Lower Melatonin Concentrations Research Articles

Plasma melatonin concentration during the early post-partum period in Thoroughbred mares and their foals.

The authors aimed to determine the plasma melatonin concentration in mares and their new-born foals in the early post-partum period. Blood samples were collected from the jugular vein of 53 mare-foal pairs within twelve hours after parturition. Plasma melatonin levels were measured by ELISA. The melatonin concentration, adjusted for the moment of parturition using a generalised linear model, was34.58 pg mL-1 in mares. It was significantly lower (27.63 pg mL-1) in the new-born foals. However, the melatonin concentration declined differently by the end of the twelve hours, it decreased less in theoffspring than in the mothers. An artificial light supplementation at the end of gestation reduced the melatonin concentration both in mares and their foals by about 10 pg mL-1, compared to the controls. An elevated melatonin production may be related to preparation of mares for parturition and ensures the chances of survival of offspring, therefore the melatonin may reach its peak at the moment of foalingregardless of its actual time. The effect of low melatonin concentration in new-born foals might be associated with the foal's health and subsequent performance. The need to monitor the melatonin concentration in the offspring justifies further studies.

Selective extraction and preconcentration of melatonin mediated by hydrophobic natural deep eutectic systems

BackgroundSample preparation is the key to achieve the goals of Green Chemistry and represents the highest source of the total negative impact of analytical methodologies on the environment. Therefore, assessing the greenness of analytical procedures and, if possible, reducing their impact on the environment and workers is necessary. In this sense, the alternative analytical techniques must be cost-effective, eco-friendly, and sustainable, to focus these issues and ensure efficient extraction, preconcentration, and analysis of the targeted compounds. ResultsThe selective extraction and preconcentration of melatonin (MEL) mediated by hydrophobic natural deep eutectic systems (HPB-NADES) from three native plant growth-promoting rhizobacteria (PGPR) cultures was studied and performed. The HPB-NADES based on menthol:thymol showed excellent selectivity for MEL, evaluated through the 3-indole acetic acid and tryptophan recovery and the matrix effect values. The extraction recovery of MEL was 97.9 to 100.3%, with an RSD of less than 3% and an interesting enrichment factor of 14.7–15.2. Finally, the LOD and LOQ for MEL were 23.4 and 71.4 ng/L, with a linear range of 71.4–5000 ng/L showing good linearity (F less than 1.16). The sustainability of the methodology showed scores of 0.71 and 0.66, and a chemical risk corresponding to the use of 0.812 g of chloroform per extracted and analyzed sample. ConclusionsThe present study introduced, for the first time, a green analytical method for selectively extracting and quantifying MEL from three PGPR cultures using HPB-NADES. The methodology exhibited favorable linearity, LOD, LOQ, and extraction recoveries with negligible matrix effects. The use of environmentally friendly solvents and sample preparation techniques ensures the sustainability of the approach. Additionally, the present work highlighted the potential of PGPR in improving plant adaptation to abiotic stress under low MEL concentrations.

Development of an impedimetric sensor for susceptible detection of melatonin at picomolar concentrations in diverse pharmaceutical and human specimens

Our investigation aimed to create and manufacture an electrochemical impedance sensor with the purpose of improving the detection efficiency of melatonin (ME). To achieve this objective, we employed gold nanoparticles coated on polydopamine formed in glassy carbon electrodes (AuNPs/PDA/GCE) as a means to enhance the sensor's capabilities. A novel approach employing the signal-off strategy and electrochemical impedance spectroscopy (EIS) technique was utilized to determine ME. When the AuNPs/PDA/GCE electrode was immersed in a buffered solution containing ME, and the oxidation current of AuNPs was recorded, it was observed that the oxidation current of AuNPs decreased upon the introduction of ME molecules. The decrease in electrical current can be ascribed to the inhibitory impact of ME molecule adsorption on the electrode surface with applying −0.2 V for 150 s in acetate buffer solution (ABS) (pH, 5) through various mechanisms, which hinders the electron transfer process crucial for AuNPs oxidation. Consequently, by utilizing EIS, various concentrations of ME were quantified spanning from 1 to 18 pM. Moreover, the ME sensor achieved an impressive detection limit of 0.32 pM, indicating its remarkable sensitivity in detecting low concentrations of ME. Importantly, these novel sensors demonstrated exceptional attributes in terms of sensitivity, specificity, stability, and repeatability. The outstanding performance of these sensors, coupled with their desirable attributes, establishes their considerable potential for a wide range of practical applications. These applications encompass various fields such as clinical diagnostics, pharmaceutical analysis, environmental monitoring, and industrial quality control, where accurate and sensitive detection of ME is of utmost importance.

