Multiple-photon bundle emission in the n-photon Jaynes-Cummings model.

We study the multiple-photon bundle emission in the n-photon Jaynes-Cummings model composed of a two-level system coupled to a single-mode optical field via the n-photon exciting process. Here, the two-level system is strongly driven by a near-resonant monochromatic field, and hence the system can work in the Mollow regime, in which a super-Rabi oscillation between the zero-photon state and the n-photon state can take place under proper resonant conditions. We calculate the photon number populations and the standard equal-time high-order correlation functions, and find that the multiple-photon bundle emission can occur in this system. The multiple-photon bundle emission is also confirmed by investigating the quantum trajectories of the state populations and both the standard and generalized time-delay second-order correlation functions for multiple-photon bundle. Our work paves the way towards the study of multiple-photon quantum coherent devices, with potential application in quantum information sciences and technologies.

NTN1 gene was risk to non-syndromic cleft lip only among Han Chinese population.

OBJECTIVE: Genome-wide association studies (GWAS) found NTN1, NOG and the region between CREBBP and ADCY9 were risks to non-syndromic cleft lip with or without cleft palate (NSCL/P). However, the association of single nucleotide polymorphisms (SNPs) in these genes with NSCL/P in Western China is unknown. SUBJECTS AND METHODS: We selected seven SNPs in NTN1, NOG and between CREBBP and ADCY9, and then performed transmission disequilibrium test (TDT), parent-of-origin effect and sliding window haplotype analysis to test the associations among 302 NSCL/P case-parent trios from Western Han Chinese. RESULTS: We found allele G at rs4791774 in NTN1 was significantly overtransmitted among non-syndromic cleft lip only (NSCLO) (p = 0.0067, OR = 1.79, 95% CI: 1.17-2.74); rs4791774 and rs9915089 tightly linked with each other among NSCL/P (D' = 0.87, r2  = 0.67) and haplotypes carrying the risk allele G at rs4791774 were always found to be overtransmitted from parents to cases. Motif analysis indicated that allele G at rs4791774 could greatly alter the affinity of Myc_disc7, so allele G at rs4791774 in NTN1 might modulate C-MYC transcription to participate in the aetiology of NSCLO. CONCLUSIONS: Our study suggested allele G at rs4791774 in NTN1 gene is risk of NSCLO, which could greatly increase the risk to have a baby with cleft.

Involvement of nerve growth factor in mouse hippocampal neuronal cell line (HT22) differentiation and underlying role of DNA methyltransferases.

DNA methylation is an epigenetic event involved in regulation of gene transcription during cell differentiation. DNA methyltransferases (DNMT) play a role in differentiation of neural stem cells into neurons. The aim of this study was to determine whether nerve growth factor (NGF) was involved in differentiation of mouse hippocampal neuronal cell line (HT22) as assessed by IncuCyte. Quantitative PCR and western blot were used to measure gene and protein expression of DNMT as well as the activity of DNMTs. Treatment with NGF was found to upregulate both gene and protein expressions as well as total activity of DNMTs in differentiating HT22 cells. Compared to undifferentiating cells, the percentage of differentiating cells at S phase increased significantly when incubated with NGF. In undifferentiated cells, NGF failed to induce gene and protein expressions and activity of DNMTs. Data demonstrate that differentiation of HT22 cells by exposure to NGF involve the activation of DNMTs pathway.

5-Aza-2'-deoxycytidine, a DNA methylation inhibitor, induces cytotoxicity, cell cycle dynamics and alters expression of DNA methyltransferase 1 and 3A in mouse hippocampus-derived neuronal HT22 cells.

