A Complex Interplay Between Melatonin and RORß: RORß is Unlikely a Putative Receptor for Melatonin as Revealed by Biophysical Assays.

A nuclear retinoic acid receptor (RAR)-related orphan receptor ß (RORß) is strictly expressed in the brain, particularly in the pineal gland where melatonin is primarily synthesized and concentrated. The controversial issues regarding the direct interaction of melatonin toward ROR receptors have prompted us to investigate the potential melatonin binding sites on different ROR isoforms. We adopted computational and biophysical approaches to investigate the potential of melatonin as the ligand for RORs, in particular RORß. Herein, possible melatonin binding sites were predicted by molecular docking on human RORs. The results showed that melatonin might be able to bind within the ligand-binding domain (LBD) of all RORs, despite their difference in sequence homology. The predicted melatonin binding scores were comparable to binding energies with respect to those of melatonin interaction to the well-characterized membrane receptors, MT1 and MT2. Although the computational analyses suggested the binding potential of melatonin to the LBD of RORß, biophysical validation failed to confirm the binding. Melatonin was unable to alter the stability of human RORß as shown by the unaltered melting temperatures upon melatonin administration in differential scanning fluorometry (DSF). A thermodynamic isothermal titration calorimetry (ITC) profile showed that melatonin did not interact with human RORß in solutions, even in the presence of SRC-1 co-activator peptide. Although the direct interaction between the LBD of RORß could not be established, RORα and RORß gene expressions were increased upon 24 h treatment with µM-range melatonin. Our data, thus, support the studies that the nuclear effects of melatonin may not be directly mediated via its interaction with the RORß. These findings warrant further investigation on how melatonin interacts with ROR signaling and urge the melatonin research community for a paradigm shift in the direct interaction of melatonin toward RORs. The quest to identify nuclear receptors for melatonin in neuronal cells remains valid for the community to achieve.

Proteomic profiling reveals neuronal ion channel dysregulation and cellular responses to DNA damage-induced cell cycle arrest and senescence in human neuroblastoma SH-SY5Y cells exposed to cypermethrin.

Cypermethrin (CYP), a synthetic pyrethroid of class II, is widely used as a pesticide worldwide. The primary target of cypermethrin is a voltage-gated sodium channel. The neurotoxicity of CYP has been extensively studied in terms of affecting neuronal development, increasing cellular oxidative stress, and apoptosis. However, little is known about how it affects the expression of channel proteins involved in synaptic transmission, as well as the effects of cypermethrin on DNA damage and cell cycle processes. We found that the ligand and voltage-gated calcium channels and proteins involved in synaptic transmission including NMDA 1 receptor subunit, alpha 1A-voltage-dependent calcium channel, synaptotagmin-17, and synaptojanin-2 were downregulated in CYP-treated cells. After 48 h of CYP exposure, cell viability was reduced with flattened and enlarged morphology. The levels of 23 proteins regulating cell cycle processes were altered in CYP-treated cells, according to a proteomic study. The cell cycle analysis showed elevated G0/G1 cell cycle arrest and DNA fragmentation at the sub-G0 stage after CYP exposure. CYP treatment also increased senescence-associated ß-galactosidase positive cells, DNA damage, and apoptotic markers. Taken together, the current study showed that cypermethrin exposure caused DNA damage and hastened cellular senescence and apoptosis via disrupting cell cycle regulation. In addition, despite its primary target sodium channel, CYP might cause synaptic dysfunction via the downregulation of synaptic proteins and dysregulation of synapse-associated ion channels.

Maternal stress induced autophagy dysfunction and immune activation in the hippocampus of adolescence rat pups.

Maternal stress (MS) has long-term effects on fetal brain development and consequently increases the risk of neuropsychiatric diseases in the offspring, however, the mechanism that links between early life stress and subsequent neuropsychiatric diseases is still not clear. It is well known that both neuroinflammation and autophagy dysfunction contributes to the pathology of psychiatric disorders. We hypothesized that MS might alter autophagy function and activate the neuroimmune response in the pup's brain. To test this hypothesis, we investigated the effects of MS on the expression of the autophagy biomarker and neuroimmune response in the hippocampus of rat pups. Results revealed that MS-induced a long-term decrease of LC3B-II throughout the postnatal periods, together with an increase of IL-6 and IL-10 in the hippocampus of rat pups during adolescence. These changes lasted at least until adulthood. Results from the In vitro studies showed that a partially toxic dose of corticosterone (CORT) induced a significant decrease of LC3B-II, together with an increase of IL-6 and IL-10, in the SH-SY5Y cells. Moreover, suppression of autophagy by mycophenolic acid (MPA) leads to an increased IL-6 and IL-10 expression in the CORT-treated SH-SY5Y cells. Findings suggested that CORT decreased autophagy dysfunction could activate neuroimmune response in the SH-SY5Y cells. Results from this study provides initial evidence for the relationship between stress hormone, autophagy dysfunction, and neuroimmune activation, which may be the linking mechanism between early-life stress and subsequent neuropsychiatric disorders.

The potential of Mangifera indica Linn. and Musa acuminata extracts to attenuate 4-hydroxyestradiol (4-OHE2 )-induced DNA oxidative damage in MCF-10A cells by upregulating detoxifying and antioxidant enzymes.