Effects of Exogenous Antioxidant Melatonin on Physiological and Biochemical Characteristics of Populus cathayana × canadansis ‘Xin Lin 1’ under Salt and Alkaline Stress

Salt and alkaline stress is one of the important problems restricting agricultural production and afforestation projects. This kind of stress will greatly limit the growth and development of forest trees. Recently, melatonin has been gradually realized as a strong kind of antioxidant due to its important regulatory and protective roles in the process of plant growth and development. This study takes Xin Lin 1 (Populus cathayana × canadansis ‘Xin Lin 1’) as the research object, and measures the changes of physiological indexes at different time points to verify the alleviation effect of melatonin under salt and alkaline stress. In this experiment, plants have different behaviors in the face of different levels of exogenous melatonin. Among them, low concentrations of melatonin (50 μM and 100 μM) were more helpful to reduce the levels of MDA and osmotic regulators in leaves. At this level, the SOD and CAT content in the leaves increased significantly. Melatonin at 800 μM was more inclined to induce POD, but its activity was not significantly induced. Overall, melatonin contributes to the secretion of ABA in plants and has a tendency to inhibit the content of SA. It is worth mentioning that the 100 μM melatonin treatment was more conducive to the secretion of IAA. To sum up, this experiment proves that melatonin has a dose effect in alleviating stress.

Salivary Biomarkers Associated with Psychological Alterations in Patients with Diabetes: A Systematic Review.

The care of individuals with diabetes needs a holistic perspective, taking into account both the physical disease and the mental health problems that may be associated. Different studies show a higher prevalence of depression or anxiety issues in diabetes patients than in the general population, which is why diabetes can be considered one of the chronic diseases in which psychological care is crucial to maintain quality of life. The objective of this review is to examine the published articles that relate the bidirectional associations between objective and subjective measures of anxiety, depressive symptomatology, stress, sleep quality, and salivary biomarkers in patients with diabetes. For this, a search was carried out in the electronic databases PubMed, Cochrane, and SCOPUS using the keywords “diabetes”, “saliva”, “sleep”, “anxiety”, “depression”, and “stress” for works published up until May 2022 and limited to the English and Spanish languages. The sample comprised 14 articles, 5 of which analysed the associations between depressive symptomatology and salivary biomarkers in people with diabetes. Among the salivary biomarkers most frequently used to evaluate psychological alterations in persons with diabetes are cortisol and melatonin. Thus, significant changes in the levels of these biomarkers were observed in most studies. Four out of five studies reported a statistically significant relationship between increased salivary cortisol in the evening/midnight or the cortisol awakening response and depressive symptoms. In contrast, lower cortisol levels upon waking in the morning were observed when there was no depression or anxiety. Regarding the association between salivary cortisol values and sleep quality in patients with diabetes, lower morning cortisol values related to prolonged nighttime sleep were common in the analysed studies. Low melatonin concentrations showed a negative correlation with sleep quality. As it is an easy-to-apply and non-invasive method, the measurement of salivary biomarkers can be very useful for predicting psychological alterations in patients with diabetes. Further scientific studies are required to determine the sensitivity of these biological substances acting as biomarkers for detecting sleep disorders and psychological alterations.

Diurnal variation of clock genes expression and other sleep-wake rhythm biomarkers among acute ischemic stroke patients