Epigenetic processes such as DNA methylation are essential for processes of gene expression in normal mammalian development. DNA methyltransferases (DNMT) are responsible for initiating and maintaining DNA methylation. It is known that 5-Aza-CdR, an inhibitor of DNMT induces cytotoxicity by reducing DNMT activity in various tumor cell lines. However, disturbances in neuronal DNA methylation may also play a role in altered brain functions. Thus, it was of interest to determine whether alterations in DNA methylation might be associated with neuronal functions by using 5-Aza-CdR, on mouse hippocampus-derived neuronal HT22 cell line. In particular, the aim of this study was to investigate the effects of 5-Aza-CdR on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of DNMT in HT22 cells. HT22 cells were incubated with 5 or 20 µmol/L 5-Aza-CdR for 24 h. Data showed that 5-Aza-CdR at both concentrations significantly inhibited proliferation of HT22 cells and exacerbated cytoplasmic vacuolization. Flow cytometry analysis demonstrated that 5-Aza-CdR treatment at both concentrations decreased early apoptosis but enhanced late apoptosis. Cell cycle analysis illustrated that 5-Aza-CdR treatment induced S phase arrest. Further, incubation with 5-Aza-CdR produced a down-regulation in expression of mRNA and protein DNMT1 and 3A but no marked changes were noted in DNMT 3B and p21 expression. In addition, DNMT1 activity was significantly decreased at both 5-Aza-CdR concentrations. Evidence indicates that 5-Aza-CdR induced cytotoxicity was associated with altered mRNA and protein expression of DNMT 1 and 3A associated with reduced DNMT1 activity in HT22 cells which might affect brain functions.

Vanadate-induced antiproliferative and apoptotic response in esophageal squamous carcinoma cell line EC109.

Vanadate is a transition element that present in nature and was shown to be a nonspecific inhibitor of protein tyrosine phosphatases. It was reported that vanadium (Vd) compounds exhibit antitumor actions in several cancer cell lines. This study aimed to examine the antiproliferative and apoptotic actions of different concentrations of sodium vanadate (NaVd) (+5) in esophageal squamous carcinoma cell line EC109 by determining the protein expression levels of cyclin D1 and caspase-3 following incubation for various times from 15 min up to 4 h. In addition, cell proliferation of EC109 treated with different concentrations (NaVd) was also measured using the MTT assay at 4, 12, 24, and 48 h. The cell cycle of EC109 cells exposed to different concentrations of NaVd was detected using flow cytometry determination at 24 h. Data showed that NaVd greater than 100 µM significantly increased cyclin D1. In contrast, reduced caspase-3 protein expression levels occurred at 50 µM. Cellular proliferation was significantly decreased at 50uM. The cell cycle was arrested at S phase with 100 µM NaVd. Taken together, data indicate that NaVd produced concentration- and time-dependent antitumor actions in EC109 cell line.

Mutant kri1l causes abnormal retinal development via cell cycle arrest and apoptosis induction.

Damage to the ribosome or an imbalance in protein biosynthesis can lead to some human diseases, such as diabetic retinopathy (DR) and other eye diseases. Here, we reported that the kri1l gene was responsible for retinal development. The kri1l gene encodes an essential component of the rRNA small subunit processome. The retinal structure was disrupted in kri1l mutants, which resulted in small eyes. The boundaries of each layer of cells in the retina were blurred, and each layer of cells was narrowed and decreased. The photoreceptor cells and Müller glia cells almost disappeared in kri1l mutants. The lack of photoreceptor cells caused a fear of light response. The development of the retina started without abnormalities, and the abnormalities began two days after fertilization. In the kri1l mutant, retinal cell differentiation was defective, resulting in the disappearance of cone cells and Müller cells. The proliferation of retinal cells was increased, while apoptosis was also enhanced in kri1l mutants. γ-H2AX upregulation indicated the accumulation of DNA damage, which resulted in cell cycle arrest and apoptosis. The kri1l mutation reduced the expression of some opsin genes and key retinal genes, which are also essential for retinal development.

Hypoxic Preconditioning Modulates BDNF and Its Signaling through DNA Methylation to Promote Learning and Memory in Mice.

