Specific inhibition of TET1 in the spinal dorsal horn alleviates inflammatory pain in mice by regulating synaptic plasticity.

DNA demethylation mediated by ten-eleven translocation 1 (TET1) is a critical epigenetic mechanism in which gene expression is regulated via catalysis of 5-methylcytosine to 5-hydroxymethylcytosine. Previously, we demonstrated that TET1 is associated with the genesis of chronic inflammatory pain. However, how TET1 participates in enhanced nociceptive responses in chronic pain remains poorly understood. Here, we report that conditional knockout of Tet1 in dorsal horn neurons via intrathecal injection of rAAV-hSyn-Cre in Tet1fl/fl mice not only reversed the inflammation-induced upregulation of synapse-associated proteins (post-synaptic density protein 95 (PSD95) and synaptophysin (SYP)) in the dorsal horn but also ameliorated abnormalities in dendritic spine morphology and alleviated pain hypersensitivities. Pharmacological blockade of TET1 by intrathecal injection of a TET1-specific inhibitor-Bobcat 339-produced similar results, as did knockdown of Tet1 by intrathecal injection of siRNA. Thus, our data strongly suggest that increased TET1 expression during inflammatory pain upregulates the expression of multiple synapse-associated proteins and dysregulates synaptic morphology in dorsal horn neurons, suggesting that Tet1 may be a potential target for analgesic strategies.

Cholesterol Metabolic Markers for Differential Evaluation of Patients with Hyperlipidemia and Familial Hypercholesterolemia.

The cholesterol metabolism in humans can be indirectly reflected by measuring cholesterol metabolism marker levels. We aimed to investigate the association of cholesterol homeostasis markers on standard lipid profiling components in familial hypercholesteremia and hyperlipidemia patients. A total of 69 hyperlipidemia patients, 25 familial hypercholesteremia (FHC) patients, and 64 healthy controls were enrolled in this study. We performed routine testing of blood lipid water. Gas chromatography was used to determine the changes in the concentration of cholesterol synthesis (squalene, desmosterol, and lathosterol) and absorption markers (campesterol, sitosterol, and stigmasterol) in the blood. Baseline hyperlipidemia patients displayed significantly higher total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels in comparison to the control group, which was reflected in the increased levels of squalene, desmosterol, campesterol, and sitosterol observed (P < 0.05) in the hyperlipidemia patients. The desmosterol, lathosterol, campesterol, stigmasterol, and sitosterol were statistically different in the FHC group than the hyperlipidemic group (P < 0.05). The proportions of squalene/cholesterol, lathosterol/cholesterol, stigmasterol/cholesterol, and sitosterol/cholesterol in the FHC group were lower than those in the hyperlipidemic group; only desmosterol/cholesterol was higher than that in the hyperlipidemic group. Correlation studies between lipid metabolic factors showed that the proportion of moderate and strong correlations was much higher in the FHC group than in the other two groups (76.92% vs. 32.50% and 31.25%). Logistic regression analysis showed that the concentrations of glucose, LDL-C, lactosterol, and sitosterol were all independent risk factors for developing hyperlipidemia. This result was further confirmed by the ROC curve. These results indicated that the study of cholesterol synthesis and decomposition markers can serve as a reference index for related diseases caused by changes in its concentration.

Gas Chromatography and Flame-Ionization Detection of Non-Cholesterol Sterols as Indicators of Cholesterol Absorption and Synthesis in 158 Chinese Individuals with Normolipidemia, Hyperlipidemia, and Familial Hypercholesterolemia.

BACKGROUND There are limited studies on the effects of cholesterol homeostasis in populations at high risk for cardiovascular disease. We aimed to use gas chromatography and flame-ionization detection (GC-FID) of non-cholesterol sterols as indicators of cholesterol absorption and synthesis. Sterol indicators of cholesterol absorption included campesterol, stigmasterol, and sitosterol. Sterol indicators of cholesterol synthesis included squalene, 7-lathosterol, and desmosterol. MATERIAL AND METHODS A total of 158 participants were enrolled in 3 groups: healthy control (n=64), hyperlipidemia (n=69), and familial hypercholesterolemia (FH, n=25). Age, sex, blood pressure, blood glucose, and lipoprotein were collected, and cholesterol absorption and synthesis markers were determined by GC-FID. RESULTS All 6 cholesterol concentration indicators, except squalene, were significantly different among the 3 groups (all P<0.05); whereas in the ratio to cholesterol (%, sterols/cholesterol), only desmosterol and lathosterol were significantly different (P<0.05). Multifactorial regression analysis showed that triglycerides, total cholesterol, and desmosterol were independent risk factors affecting the development of hyperlipidemia (P<0.05). The efficacy of the ROC curve for the diagnosis of dyslipidemia was also higher for all 3 indices (Model 1, AUC=0.960). Model 1 was superior to Model 2 for the 6 indicators of cholesterol. For the FH and dyslipidemia groups, the 6-indicator model (Model 3) was shown to have a good diagnostic value (AUC=1.000). CONCLUSIONS The 6 sterol indicators of cholesterol absorption and synthesis had a dynamic course in all study participants. Desmosterol was an indicator of dyslipidemia. The combined use of the 6 sterol indicators differentiated between healthy individuals and patients with dyslipidemia and FH.

Integration of gene expression profile data to screen and verify immune-related genes of chicken erythrocytes involved in Marek's disease virus.