There is accumulating evidence about sleep-wake rhythm disturbances as potential modifiable risk factors of both incident and recurrent stroke and less favorable outcomes after stroke. To our best knowledge this is the first study designed to investigate clock genes expression profiles in ischemic stroke patients and their relations to other biological and behavioral sleep-wake rhythm biomarkers, sleep structural and clinical stroke features. Altogether, 27 ischemic stroke patients (20 males) with the median age of 56 years and 25 gender and age matched controls were investigated with neurological and objective examination, scales, polysomnography, actigraphy and 24-h blood sampling for melatonin and clock genes profiles. Median melatonin plasma concentrations at four time points at 7, 11 p.m., 3 a.m. and 12 p.m. did not differ significantly between patients and controls, only early morning melatonin concentration at 7 a.m. was significantly lower and cortisol plasma concentration - significantly higher among stroke patients. All four clock genes (ARNTL (BMAL1), NR1D1 (Rev-erbα/β), PER1, and PER3) showed significant time-of-day variation in both patients' and controls’ groups, except expression of NR1D1 (Rev-erbα/β) at 7 a.m. and PER1 at 12 p.m. differed significantly. In conclusion, acute ischemic stroke patients tended to preserve most of diurnal variation of sleep-wake rhythm molecular patterns. Nevertheless, early morning time point showing higher cortisol and lower melatonin concentrations and lower NR1D1 (Rev-erbα/β) expression, as well as lower PER1 midday expression reflect specific circadian desynchrony features in different loops of the molecular circadian clock system.

0035 Enrichment of RAI1 genetic aberrations associated with sleep disturbances in SMS, in Autism Spectrum Disorder

Abstract Introduction Autism Spectrum Disorder (ASD) comprises a complex of neurodevelopmental disorders primarily characterized by deficits in verbal communication, impaired social interactions and repetitive behaviors. The profound clinical heterogeneity of ASD poses challenges in diagnosis and treatment. Genetic studies have pointed to hundreds of presumptive causative or susceptibility variants in ASD, making it difficult to find common underlying pathogenic mechanisms and suggesting that multiple different genetic etiologies for ASDs influence a continuum of traits. Smith–Magenis syndrome is a rare genetic disorder that results from an interstitial deletion of 17p11.2 and, in rare cases, from a retinoic acid induced 1 (RAI1) gene variant. The prevalence is estimated to be 1/15,000–25,000. Haploinsufficiency of RAI1 is the primary cause of the neurobehavioral and metabolic phenotype in SMS. Individuals with SMS present with a distinct pattern of mild to moderate intellectual disability, delayed speech and language skills, distinctive craniofacial and skeletal abnormalities, behavioral disturbances, and, almost uniformly, significant sleep disturbances. Alterations in RAI1 copy number has been also linked to a number of neurodevelopmental disorders including ASD. Methods We conducted a large-scale association analysis of the ASD MSSNG whole genome sequencing data to elucidate the prevalence of RAI1 SNVs and CNVs in the ASD population. We accessed the MSSNG database hosting over 11,000 genomes (6080 probands) and queried both SNVs and CNVs. Results Specifically, we focused on the prevalence of the classic deletions, microdeletions of (exon 3) and of the causative variants. We report a single case of a classic deletion (17p11.2 critical region), and an additional 3 cases of microdeletions in exon 3. Moreover, we report 2 frameshift mutations and one splicing variant. Given that the frequency of SMS is 1 in 15000 in the general population, we observe a 2.5x enrichment of the major deletion (1 in 6080 samples) and a >5x enrichment of the frameshift variants (2 in 6080 samples). In a set of 6080 probands we also observed 54 unique missense variants in 84 individuals within exon 3 of RAI1 gene. Conclusion Both ASD patients and SMS patients suffer from sleep disturbances. In this population of individuals with ASD, we report here an enrichment of variants known to cause SMS. We estimate the enrichment to be at least 2.5-fold and potentially higher than 10-fold enrichment, considering the types of variants observed in the population. Currently, the prevailing theory is that there is an underlying circadian pathophysiology causing sleep disturbances in SMS associated with RAI1 haploinsufficiency, as these patients exhibit low overall melatonin concentrations and abnormal timing of peak plasma melatonin concentrations. This abnormal inverted circadian rhythm is estimated to occur in 95% of individuals with SMS. The sleep disturbance seen in individuals with SMS may be also the underlying mechanism in at least a subset of individuals with ASD, especially in those individuals with consequential variants in the RAI1 gene Further studies will help delineate the role of RAI1 variants in sleep physiology. Support (If Any)

Women with polycystic ovary syndrome (PCOS) have reduced melatonin concentrations in their follicles and have mild sleep disturbances