Hypoxic preconditioning (HPC) as an endogenous mechanism can resist hypoxia/ischemia injury and exhibit protective effects on neurological function including learning and memory. Although underlying molecular mechanisms remain unclear, HPC probably regulates the expression of protective molecules by modulating DNA methylation. Brain-derived neurotrophic factor (BDNF) activates its signaling upon binding to the tropomyosin-related kinase B (TrkB) receptor, which is involved in neuronal growth, differentiation, and synaptic plasticity. Therefore, this study focused on the mechanism by which HPC regulates BDNF and BDNF/TrkB signaling through DNA methylation to influence learning and memory. Initially, the HPC model was established by hypoxia stimulations on ICR mice. We found that HPC downregulated the expression of DNA methyltransferase (DNMT) 3A and DNMT3B. Then, the upregulation of BDNF expression in HPC mice was generated from a decrease in DNA methylation of the BDNF gene promoter detected by pyrophosphate sequencing. Subsequently, upregulation of BDNF activated BDNF/TrkB signaling and ultimately improved learning and spatial memory in HPC mice. Moreover, after mice were intracerebroventricularly injected with the DNMT inhibitor, the restraint of DNA methylation accompanied by an increase of BDNF and BDNF/TrkB signaling was also discovered. Finally, we observed that the inhibitor of BDNF/TrkB signaling prevented HPC from ameliorating learning and memory in mice. However, the DNMT inhibitor promoted spatial cognition in mice. Thus, we suggest that HPC may upregulate BDNF by inhibiting DNMTs and decreasing DNA methylation of the BDNF gene and then activate BDNF/TrkB signaling to improve learning and memory in mice. This may provide theoretical guidance for the clinical treatment of cognitive dysfunction caused by ischemia/hypoxia disease.

Effects of 5-Aza on neurogenesis contribute to learning and memory in the mouse hippocampus.

BACKGROUND: 5-Aza-2'-deoxycytidine (5-Aza-CdR) is a demethylating agent that has various biological effects related to DNA methylation. DNA methylation plays important roles in learning and memory. We have reported that 5-Aza-CdR improved the performance of mice in the water maze and step-down tests. Some behaviours have been well recognized to be mediated by neurogenesis in the hippocampus. The Notch signalling pathway plays a key role in adult hippocampal neurogenesis. In this study, we examined whether 5-Aza-CdR (DNA methyltransferase inhibitor) affects neurogenesis and Notch1 expression. METHODS: The learning and memory behaviour of mice was evaluated by a conditioned avoidance learning 24 h after 5-Aza-CdR treatment. The mRNA and protein expression levels of Notch1 and HES1 were measured by real-time PCR and Western blotting. The 5-bromo-2'-deoxyuridine (BrdU)-positive cells and the expression of Notch1 in the hippocampal DG were observed through laser confocal microscopy. To further clarify whether 5-Aza-CdR affects behaviour through neurogenesis, the expression level of Notch1, cell viability and cell cycle were analysed using the HT22 cell line. RESULTS: The behaviour in conditioned avoidance learning was improved, while neurogenesis and the Notch1 pathway were increased in the hippocampus of mice that were injected with 5-Aza-CdR. In vitro experiments showed that 5-Aza-CdR increased the expression of the Notch1 pathway and upregulated S-phase in the cell cycle and cell viability. CONCLUSIONS: Our results suggest that the effect of 5-Aza-CdR on behaviour may be related to an increase in neurogenesis with upregulation of the Notch1 pathway in the hippocampus.

Possible Involvement of DNA Methylation in TSC1 Gene Expression in Neuroprotection Induced by Hypoxic Preconditioning.