Nowadays, there have been attempts to use phytochemicals in fruits to reduce the risk of suffering a given sickness. In this work, we studied the potential effects of mango (cultivar "Nam Dok Mai") and banana (cultivar "Khai") to attenuate DNA oxidative damage in MCF-10A cells induced by 4-hydroxyestradiol (4-OHE2 ). The effects of mango extract (MNE) and banana extract (BKE) were comparable with three carotenoid compounds, ß-carotene, lycopene, and lutein. The oxidative-induced DNA damage was evaluated by 8-hydroxy-2-deoxyguanosine (8-OHdG) reduction. 4-OHE2 -induced DNA oxidative damage in MCF-10A cells showed a decrease in 8-OHdG formation when treated with MNE and BKE. Both fruit extracts also enabled the regaining production of Phase II detoxifying (GSTs and NQO1) and antioxidant (SOD, GPx, and CAT) enzymes during 4-OHE2 -induced DNA oxidative damage in the MCF-10A cells when compared with the untreated control. These results indicate that MNE and BKE can exert potential mitigating effects against 4-OHE2 -induced DNA oxidative damage in MCF-10A cells by enhancing the activities of detoxifying and antioxidant enzyme. PRACTICAL APPLICATIONS: Long-term exposure to estrogen increases the risk of sickness since oxidative stress via the estrogen pathway, leading to DNA damage. This study indicated that mango (cultivar "Nam Dok Mai") extract contains ß-carotene and lycopene, while banana (cultivar "Khai") extract contains ß-carotene and lutein, which act as natural antioxidants. Both fruit extracts have preventive properties against oxidative DNA damage and are potentially good supplements for women taking E2 between HRT.

Generation of human induced pluripotent stem cell line (MUi026-A) from a patient with autosomal dominant polycystic kidney disease carrying PKD1 point mutation.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the common genetic kidney disorders that are caused by mutations in PKD1 or PKD2 gene. In this report, the MUi026-A human induced pluripotent stem cell (hiPSC) line was established from the skin fibroblasts of a female ADPKD patient who had the PKD1 mutation with c.5878C > T. The iPSC line retained normal karyotype. The cells displayed embryonic stem cell-like characteristics with pluripotency marker expression and were able to differentiate into three germ layers.

Proteome profiling of bovine follicular fluid-specific proteins and their effect on in vitro embryo development.

The objective of this study was to determine the effect of bovine follicular fluid proteins (bFF) and their differently charged groups as maturation media supplements for in vitro embryo development. bFF was obtained by aspiration from large healthy follicles (4-10 mm in diameter) and was precipitated by 30-50% (NH4)2SO4. The precipitated protein was fractionated into basic and acidic fractions by ion-exchanger columns. In experiment 1, the oocytes were matured in TCM-199 with 1) FBS+hormones (control) and 2) 10% bFF. The oocyte maturation rate, the development to the blastocyst stage rate and blastocyst cell number were not significantly different between the groups. However, the INFα and IGF-2r expression levels in the 10% bFF were higher than in the control (P<0.05). In experiment 2, the specific charge proteins of bFF (basic and acidic) were also used as media supplements in the maturation medium. The basic fraction had higher oocyte maturation rate and blastocyst cell number when compared with addition of acidic fraction (P<0.05). The expression levels for almost all developmentally important genes in the basic fraction were greater than those in the acidic fraction, particularly INFα (P<0.05). Most of the protein in the basic fraction was associated with the immune response and mRNA processing. In conclusion, supplementation of 10% bFF alone in maturation medium can support oocyte maturation and embryo development. The basic fraction in bFF seemed to have effect on oocyte maturation rate and blastocyst cell number.

The scorpion venom peptide BmKn2 induces apoptosis in cancerous but not in normal human oral cells.

AIM: This study aimed to investigate the mechanism of the induction of apoptosis of human oral cancer cells by the scorpion venom peptide BmKn2. METHODS: Human oral squamous carcinoma cells (HSC4), mouth epidermoid carcinoma cells (KB), human normal gingival cells (HGC) and dental pulp cells (DPC) were treated with BmKn-2 peptide for 24h. Cell viability was determined by the MTT assay. Apoptosis was assessed using phase contrast microscopy, by propidium iodide (PI) staining to assess nuclear morphology and by Annexin V staining. Apoptotic signaling pathways were investigated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blotting. RESULTS: BmKn-2 showed potent cytotoxic effects towards both HSC4 and KB cells with the associated induction of apoptosis. The cells showed distinct morphological changes, nuclear disintegration and an increase in the number of Annexin V-positive cells. Interestingly, at concentrations which kill cancerous cells, BmKn-2 did not affect cell viability or mediate the induction of apoptosis in normal HGC or DPC. Induction of apoptosis by BmKn-2 in HSC4 and KB cells was associated with the activation of tumor suppress p53. Pro-apoptotic BAX expression was increased, whereas antiapoptotic BCL-2 expression was decreased in BmKn-2 exposed HSC4 and KB cells. BmKn-2 treated-oral cancer cells showed distinct upregulation of initiator caspase-9, with no effect on caspase-8 expression. Increased expression levels of executor caspases-3 and -7 were also found in treated cells for both oral cancers. CONCLUSION: This study has suggested for the first time that BmKn-2 exerts selective cytotoxic effects on human oral cancer cells by inducting apoptosis via a p53-dependent intrinsic apoptotic pathway. BmKn-2 peptide originally derived from a natural source shows great promise as a candidate treatment for oral cancer, with minimal effects on healthy tissue.