsy. However, the causal relationship between these microbial/metabolite components and the SRS occurrence still needs further exploration.

Maternal crack cocaine use in rats leads to depressive- and anxiety-like behavior, memory impairment, and increased seizure susceptibility in the offspring.

Crack users suffer the effects of cocaine present in the drug and the action of other active compounds from its pyrolysis. An emergent fact is an increase in the number of pregnant crack cocaine users. Studies suggest that crack cocaine and its metabolites cross the placenta, promoting premature birth, fever, irritability, sweating, and seizures in the early months of life. In children, the effects of crack cocaine have been associated with cognitive deficits, difficulty in verbalization, aggressiveness, and depression, besides enhancing the susceptibility to epileptic seizures, including status epilepticus (SE) in adulthood. Therefore, we investigated the effect of maternal exposure to smoke crack cocaine on several behavioral parameters in the offspring during adulthood. A series of behavioral tests and intrahippocampal pilocarpine (H-PILO) microinjection at sub-convulsive and convulsive doses in a rat model demonstrated that exposure to crack cocaine during the embryonic period leads to anxiogenic-like behavior and long-term memory impairment in both genders and promotes depressive-like behavior in the female. Besides, crack cocaine offspring exposed to a sub-convulsive H-PILO dose showed higher susceptibility to SE, increased seizure frequency, and neurodegeneration, while animals that received a convulsive dose of H-PILO displayed no alteration in SE severity. Taken together, our data suggest that crack cocaine exposure during the gestational period leads to an increased predilection for anxiety and depression, long-term memory deficits, and reduction in the threshold for developing epileptic seizures associated with neuronal death, which predispose crack cocaine babies to develop neuropsychological disorders.

Modulation of Glucose Availability and Effects of Hypo- and Hyperglycemia on Status Epilepticus: What We Do Not Know Yet?

Status epilepticus (SE) can lead to serious neuronal damage and act as an initial trigger for epileptogenic processes that may lead to temporal lobe epilepsy (TLE). Besides promoting neurodegeneration, neuroinflammation, and abnormal neurogenesis, SE can generate an extensive hypometabolism in several brain areas and, consequently, reduce intracellular energy supply, such as adenosine triphosphate (ATP) molecules. Although some antiepileptic drugs show efficiency to terminate or reduce epileptic seizures, approximately 30% of TLE patients are refractory to regular antiepileptic drugs (AEDs). Modulation of glucose availability may provide a novel and robust alternative for treating seizures and neuronal damage that occurs during epileptogenesis; however, more detailed information remains unknown, especially under hypo- and hyperglycemic conditions. Here, we review several pathways of glucose metabolism activated during and after SE, as well as the effects of hypo- and hyperglycemia in the generation of self-sustained limbic seizures. Furthermore, this study suggests the control of glucose availability as a potential therapeutic tool for SE.

Extracellular vesicles from human iPSC-derived neural stem cells: miRNA and protein signatures, and anti-inflammatory and neurogenic properties.

Grafting of neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise for brain repair after injury or disease, but safety issues have hindered their clinical application. Employing nano-sized extracellular vesicles (EVs) derived from hiPSC-NSCs appears to be a safer alternative because they likely have similar neuroreparative properties as NSCs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. However, reliable methods for isolation, characterization and testing the biological properties of EVs are critically needed for translation. We investigated signatures of miRNAs and proteins and the biological activity of EVs, isolated from hiPSC-NSCs through a combination of anion-exchange chromatography (AEC) and size-exclusion chromatography (SEC). AEC and SEC facilitated the isolation of EVs with intact ultrastructure and expressing CD9, CD63, CD81, ALIX and TSG 101. Small RNA sequencing, proteomic analysis, pathway analysis and validation of select miRNAs and proteins revealed that EVs were enriched with miRNAs and proteins involved in neuroprotective, anti-apoptotic, antioxidant, anti-inflammatory, blood-brain barrier repairing, neurogenic and Aß reducing activities. Besides, EVs comprised miRNAs and/or proteins capable of promoting synaptogenesis, synaptic plasticity and better cognitive function. Investigations using an in vitro macrophage assay and a mouse model of status epilepticus confirmed the anti-inflammatory activity of EVs. Furthermore, the intranasal administration of EVs resulted in the incorporation of EVs by neurons, microglia and astrocytes in virtually all adult rat and mouse brain regions, and enhancement of hippocampal neurogenesis. Thus, biologically active EVs containing miRNAs and proteins relevant to brain repair could be isolated from hiPSC-NSC cultures, making them a suitable biologic for treating neurodegenerative disorders.