Background: It has been reported that ischemia and ischemic preconditioning (IPC) have different effects on the expression of tuberous sclerosis complex 1 (TSC1), which may contribute to the tolerance to ischemia/hypoxia with the increase of autophagy. The mechanisms of TSC1 differential expression are still unclear under ischemia/IPC conditions in hippocampal Cornu Ammon 1 (CA1) and Cornu Ammon 3 (CA3) area neuronal cells. While we have shown that 5-Aza-CdR, a DNA methyltransferase inhibitor, can upregulate TSC1 and increase hypoxic tolerance by autophagy in vivo and in vitro, in this study, we examined whether DNA methylation was involved in the differential expression of TSC1 in the CA1 and CA3 regions induced by hypoxic preconditioning (HPC). Methods: Level of rapamycin (mTOR) autophagy, a downstream molecular pathway of TSC1/TSC2 complex, was detected in HPC mouse hippocampal CA1 and CA3 areas as well as in the HPC model of mouse hippocampal HT22 cells. DNA methylation level of TSC1 promoter (-720 bp~ -360 bp) was determined in CA1 and CA3 areas by bisulfite-modified DNA sequencing (BMDS). At the same time, autophagy was detected in HT22 cells transfected with GFP-LC3 plasmid. The role of TSC1 in neuroprotection was measured by cell viability and apoptosis, and the role of TSC1 in metabolism was checked by ATP assay and ROS assay in HT22 cells that overexpressed/knocked down TSC1. Results: HPC upregulated the expression of TSC1, downregulated the level of P-mTOR (Ser2448) and P-p70S6K (Thr389), and enhanced the activity of autophagy in both in vivo and in vitro. The increased expression of TSC1 in HPC may depend on its DNA hypomethylation in the promoter region in vivo. HPC also could reduce energy consumption in HT22 cells. Overexpression and knockdown of TSC1 can affect cell viability, cell apoptosis, and metabolism in HT22 cells exposed to hypoxia. Conclusion: TSC1 expression induced by HPC may relate to the downregulation of its DNA methylation level with the increase of autophagy and the decrease of energy demand.

MicroRNA-155 deficiency attenuates inflammation and oxidative stress in experimental autoimmune prostatitis in a TLR4-dependent manner.

To explore the mechanism of microRNA-155 (miR-155) deficiency, protecting against experimental autoimmune prostatitis (EAP) in a toll-like receptor 4 (TLR4)-dependent manner. After wild-type (WT) and miR-155-/- mice were injected with complete Freund's adjuvant and prostate antigen to establish EAP model, half were randomly selected for injection with lipopolysaccharide (LPS, a TLR4 ligand). The following experiments were then performed: von Frey filaments, hematoxylin-eosin (HE) staining, real time quantitative polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). And the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the level of Malondialdehyde (MDA) were detected by corresponding kits.miR-155-/- mice with prostatitis exhibited the attenuated pelvic tactile allodynia/hyperalgesia and the suppressed TLR4/nuclear factor-kappa B (NF-κB) pathway as compared with the WT mice with prostatitis. In addition, LPS enhanced the upregulation of miR-155 and the activation of the TLR4/NF-κB pathway in the prostatic tissues of WT mice with EAP. Furthermore, prostatitis mice had aggravated inflammation scores accompanying the increased interleukin (IL)-1ß, tumor necrosis factor-α, IL-6, interferon-γ, IL-12, and MDA in prostatic tissues with the decreased IL-10, SOD and GSH-Px, and the unaltered IL-4. Compared with the mice from the WT + EAP group and the miR-155-/- + EAP + LPS group, mice from the miR-155-/- + EAP group had decreased inflammation and oxidative stress. miR-155 deficiency ameliorated pelvic tactile allodynia/hyperalgesia in EAP mice and improved inflammation and oxidative stress in prostatic tissues in a TLR4-dependent manner involving NF-κB activation, thereby exerting a therapeutic effect in chronic prostatitis treatment.

Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning.