BackgroundPolycystic ovary syndrome (PCOS) is a common gynecologic disorder related to abnormal circadian rhythm. Therefore, we aimed to find whether the level of melatonin, a rhythm regulating hormone changed in the ovarian microenvironment in this disease.MethodsThe melatonin concentrations in follicular fluid (FF) were measured in 35 PCOS and 36 non-PCOS women undergoing in vitro fertilization (IVF) treatment.ResultsThe FF melatonin concentration was significantly lower in PCOS women than non-PCOS women (p = 0.045) and it was found positively correlated with serum basal FSH level (r = 0.308, p = 0.013). In IVF procedures, there was no significant difference in the fertilization rate of oocytes between the two groups, but the high-quality embryogenesis rate on the third day of the PCOS group was significantly lower than that of the control group (p = 0.042), which showed a weak positive correlation with the FF melatonin concentration (rs = 0.240, p = 0.044). Furthermore, there was no significant difference in overall pregnancy outcome. The PSQI questionnaire showed that sleep disorders were more likely to exist in the PCOS group, though there was no significant difference.ConclusionThe obtained results suggested PCOS women had lower melatonin concentrations in the ovarian microenvironment.

Effects of melatonin supplementation on the quality of cryopreserved sperm in the neotropical fish Prochilodus lineatus

The cryopreservation process causes damage to sperm structures and supplementation of the cryoprotective medium is an alternative to reduce these damages. The aim of this study was to determine the effect of melatonin supplementation on post-thaw sperm quality in Prochilodus lineatus. The cryoprotective medium was supplemented with 2.00, 2.75, 3.50, and 4.25 mM melatonin, and the control group (without melatonin). Sperm motility parameters, membrane integrity, sperm morphology, oxidative stress (lipid peroxidation and enzymatic activity), and fertilization capacity were analyzed in the post-thaw sperm. Samples cryopreserved with 2.00 mM melatonin yielded higher sperm motility rate than other treatments with the addition of melatonin. Sperm curvilinear velocity (VCL) and average path velocity (VAP) were higher in samples containing 2.00 mM melatonin than in other treatments. Samples from control and with 2.00 mM melatonin presented higher membrane integrity and morphological normality than samples containing 4.25 and 2.75 mM melatonin, respectively. Regarding oxidative stress, lower lipid peroxidation (LPO) occurred in 2.75 and 3.50 mM melatonin, compared to control. While higher enzyme activity of catalase (CAT) occurred in the control than in other treatments, no differences were observed in the activity of superoxide dismutase (SOD). Higher fertilization and hatching rates occurred at 2.75 mM melatonin compared to 4.25 mM. Although no significant differences were observed in LPO between the control and samples supplemented with 2 mM melatonin, it was observed that this dosage of melatonin allowed higher VCL and VSL and reduced values of CAT. However, as there are no differences in motility and fertilization rates between the control and the 2 mM concentration, it is suggested that further studies be carried out with lower concentrations of melatonin in order to determine its effectiveness as an antioxidant in the sperm of this species.

Melatonin rescues glucocorticoid-induced inhibition of osteoblast differentiation in MC3T3-E1 cells via the PI3K/AKT and BMP/Smad signalling pathways

AimsHigh-dose glucocorticoid (GC) administration causes osteoporosis. Many previous studies from our group and other groups have shown that melatonin participates in the regulation of osteoblast proliferation and differentiation, especially low concentrations of melatonin, which enhance osteoblast osteogenesis. However, the role of melatonin in glucocorticoid-induced osteoblast differentiation remains unknown. Materials and methodsAn examination of the expression of osteoblast differentiation markers (ALP, OCN, COLL-1), as well as alkaline phosphatase staining and alkaline phosphatase enzymatic activity assay to measure osteoblast differentiation and quantifying Alizarin red S staining to measure mineralization, were performed to determine the effects of dexamethasone (Dex) and melatonin on the differentiation of MC3T3-E1 cells. We used immunofluorescence staining to detect the expression of Runx2 in melatonin-treated MC3T3-E1 cells. The expression of mRNA was determined by qRT-PCR, and protein levels were measured by western blotting. Key findingsIn the present study, we found that 100 μM Dex significantly reduced osteoblast differentiation and mineralization in MC3T3-E1 cells and that 1 μM melatonin attenuated these inhibitory effects. We found that only inhibition of PI3K/AKT (MK2206) and BMP/Smad (LDN193189) signalling abolished melatonin-induced differentiation and mineralization. Meanwhile, MK2206 decreased the expression of P-AKT and P-Smad1/5/9 and LDN193189 decreased the expression of P-Smad1/5/9 but had no obvious effect on P-AKT expression in melatonin-treated and Dex-induced MC3T3-E1 cells. SignificanceThese findings suggest that melatonin rescues Dex-induced inhibition of osteoblast differentiation in MC3T3-E1 cells via the PI3K/AKT and BMP/Smad signalling pathways and that PI3K/AKT signalling may be the upstream signal of BMP/Smad signalling.