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.

Extracellular Vesicles in the Forebrain Display Reduced miR-346 and miR-331-3p in a Rat Model of Chronic Temporal Lobe Epilepsy.

An initial precipitating injury in the brain, such as after status epilepticus (SE), evolves into chronic temporal lobe epilepsy (TLE). We investigated changes in the miRNA composition of extracellular vesicles (EVs) in the forebrain after the establishment of SE-induced chronic TLE. We induced SE in young Fischer 344 rats through graded intraperitoneal injections of kainic acid, which resulted in consistent spontaneous recurrent seizures at ~ 3 months post-SE. We isolated EVs from the entire forebrain of chronically epileptic rats and age-matched naïve control animals through an ultracentrifugation method and performed miRNA-sequencing studies to discern changes in the miRNA composition of forebrain-derived EVs in chronic epilepsy. EVs from both naïve and epileptic forebrains displayed spherical or cup-shaped morphology, a comparable size range, and CD63 expression but lacked the expression of a deep cellular marker GM130. However, miRNA-sequencing studies suggested downregulation of 3 miRNAs (miR-187-5p, miR-346, and miR-331-3p) and upregulation of 4 miRNAs (miR-490-5p, miR-376b-3p, miR-493-5p, and miR-124-5p) in EVs from epileptic forebrains with fold changes ranging from 1.5 to 2.4 (p < 0.0006; FDR < 0.05). By using geNorm and Normfinder software, we identified miR-487 and miR-221 as the best combination of reference genes for measurement of altered miRNAs found in the epileptic forebrain through qRT-PCR studies. The validation revealed that only miR-346 and miR-331-3p were significantly downregulated in EVs from the epileptic forebrain. The enrichment pathway analysis of these miRNAs showed an overrepresentation of signaling pathways that are linked to molecular mechanisms underlying chronic epilepsy, including GABA-ergic (miR-346 targets) and mTOR (miR-331-3p targets) systems. Thus, the packaging of two miRNAs into EVs in neural cells is considerably altered in chronic epilepsy. Functional studies on these two miRNAs may uncover their role in the pathophysiology and treatment of TLE.

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.

Maternal, fetal and neonatal consequences associated with the use of crack cocaine during the gestational period: a systematic review and meta-analysis.

OBJECTIVE: Crack cocaine consumption is one of the main public health challenges with a growing number of children intoxicated by crack cocaine during the gestational period. The primary goal is to evaluate the accumulating findings and to provide an updated perspective on this field of research. METHODS: Meta-analyses were performed using the random effects model, odds ratio (OR) for categorical variables and mean difference for continuous variables. Statistical heterogeneity was assessed using the I-squared statistic and risk of bias was assessed using the Newcastle-Ottawa Quality Assessment Scale. Ten studies met eligibility criteria and were used for data extraction. RESULTS: The crack cocaine use during pregnancy was associated with significantly higher odds of preterm delivery [odds ratio (OR), 2.22; 95% confidence interval (CI), 1.59-3.10], placental displacement (OR, 2.03; 95% CI 1.66-2.48), reduced head circumference (- 1.65 cm; 95% CI - 3.12 to - 0.19), small for gestational age (SGA) (OR, 4.00; 95% CI 1.74-9.18) and low birth weight (LBW) (OR, 2.80; 95% CI 2.39-3.27). CONCLUSION: This analysis provides clear evidence that crack cocaine contributes to adverse perinatal outcomes. The exposure of maternal or prenatal crack cocaine is pointedly linked to LBW, preterm delivery, placental displacement and smaller head circumference.

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process.