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice, accompanied by the downregulation of DNA methyltransferases (DNMTs) in the brain. However, the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood. This study aimed to establish an in vitro model of hypoxic preconditioning, using a cultured mouse hippocampal neuronal cell line (HT22 cells), to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning. HT22 cells were divided into a control group, which received no exposure to hypoxia, a hypoxia group, which was exposed to hypoxia once, and a hypoxic preconditioning group, which was exposed to four cycles of hypoxia. To test the ability of hypoxic preadaptation to induce hypoxic tolerance, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group. The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry. Compared with the hypoxia group, the expression levels of caspase-3 and spectrin, which are markers of early apoptosis and S-phase arrest, respectively, noticeably reduced in the hypoxic preconditioning group. Finally, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning. The results showed that compared with the control group, hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B. In conclusion, hypoxic preconditioning may exert anti-hypoxic neuroprotective effects, maintaining HT22 cell viability and inhibiting cell apoptosis. These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

Nd2O3 Nanoparticles Induce Toxicity and Cardiac/Cerebrovascular Abnormality in Zebrafish Embryos via the Apoptosis Pathway.

INTRODUCTION: Rare-earth nanoparticles in the environment and human body pose a potential threat to human health. Although toxic effects of rare-earth nanoparticles have been extensively studied, the effects on the early development are not well understood. In this study, we attempted to explain the toxic effects of neodymium oxide (Nd2O3) nanoparticles on early development. METHODS: We added the Nd2O3 nanoparticles at different concentrations and recorded the mortality and malformation rate per 24 hrs under a microscope. The live embryos treated with Nd2O3 nanoparticles were imaged as movies and Z step lapses with a confocal microscope, and heart rates were counted for 30 s to measure the cardiac function. The live Tg (Flk1:EGFP) transgenic embryos exposed to Nd2O3 nanoparticles were observed under confocal microscope to measure the cerebrovascular development. Subsequently, we extracted the total protein for Western blot at 5 days post-fertilisation (dpf). Embryos were collected to undergo TUNEL staining for apoptosis detection. RESULTS: Nd2O3 nanoparticles disturbed embryo development at high concentrations (>200 µg/mL). The mortality and malformation rate gradually increased in a dose-dependent manner by morphological observation, while the Nd2O3 median lethal concentration (LD50) was 203.4 µg/mL at 120 hrs post-fertilisation (hpf). Furthermore, the Nd2O3-treated embryos showed severe arrhythmia and reduced heart rate. We also observed the markedly cerebrovascular disappearance at middle concentration (100 and 200 µg/mL). The downregulated autophagy flux in brain blood vessels and increased apoptosis level in neurons might affect vessels sprouting and contribute to the vanished cerebrovascular. CONCLUSION: The results suggested that the embryos exposed to Nd2O3 activated the apoptosis pathway and induced toxicity and abnormal cardiac/cerebrovascular development.

Exosomal MicroRNA-126 from RIPC Serum Is Involved in Hypoxia Tolerance in SH-SY5Y Cells by Downregulating DNMT3B.

Ischemic tolerance in the brain can be induced by transient limb ischemia, and this phenomenon is termed remote ischemic preconditioning (RIPC). It still remains elusive how this transfer of tolerance occurs. Exosomes can cross the blood-brain barrier, and some molecules may transfer neuroprotective signals from the periphery to the brain. Serum miRNA-126 is associated with ischemic stroke, and exosomal miRNA-126 has shown protective effects against acute myocardial infarction. Therefore, this study aims to explore whether exosomal miRNA-126 from RIPC serum can play a similar neuroprotective role. Exosomes were isolated from the venous serum of four healthy young male subjects, both before and after RIPC. Exosomal miRNA-126 was measured by real-time PCR. The miRNA-126 target sequence was predicted by bioinformatics software. SH-SY5Y neuronal cells were incubated with exosomes, and the cell cycle was analyzed by flow cytometry. The expression and activity of DNA methyltransferase (DNMT) 3B, a potential target gene of miRNA-126, were examined in SH-SY5Y cells. The cell viability of SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD) was also investigated. To confirm the association between miRNA-126 and DNMT3B, we overexpressed miRNA-126 in SH-SY5Y cells using lentiviral transfection. miRNA-126 expression was upregulated in RIPC exosomes, and bioinformatics prediction showed that miRNA-126 could bind with DNMT3B. DNMT levels and DNMT3B activity were downregulated in SH-SY5Y cells incubated with RIPC exosomes. After overexpression of miRNA-126 in SH-SY5Y cells, global methylation levels and DNMT3B gene expression were downregulated in these cells, consistent with the bioinformatics predictions. RIPC exosomes can affect the cell cycle and increase OGD tolerance in SH-SY5Y cells. RIPC seems to have neuroprotective effects by downregulating the expression of DNMTs in neural cells through the upregulation of serum exosomal miRNA-126.