МОРФО-ФУНКЦІОНАЛЬНИЙ СТАН ЕПІФІЗА ТА СУПРАХІАЗМАТИЧНОГО ЯДРА ГІПОТАЛАМУСА ЩУРІВ ЗА РІЗНИХ РЕЖИМІВ УВЕДЕННЯ ЕКЗОГЕННОГО МЕЛАТОНІНУ

In modern society increase of digitalization associated with grown exceed level of light at night – a new type of pollution. Presence of light at night inhibited endogenous melatonin synthesis by pineal gland, that influence on circadian system work cycles, so organism oftenbroken regime of wake/sleep, meals, physical activity. Also, a lack of melatonin in some certain time of dayand low melatonin concentration both, were shown take some intervention in diseases development through incorrect regulation of clock-depended genes expression. In connect with this, some latest clinical protocol in therapy or clinical trials of many different pathologies (for example, insomnia, metabolic syndrome, cardiovascular diseases, central nervous and immune system trouble, cancer, viral infection, etc.) include exogenous melatonin usage. As melatonin perform his function via endocrine and paracrine ways in variety types of cell, his application take place in wide range of doses and in different time of day (chronotherapeutic approach). Therefore, important to control state of circadian system central elements – pineal gland (main producer of endogenous melatonin) and suprachiasmatic nucleus (SCN) of hypothalamus (central pacemaker of circadian rhythm) in conditionsof exogenous melatonin treatment. Thus, the main goal of our research were analysis of rats pineal gland and hypothalamic SCN morpho-functional state after different time (morning, evening and continuously with drinking water) melatonin daily administration. Melatonin was administered by gavage for 7 weeks in dose 30 mg/kg 1 h before lights-off (M ZT11, evening), or 1 h after lights-on (M ZT01, morning), or continuously with drinking water during day-night period (MW). After melatonin use only in MW group pineal gland demonstrates changes in morphology (pinealocytes nucleus had mild basophilic color) and morphometric (increased cross-sectional area of the pinealocytes nucleus in compare with control group) analysis data. Besides, some similar changes were observed in SCN: the cross-sectional area of the SCN neurons nucleus grown in case of usage each of regime melatonin administration, while morphology characteristic remains without any alteration. In general, it suggesting about having by melatonin non-inhibiting features in context of circadian system feedback loop and supposing wide potential for melatonin use with absent huge side effect on central elements of above mentioned system.

Lower melatonin concentrations and sleep quality in patients with type 2 diabetes and obesity

Lower melatonin concentrations and sleep quality in patients with type 2 diabetes and obesity

Exogenous melatonin accelerates seed germination in cotton (Gossypium hirsutum L.).

Seed germination is considered the beginning of the spermatophyte lifecycle, and it is a crucial stage in determining subsequent plant growth and development. Although many previous studies have found that melatonin can promote seed germination, the role of melatonin in cotton germination remains unexamined. The main objective of this study is the characterization of potential promotional effects of melatonin (at doses of 0, 10, 20, 50, 100 and 200 μM) on cotton seed germination. This experiment demonstrated that low concentrations of melatonin can promote germination, while high concentrations failed to promote germination and even inhibited germination. Together, these results indicate that a 20 μM melatonin treatment optimally promotes cotton seed germination. Compared with the control, germination potential (GP), germination rate (GR), and final fresh weight (FW) increased by 16.67%, 12.30%, and 4.81%, respectively. Although low concentrations of melatonin showed some improvement in vigor index (VI), germination index (GI), and mean germination time (MGT), these effects were not statistically significant. Antioxidant enzyme activity during seed germination was most prominent under the 20 μM melatonin treatment. Superoxide dismutase (SOD) and peroxidase (POD) activities were significantly increased by 10.37–59.73% and 17.79–47.68%, respectively, compared to the melatonin-free control. Malondialdehyde (MDA) content was reduced by 16.73–40.33%. Two important plant hormones in seed germination, abscisic acid (ABA) and gibberellins (GAs), were also studied. As melatonin concentration increased, ABA content in seeds decreased first and then increased, and GA3 content showed a diametrically opposite trend, in which the 20 μM melatonin treatment was optimal. The 20 μM melatonin treatment reduced ABA content in seeds by 42.13–51.68%, while the 20 μM melatonin treatment increased GA3 content in seeds to about 1.7–2.5 times that of seeds germinated without melatonin. This study provides new evidence suggesting that low concentrations of melatonin can promote cotton seed germination by increasing the activity of antioxidant enzymes, thereby reducing the accumulation of MDA and regulating plant hormones. This has clear applications for improving the germination rate of cotton seeds using melatonin.