Neuropathological studies often use autopsy brain tissue as controls to evaluate changes in protein or RNA levels in several diseases. In mesial temporal lobe epilepsy (MTLE), several genes are up or down regulated throughout the epileptogenic and chronic stages of the disease. Given that postmortem changes in several gene transcripts could impact the detection of changes in case-control studies, we evaluated the effect of using autopsy specimens with different postmortem intervals (PMI) on differential gene expression of the Pilocarpine (PILO)induced Status Epilepticus (SE) of MTLE. For this, we selected six genes (Gfap, Ppia, Gad65, Gad67, Npy, and Tnf-α) whose expression patterns in the hippocampus of PILO-injected rats are well known. Initially, we compared hippocampal expression of naïve rats whose hippocampi were harvested immediately after death (0h-PMI) with those harvested at 6h postmortem interval (6h-PMI): Npy and Ppia transcripts increased and Tnf-α transcripts decreased in the 6h-PMI group (p<0.05). We then investigated if these PMI-related changes in gene expression have the potential to adulterate or mask RT-qPCR results obtained with PILO-injected rats euthanized at acute or chronic phases. In the acute group, Npy transcript was significantly higher when compared with 0h-PMI rats, whereas Ppia transcript was lower than 6h-PMI group. When we used epileptic rats (chronic group), the RT-qPCR results showed higher Tnf-α only when compared to 6h-PMI group. In conclusion, our study demonstrates that PMI influences gene transcription and can mask changes in gene transcription seen during epileptogenesis in the PILO-SE model. Thus, to avoid erroneous conclusions, we strongly recommend that researchers account for changes in postmortem gene expression in their experimental design.

Genetic susceptibility in Juvenile Myoclonic Epilepsy: Systematic review of genetic association studies.

BACKGROUND: Several genetic association investigations have been performed over the last three decades to identify variants underlying Juvenile Myoclonic Epilepsy (JME). Here, we evaluate the accumulating findings and provide an updated perspective of these studies. METHODOLOGY: A systematic literature search was conducted using the PubMed, Embase, Scopus, Lilacs, epiGAD, Google Scholar and Sigle up to February 12, 2016. The quality of the included studies was assessed by a score and classified as low and high quality. Beyond outcome measures, information was extracted on the setting for each study, characteristics of population samples and polymorphisms. RESULTS: Fifty studies met eligibility criteria and were used for data extraction. With a single exception, all studies used a candidate gene approach, providing data on 229 polymorphisms in or near 55 different genes. Of variants investigating in independent data sets, only rs2029461 SNP in GRM4, rs3743123 in CX36 and rs3918149 in BRD2 showed a significant association with JME in at least two different background populations. The lack of consistent associations might be due to variations in experimental design and/or limitations of the approach. CONCLUSIONS: Thus, despite intense research evidence established, specific genetic variants in JME susceptibility remain inconclusive. We discussed several issues that may compromise the quality of the results, including methodological bias, endophenotype and potential involvement of epigenetic factors. PROSPERO REGISTRATION NUMBER: CRD42016036063.

Diurnal Variation Has Effect on Differential Gene Expression Analysis in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy.

The molecular mechanisms underlying epileptogenesis have been widely investigated by differential gene expression approach, especially RT-qPCR methodology. However, controversial findings highlight the occurrence of unpredictable sources of variance in the experimental designs. Here, we investigated if diurnal rhythms of transcript's levels may impact on differential gene expression analysis in hippocampus of rats with experimental epilepsy. For this, we have selected six core clock genes (Per1, Per3, Bmal1, Clock, Cry1 and Cry2), whose rhythmic expression pattern in hippocampus had been previously reported. Initially, we identified Tubb2a/Rplp1 and Tubb2a/Ppia as suitable normalizers for circadian studies in hippocampus of rats maintained to 12:12 hour light:dark (LD) cycle. Next, we confirmed the temporal profiling of Per1, Per3, Bmal1, Cry1 and Cry2 mRNA levels in the hippocampus of naive rats by both Acrophase and CircWave statistical tests for circadian analysis. Finally, we showed that temporal differences of sampling can change experimental results for Per1, Per3, Bmal1, Cry1 and Cry2, but not for Clock, which was consistently decreased in rats with epilepsy in all comparison to the naive group. In conclusion, our study demonstrates it is mandatory to consider diurnal oscillations, in order to avoid erroneous conclusions in gene expression analysis in hippocampus of rats with epilepsy. Investigators, therefore, should be aware that genes with circadian expression could be out of phase in different animals of experimental and control groups. Moreover, our results indicate that a sub-expression of Clock may be involved in epileptogenicity, although the functional significance of this remains to be investigated.

Lack of association between the prothrombin rs1799963 polymorphism and juvenile myoclonic epilepsy.