5-Aza-2'-deoxycytidine increases hypoxia tolerance-dependent autophagy in mouse neuronal cells by initiating the TSC1/mTOR pathway.

BACKGROUND: Our previous study found that 5-Aza-2'-deoxycytidine (5-Aza-CdR) can repress the expression and activity of protein serine/threonine phosphatase-1γ (PP1γ) in mouse hippocampus. It is well known that PP1γ regulates cell metabolism, which is related to hypoxia/ischaemia tolerance. It has been reported that it can also induce autophagy in cancer cells. Autophagy is important for maintaining cellular homeostasis associated with metabolism. In this study, we examined whether 5-Aza-CdR increases hypoxia tolerance-dependent autophagy by initiating the TSC1/mTOR/autophagy signalling pathway in neuronal cells. METHODS: 5-Aza-CdR was either administered to mice via intracerebroventricular injection (i.c.v) or added to cultured hippocampal-derived neuronal cell line (HT22 cell) in the medium for cell culture. The hypoxia tolerance of mice was measured by hypoxia tolerance time and Perl's iron stain. The mRNA and protein expression levels of tuberous sclerosis complex 1 (TSC1), mammalian target of rapamycin (mTOR) and autophagy marker light chain 3 (LC3) were measured by real-time PCR and western blot. The p-mTOR and p-p70S6k proteins were used as markers for mTOR activity. In addition, the role of autophagy was determined by correlating its intensity with hypoxia tolerance in a time-dependent manner. At the same time, the involvement of the TSC1/mTOR pathway in autophagy was also examined through transfection with TSC1 (hamartin) plasmid. RESULTS: 5-Aza-CdR was revealed to increase hypoxia tolerance and induce autophagy, accompanied by an increase in mRNA and protein expression levels of TSC1, reduction in p-mTOR (Ser2448) and p-p70S6k (Thr389) protein levels, and an increase in the ratio of LC3-II/LC3-I in both mouse hippocampus and hippocampal-derived neuronal cell line (HT22). The fluorescence intensity of hamartin was enhanced in the hippocampus of mice exposed to 5-Aza-CdR. Moreover, HT22 cells that over-expressed TSC1 showed more autophagy. CONCLUSIONS: 5-Aza-CdR can increase hypoxia tolerance by inducing autophagy by initiating the TSC1/mTOR pathway.

Potassium bisperoxo (1,10-phenanthroline) oxovanadate suppresses proliferation of hippocampal neuronal cell lines by increasing DNA methyltransferases.

Bisperoxo (1,10-phenanthroline) oxovanadate (BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases (DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells (HT22) were treated with 0.3 or 3 µM BpV. Proliferation, morphology, and viability of HT22 cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT22 cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 µM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 µM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT22 cells; and 3 µM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.

Effects of 5-Aza on p-Y1472 NR2B related to learning and memory in the mouse hippocampus.