Dual-functional melatonin releasing device loaded with PLGA microparticles and cyclodextrin inclusion complex for osteosarcoma therapy

The aim of this study is to develop a scaffold-based device in which both osteoinductive and anticarcinogenic properties of melatonin can be combined for osteosarcoma therapy. While a high melatonin concentration and fast release are needed to achieve an anticancer effect, a low melatonin concentration and long-term release are necessary for bone regeneration. Long-term controlled release of melatonin was provided by the incorporation of melatonin-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles into the chitosan/hydroxyapatite (chitosan/HAp) scaffolds. This system showed a biphasic melatonin release-pattern with a burst release within 24 h, and then, a sustained release for 40 days that resulted into ∼40–70 μM concentration. Preosteoblastic MC3T3-E1 cells were cultured on this 3D device, and released melatonin caused increase in the expressions of osteogenic differentiation markers. For the inhibition of MG-63 human osteosarcoma cells, a melatonin/2-hydroxypropyl-β-cyclodextrin (HPβCD) inclusion complex was prepared by microwave technology to load a high amount of melatonin and provide fast release. Melatonin/HPβCD inclusion complex was incorporated into the chitosan/HAp scaffolds and loaded melatonin was rapidly released within 5 h and inhibited the proliferation of MG-63 cells in the G0/G1 phase. In conclusion, this melatonin-releasing scaffold-based device including melatonin-loaded PLGA microparticles and melatonin/HPβCD inclusion complex can be considered as a promising system for osteosarcoma therapy.

Melatonin reduces oxidative stress and promotes drought tolerance in young Coffea arabica L. plants

Water deficit severely compromises the homeostatic balance of plants and suggests a redox imbalance in cells. Melatonin is described as a promoter of stress tolerance in plants that induces the expression of stress-related genes, reduces lipid peroxidation and increases the antioxidant system. To analyze the influence of exogenous melatonin on the promotion of tolerance to water deficit, we applied concentrations of 300 μM and 500 μM to Coffea arabica L. seedlings. Here, we show that the intensity of the short-term responses to melatonin may vary according to the concentration of melatonin. We also report that lower concentrations of melatonin (300 μM) promote an increase in the root system and the protection of the photosynthetic apparatus, allowing greater gas exchange, greater carboxylation efficiency and higher chlorophyll contents. Moreover, melatonin improved the activity of the enzymatic and nonenzymatic antioxidant systems and reduced lipid peroxidation. In addition, we demonstrated that higher concentrations (500 μM) caused negative effects on stress tolerance, thus demonstrating a toxic level. Overall, these results demonstrated that melatonin mediates the signaling for water deficit responses by acting as an inductor of tolerance, most likely enhanced by increased carboxylation efficiency and antioxidant systems. This study provides evidence that exogenous melatonin protects coffee against water deficit.