UNLABELLED: Juvenile myoclonic epilepsy (JME) accounts for 26% of generalized idiopathic epileptic syndromes. The highest levels of thrombin activity are closely involved in the development of neurological diseases, including epilepsy. The prothrombin c.20210G>A (rs1799963) variation, which alters prothrombin mRNA stability, is associated with high plasma prothrombin levels. OBJECTIVE: The present study was designed to investigate whether the SNP rs1799963 is a risk factor for JME in the northeastern Brazilian population. RESULTS: The polymorphism was genotyped in 207 controls and 123 patients using polymerase chain reaction-restriction fragment length polymorphism method. No significant differences were observed in the genotype and allele frequencies of this polymorphism between cases and controls. CONCLUSION: These results present no evidence for an association of rs1799963 with JME. Further studies including other types of epilepsy are required to investigate the involvement of prothrombin gene in the genetic susceptibility to chronic seizure.

Lack of association between the prothrombin rs1799963 polymorphism and juvenile myoclonic epilepsy / Ausência de associação entre o polimorfismo G20210A (rs1799963) da protrombina e epilepsia mioclônica juvenil

Juvenile myoclonic epilepsy (JME) accounts for 26% of generalized idiopathic epileptic syndromes. The highest levels of thrombin activity are closely involved in the development of neurological diseases, including epilepsy. The prothrombin c.20210G>A (rs1799963) variation, which alters prothrombin mRNA stability, is associated with high plasma prothrombin levels. Objective : The present study was designed to investigate whether the SNP rs1799963 is a risk factor for JME in the northeastern Brazilian population. Results : The polymorphism was genotyped in 207 controls and 123 patients using polymerase chain reaction-restriction fragment length polymorphism method. No significant differences were observed in the genotype and allele frequencies of this polymorphism between cases and controls. Conclusion : These results present no evidence for an association of rs1799963 with JME. Further studies including other types of epilepsy are required to investigate the involvement of prothrombin gene in the genetic susceptibility to chronic seizure. .

Epilepsia mioclônica juvenil (EMJ) representa 26% das síndromes epilépticas idiopáticas generalizadas. Níveis elevados de atividade da trombina estão intimamente envolvidos no desenvolvimento de distúrbios neurológicos, incluindo epilepsia. A variante c.20210G>A (rs1799963) do gene de protrombina, que altera a estabilidade do RNAm, está associada com altos níveis de protrombina no plasma. Objetivo: Investigar se o SNP rs1799963 é um fator de risco para EMJ em uma amostra da população do nordeste brasileiro. Resultados : O polimorfismo foi genotipado em 123 pacientes e 207 controles usando a reação de polimerase em cadeia com restrição de polimorfismo. Não observamos diferença significativa nas frequências alélicas e genotípicas deste polimorfismo, entre as populações de pacientes e controle. Conclusão : Estes resultados não demonstram evidências para uma associação do polimorfismo rs1799963 com EMJ. Estudos posteriores, incluindo outros tipos de epilepsia, são necessários para investigar o envolvimento do gene protrombina na susceptibilidade genética a crises crônicas. .

Daily variations in the expression of miR-16 and miR-181a in human leukocytes.

Circadian rhythms are controlled by a molecular mechanism that is organized in transcriptional and translational feedback loops of gene expression. Recent studies have been demonstrating the involvement of microRNAs (miRs) in post-transcriptional/translational control of circadian rhythms. In the present study we aimed to analyze the daily variations of miR-16 and miR-181a expression in human leukocytes. These miRs were independently associated with hematopoiesis and circadian rhythms in previous studies using experimental models. Peripheral blood from 6 subjects was sampled in a 24 hour period for expression analysis using quantitative real-time PCR (RT-qPCR). Initially, we evaluated the expression stability of RNU6-2, RNU1A-1, RNU5A-1, SNORD-25, SCARNA-17 and SNORA-73A as candidate genes for normalization of RT-qPCR data. The combination of the four most stable genes (SNORA-73A/SCARNA-17/SNORD-25/RNU6-2) was indicated to provide a better normalization of miRs expressions. The results show a daily variation of miR-181a and miR-16 expression in human leukocytes, suggesting a potential participation of these genes in the modulation of the circadian rhythms present in blood cells.

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.