BACKGROUND: We have previously reported that 5-Aza-2-deoxycytidine (5-Aza-cdR) can repress protein serine/threonine phosphatase-1γ (PP1γ) expression and activity in the mouse hippocampus and affect the behaviour of mice in a water maze. It is well known that the phosphorylation of N-methyl-d-aspartate receptor 2B subunit (NR2B) plays a role in behaviour. In this study, we examined whether 5-Aza-cdR affects NR2B phosphorylation at tyrosine 1472 (p-Y1472 NR2B) and whether it affected the responses of the mice in a passive avoidance test. METHODS: 5-Aza-cdR (10 µM) was administered to mice via intracerebroventricular injection (i.c.v). The learning and memory behaviour of the mice were evaluated by measuring their response in a step-down type passive avoidance test 24 h after the injection. The mRNA level of NR2B was measured by real-time PCR. NR2B and p-Y1472 NR2B protein expression in the mouse hippocampus was detected by western blot and immunofluorescence. CDK5 activity was detected by the ADP-Glo™ + CDK5/p35 Kinase Enzyme System. To further clarify whether the 5-Aza-cdR effects on behaviour were dependent on cellular proliferation or not, the effect of 5-Aza-cdR on the expression level of NR2B, the phosphorylation level of p-Y1472 NR2B, cell viability and the cell cycle were analysed using the immortalized mouse hippocampal neuronal cells neural cell line HT22 treated with 10 µM 5-Aza-cdR compared with an untreated control group. RESULTS: After injection with 5-Aza-cdR, the behaviour of the mice in the step-down test was improved, while their phosphorylation level of p-Y1472 NR2B was increased and their CDK5 activity was decreased in the hippocampus. In vitro experiments showed 10 µM 5-Aza-cdR increased the p-Y1472 NR2B phosphorylation level with inhibition of cell viability and cell cycle arrest. CONCLUSIONS: Our results suggested that the effect of 5-Aza-cdR on behaviour may be related to the increase in phosphorylation of p-Y1472 NR2B in the hippocampus.

Family-based study of association between MAFB gene polymorphisms and NSCL/P among Western Han Chinese population.

BACKGROUND: Non-syndromic cleft lip with or without cleft palate (NSCL/P) are the most common human congenital birth defects with a complex etiology. MAFB has been reported as a candidate gene involved in the pathogenesis of NSCL/P from genome-wide association study (GWAS) findings, and no replication studies have been performed in Western Han Chinese. OBJECTIVES: The aim of this study was to investigate the associations of MAFB among NSCL/P trios in Western Han Chinese. MATERIAL AND METHODS: We selected 6 single nucleotide polymorphisms (SNPs) (rs6072081, rs6065259, rs17820943, rs13041247, rs11698025 and rs6102085) near MAFB based on previous GWAS findings and recruited 298 case-parents trios with NSCL/P from Western Han Chinese population, while genotypes were done by SNPscan technology. RESULTS: Strong evidence of an association was found at rs17820943 (p = 0.0023; odds ratio - ORtranmission = 0.7 and 95% confidence interval [CI]: 0.55-0.88) and rs13041247 (p = 0.0023; ORtranmission = 0.7 and 95% CI: 0.55-0.88) among NSCL/P; genotypic transmission-disequilibrium test (TDT) analysis further confirmed this. C/C homozygote at rs17820943 (z = 3.44 and p = 0.00058) and T/T homozygote at rs13041247 (z = 3.14 and p = 0.0017) was over-transmitted among NSCL/P, which indicated they could increase the risk of having an affected baby. Sliding window haplotype analysis showed that haplotypes consisting of C allele at rs17820943 and T allele at rs13041247 were still over-transmitted among NSCL/P (lowest p = 0.0021). CONCLUSIONS: This study further confirmed that the targeted SNPs at MAFB were associated with NSCL/P trios from Western Han Chinese population, which provides more scientific evidence for the future research and genetic counseling.

Rs12941170 at SOX9 gene associated with orofacial clefts in Chinese.