172 Effect of Melatonin on Meiosis Progression and Lipid Content in Bovine Oocytes Matured In Vitro

Melatonin (MLT) may have positive effects on oocyte nuclear maturation and may influence their lipid metabolism. The aim of this study was to assess the effect of MLT on nuclear maturation and lipid content inbovine oocytes during in vitro maturation (IVM). Cumulus-oocyte complexes (30-35/group) were submitted to IVM in TCM-199 supplemented with 3 mg mL−1 BSA (negative control; NC) or with added hormones: 1 µg mL−1 FSH (positive control; PC) or MLT (10−11, 10−9 and 10−7 M), to evaluate the individual action of each treatment on meiosis resumption (metaphase I, MI) at 9 h and progression to metaphase II (MII) at 24 h IVM. Lipid content in oocytes was also assessed. Oocytes were denuded and stained with Hoechst 33342 and Nile Red, and evaluated by epifluorescence microscopy to determine the nuclear maturation stage and lipid content, respectively. The fluorescence intensity (FI) was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by ANOVA followed by Tukey’s test to compare effects of treatments on meiosis resumption and lipid contents at 9 h and on meiosis progression and lipid contents at 24 h; and t-test to compare the same treatment at 9 and 24 h IVM regarding lipid contents (4 replicates/treatment). Significance level was 5% (GraphPrism software; GraphPad Inc., San Diego, CA, USA). At 9 h IVM, meiosis resumption rate (MI) increased (P < 0.05) for MLT 10−9 M (32.6 ± 14.8%, 45/138) and FSH groups (33.9 ± 18.1%, 38/112) compared with NC (4.2 ± 2.7%, 5/120). The other treatments did not differ in relation to NC (20.5 ± 12.5%, 27/132 and 14.9 ± 9.0%, 20/134 to MLT 10−11 and 10−7 M, respectively). Regarding lipid contents at 9 h, MLT 10−11 M showed higher lipid content (8.43 ± 7.61FI; P < 0.0001) compared with other treatments [MLT 10−9 M (5.59 ± 4.13), MLT 10−7 M (4.87 ± 3.21), FSH (5.34 ± 3.66), and NC (4.27 ± 3.28)]. Maturation rate (MII) at 24 h IVM was highest in FSH group (70.1 ± 10.2%, 94/134). No differences were observed (P > 0.05) between groups matured in different concentrations of MLT (65.9 ± 4.2%, 83/126; 62.2 ± 0.6%, 56/90 and 53.5 ± 10.0%, 61/114 for MLT 10−11, 10−9 and 10−7 M, respectively) compared with NC (86.3 ± 1.2%, 88/102; P < 0.05). Similarly, lipid content at 24 h IVM was higher (P < 0.05) in FSH group (10.72 ± 4.71 FI) compared with NC (7.98 ± 3.45 FI) or MLT treatments with 10−11 M (8.90 ± 3.91), 10−9 M (7.85 ± 3.56), and 10−7 M (8.29 ± 3.87). However, MLT 10−7 M, FSH, and NC groups had higher (P < 0.0001) lipid content at 24 than at 9 h. In conclusion, MLT 10−9 M alone was able to stimulate meiosis resumption (9 h) at a rate similar to FSH, but was insufficient to stimulate progression to MII at 24 h, indicating that it may affect nuclear maturation only during the initial steps of meiosis. Lipid contents were increased during IVM but for lower MLT concentrations, this increase was not observed. Whether these observations have any impact on oocyte quality remains to be determined. Further studies are necessary to improve our knowledge of the role of MLT on lipid metabolism and by which mechanism it may affect meiosis resumption in bovine oocytes. This research was funded by FAPESP (HF- 2016/24884-3, scholarship; CLVL, financial support 2015/20379-0).

Clinical Uses of Melatonin in Neurological Diseases and Mental and Behavioural Disorders.

Melatonin is a molecule with numerous properties applicable to the treatment of neurological diseases. Among these properties are the following: potent scavenger of oxygen and nitrogen reactive species, anti-inflammatory features, immuno-enhancing nature, and modulation of circadian rhythmicity. Furthermore, low concentrations of melatonin are usually found in patients with neurological diseases and mental disorders. The positive results obtained in experimental models of diverse pathologies, including diseases of the nervous system (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, epilepsy, headaches, etc.) as well as mental and behavioural disordes (e.g., autism spectrum disorders, attention-deficit hyperactivity disorders, etc.), have served as a basis for the design of clinical trials to study melatonin's possible usefulness in human pathology, although the satisfactory results obtained from the laboratory "bench" are not always applicable to the patient's "bedside". In this article, we review those papers describing the results of the administration of melatonin to humans for various therapeutic purposes in the field of neuropathology. Clinical trials with strong methodologies and appropriate doses of melatonin are necessary to support or reject the usefulness of melatonin in neurological diseases.

Melatonin alleviates aluminium toxicity through modulating antioxidative enzymes and enhancing organic acid anion exudation in soybean.

Aluminium (Al) toxicity is a major chemical constraint limiting plant growth and production on acidic soils. Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous molecule that plays crucial roles in plant growth and stress tolerance. However, there is no knowledge regarding whether melatonin is involved in plant responses to Al stress. Here, we show that optimal concentrations of melatonin could effectively ameliorate Al-induced phytotoxicity in soybean (Glycine max L.). The concentration of melatonin in roots was significantly increased by the 50μM Al treatment. Such an increase in endogenous melatonin coincided with the upregulation of the gene encoding acetyltransferase NSI-like (nuclear shuttle protein-interacting) in soybean roots. Supplementation with low concentrations of melatonin (0.1 and 1μM) conferred Al resistance as evident in partial alleviation of root growth inhibition and decreased H2O2 production: in contrast, high concentrations of melatonin (100 and 200μM) had an opposite effect and even decreased root growth in Al-exposed seedlings. Mitigation of Al stress by the 1μM melatonin root treatment was associated with enhanced activities of the antioxidant enzymes and increased exudation of malate and citrate. In conclusion, melatonin might play a critical role in soybean resistance to Al toxicity.

Melatonin Increases Oligodendrocyte Differentiation in Cultured Neural Stem Cells.

Neural stem cell (NSC) culture is a remarkable tool to investigate the potential therapeutic benefits of drugs in neurological diseases. The purpose of this study was to determine the effect of melatonin on proliferation and differentiation of NSCs in vitro. NSCs were isolated and expanded from mouse embryonic E14 cortex, and the effect of various concentrations of melatonin (0.05, 0.1, 0.5, 1, 5 and 10μM) on NSC proliferation was assessed by MTT and neurosphere assay. Results showed that melatonin significantly increased NSC viability and NSC proliferation in a dose-dependent manner, in comparison to controls. Similarly, neurosphere formation frequency and cell counts increased significantly with increasing melatonin concentrations and reached its peak at 0.5μM, in comparison to controls. Moreover, NSCs treated with either low (0.05µM) or high concentrations (5µM) of melatonin showed that the mean percentage of glial fibrillary acidic protein (GFAP)positive cells were not significantly different in PDGF or melatonin at 5μM, in comparison to controls. However, low melatonin concentrations (0.05µM) showed a slight significant increase in comparison to controls and PDGF. On the other hand, both concentrations of melatonin treatment significantly increased the percentage of myelin basic protein (MBP)positive cells (oligodendrocytes), in comparison to controls and to PDGF. Our results demonstrated, for the first time, that melatonin increased oligodendrocyte differentiation from NSCs. These results suggest that melatonin might have a potential therapeutic effect for some neurological diseases that involve oligodendrocyte and myelin pathologies.

Melatonin suppression of aerobic glycolysis (Warburg effect), survival signalling and metastasis in human leiomyosarcoma.

Leiomyosarcoma (LMS) represents a highly malignant, rare soft tissue sarcoma with high rates of morbidity and mortality. Previously, we demonstrated that tissue-isolated human LMS xenografts perfused in situ are highly sensitive to the direct anticancer effects of physiological nocturnal blood levels of melatonin which inhibited tumour cell proliferative activity, linoleic acid (LA) uptake and metabolism to 13-hydroxyoctadecadienoic acid (13-HODE). Here, we show the effects of low pharmacological blood concentrations of melatonin following oral ingestion of a melatonin supplement by healthy adult human female subjects on tumour proliferative activity, aerobic glycolysis (Warburg effect) and LA metabolic signalling in tissue-isolated LMS xenografts perfused in situ with this blood. Melatonin markedly suppressed aerobic glycolysis and induced a complete inhibition of tumour LA uptake, 13-HODE release, as well as significant reductions in tumour cAMP levels, DNA content and [(3) H]-thymidine incorporation into DNA. Furthermore, melatonin completely suppressed the phospho-activation of ERK 1/2, AKT, GSK3β and NF-kB (p65). The addition of S20928, a nonselective melatonin antagonist, reversed these melatonin inhibitory effects. Moreover, in in vitro cell culture studies, physiological concentrations of melatonin repressed cell proliferation and cell invasion. These results demonstrate that nocturnal melatonin directly inhibited tumour growth and invasion of human LMS via suppression of the Warburg effect, LA uptake and other related signalling mechanisms. An understanding of these novel signalling pathway(s) and their association with aerobic glycolysis and LA metabolism in human LMS may lead to new circadian-based therapies for the prevention and treatment of LMS and potentially other mesenchymally derived solid tumours.