OBJECTIVE: Non-syndromic orofacial cleftings (NSOCs) are considered as complex trait, which results from genetic and/or environmental modifiers. Current findings could only explain small portion of the NSOCs. SOX9 gene plays an important role during craniofacial development in animal models and the Pierre Robin sequence (PRS). However, its role in non-syndromic clefts remains unknown. DESIGN: In this study, we selected eight SNPs in and around SOX9 gene to make maximum coverage, and genotyped them by using RFLP-PCR and ligase detection reaction (LDR) methods to test its associations among 151 NSOCs (53 NSCLP, 52 NSCLO and 46 NSCPO) from Western Han Chinese population. RESULTS: Allelic TDT results showed that G allele at rs12941170 of SOX9 was under-transmitted among NSOCs (p=0.00014, OR=0.55 and 95%CI: 0.40-0.75), which could indicate that the G allele is protective against NSOCs; parent-of-origin effect analysis showed that G allele at rs12941170 was maternally under-transmitted (p=0.002), while there was no statistically difference between the maternal and paternal transmission of it. To test if the adjacent SNPs travel together from parents to the affected individual, we carried out the sliding window haplotype analysis, it is interesting to find that the haplotypes carrying the G allele at rs12941170 also was under-transmitted for NSOCs, NSCL/P, NSCLP and NSCPO (lowest p=0.00033). CONCLUSIONS: This study suggested that G allele at rs12941170 was protective, which could decrease the risk for NSOCs from Western Han Chinese population, and it will provide new reference for future research and genetic counseling in NSOCs.

An association between overexpression of DNA methyltransferase 3B4 and clear cell renal cell carcinoma.

It is well known that abnormal DNA methylations occur frequently in kidney cancer. However, it remains unclear exactly which types of DNA methyltransferases (DNMT) contribute to the pathologies of kidney cancers. In order to determine the functions of DNA methyltransferase in kidney tumorigenesis on the molecular level, we examined the mRNA expression levels of DNMT1, DNMT3A, DNMT3B, and DNMT3B variants in renal cell carcinoma tissue. Both mRNA and protein levels of DNMT3B4, a splice variant of DNMT3B, were increased in renal cell carcinoma tissue compared with adjacent control tissues. Additionally, Alu elements and long interspersed nuclear elements (LINE-1) were hypomethylated in renal cell carcinoma tissue. Meanwhile, methylation of the promoter for RASSF1A, a tumor suppressor gene, was moderately increased in renal cell carcinoma tissue, while RASSF1A expression was decreased. Thus, our data suggest that the overexpression of DNMT3B4 may play an important role in human kidney tumorigenesis through chromosomal instability and methylation of RASSF1A.

The effect of hypoxia preconditioning on DNA methyltransferase and PP1γ in hippocampus of hypoxia preconditioned mice.

It is well known that hypoxia preconditioning can increase hypoxic tolerance by changing the expressions of some genes in the brain. DNA methylation is important for regulating gene expression and is catalyzed by DNA methyltransferase (DNMT), an enzyme that is abundant in the brain. However, the impact of hypoxia preconditioning on DNA methylation remains unknown. In the current study, mice were randomly divided into three groups: blank control group with no exposure to hypoxia (H0), the hypoxia control group exposed to hypoxia once (H1), and the hypoxia preconditioning group exposed to 4 runs of hypoxia (H4). The mRNA and protein levels of three kinds of DNMTs and the activity of total DMNT were measured. Protein phosphatase 1(PP1)γ, which critically regulates neuroprotective pathways in brain, was measured in mRNA and protein activity and promoter methylation. DNMT1 was unchanged in H1 and H4, while DNMT3A and DNMT3B were decreased in H4. The mRNA and protein levels of PP1γ were decreased in H4. However, there was no detectable change in the level of DNA methylation of the promoter of PP1γ (-321 bp to 95 bp). These findings suggest that DNA methylation may have a role in hypoxia neuroprotection, and the change of PP1γ, which did not depend on the change of its promoter (-321 bp to 95bp) DNA methylation, may be involved in neuroprotection.