Chicken erythrocytes participated in immunity, but the role of erythrocytes in the immunity of Marek's disease virus (MDV) has not been reported related to the immunity genes. The purpose of this study was to screen and verify the immune-related genes of chicken erythrocytes which could be proven as a biomarker in MDV. The datasets (GPL8764-Chicken Gene Expression Microarray) were downloaded from the GEO profile database for control and MDV infected chickens to obtain differentially expressed genes (DEGs) through bioinformatics methods. Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to find enriched pathways, including Gene Ontology (GO). Based on enriched pathways, the top 19 immune-related genes were screened-out and process further to construct the protein-protein interaction (PPI) networks. The screened genes were validated on RT-PCR and qPCR. Results suggested that the mRNA transcription of Toll-like receptors 2, 3, 4, 6 (TLR2, TLR3, TLR4, TLR6), major histocompatibility complex-II (MHCII), interleukin-7 (IL-7), interferon-ßeta (IFN-ß), chicken myelomonocytic growth factor (cMGF) and myeloid differentiation primary response 88 (MyD88) were significantly up-regulated. The expression of toll-like receptor 5, 7 (TLR5, TLR7) interleukin-12 (IL-12 p40), interleukin-13 (IL-13), and interferon-αlpha (IFN-α) were significantly down-regulated in the erythrocytes of the infected group (P < 0.05). In contrast, the expression of toll-like receptor-1, 15, 21 (TLR1, TLR15, TLR21), major histocompatibility complex I (MHCI) and Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) were not significant. In conclusion, it has been verified on qRT-PCR results that 19 immune-related genes, which included TLRs, cytokines and MHC have immune functions in MDV infected chickens.

miR-30c promotes Schwann cell remyelination following peripheral nerve injury.

Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination following peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.

Gas chromatography analysis of serum cholesterol synthesis and absorption markers used to predict the efficacy of simvastatin in patients with coronary heart disease.

OBJECTIVES: We investigated the changes in cholesterol absorption and synthesis markers before and after simvastatin therapy in Chinese patients with coronary heart disease. DESIGN AND METHOD: We developed a gas chromatography method to identify cholesterol synthesis and absorption markers and measured them in patients with coronary heart disease. We then tested their use in predicting the efficacy of simvastatin in lowering cholesterol. Serum samples from 45 patients and 38 healthy humans (controls) were analyzed in a gas chromatography-flame ionization detector. RESULTS: Squalene and five non-cholesterol sterols--desmosterol and lathosterol (synthesis markers) and campesterol, stigmasterol, and sitosterol (absorption markers)--were detected. The recovery rates of the markers were 95-102%. After simvastatin treatment for four weeks, the total cholesterol and low-density lipoprotein cholesterol levels had significantly decreased from the baseline values (p<0.05). The baseline lathosterol level was significantly higher in good responders than in poor responders (p<0.05), and the stigmasterol level was significantly lower in good responders than in poor responders (p<0.05). CONCLUSIONS: This method should be suitable for the detection of serum squalene and non-cholesterol markers and can be used to predict the efficacy of simvastatin in patients with coronary heart disease.

CYP3A4*1B polymorphism and cancer risk: a HuGE review and meta-analysis.

CYP450 3A4 (CYP3A4), encoded by the CYP3A4 gene, is a major enzyme catalyzing the metabolism of both endogenous and exogenous agents that may play a role in the etiology of carcinogenesis. Several potentially functional polymorphisms of the CYP3A4 gene have been implicated in cancer risk, but individually published studies have shown inconclusive results. The aim of this Human Genome Epidemiology (HuGE) review and meta-analysis was to investigate the association between CYP3A4*1B (rs2740574 A > G) polymorphism and cancer risk. Eleven studies were included with a total of 3,810 cancer patients and 3,173 healthy controls. We found that the G allele and GG genotype of CYP3A4*1B polymorphism were associated with increased risk of cancers using the fixed effects model (allele model: odds ratio (OR) = 1.24, 95 %CI: 1.09-1.42, P = 0.001; recessive model: OR = 1.77, 95 %CI: 1.30-2.41, P < 0.001; homozygous model: OR = 1.72, 95 %CI: 1.19-2.47, P = 0.004). Subgroup analyses by cancer type showed that the G allele and G carrier (AG + GG) of CYP3A4*1B polymorphism had significant associations with increased risk of prostate cancer, but not with breast cancer, leukemia, or other cancers. With further subgroup analysis based on different ethnicities, the results indicated that the GG genotype of CYP3A4*1B polymorphism might increase the risk of cancer among African populations. However, similar associations were not observed among Caucasian and Asian populations. Results from the current meta-analysis indicate that the G allele and GG genotype of CYP3A4*1B polymorphism might be associated with increased cancer risk, especially for prostate cancer among African populations.

Effects of Tat peptide on intracellular delivery of arsenic trioxide albumin microspheres.

The current study was designed to evaluate the ability of cell-penetrating peptides to deliver arsenic trioxide albumin microspheres (AsAMs) into bladder cancer cells. The transactivating transcriptional activator (Tat) peptide was labeled with the enhanced green fluorescent protein (EGFP) using eukaryotic vector construction and fusion gene expression techniques. Arsenic trioxide albumin mirospheres were prepared using the chemical crosslink and solidification method. The conjugate, Tat-EGFP-As2O3-AMs (TEAsAMs), was synthesized using the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio) propionate and verified by electrophoresis under reducing conditions and fluorescence microscopy. The intracellular delivery of TEAsAMs was evaluated by laser confocal microscopy and transmission electron microscopy. The arsenic content in the bladder cancer EJ cells was assayed to evaluate the efficiency of delivery. Gene sequencing showed that the pET-Tat-EGFP expression vector was constructed successfully. The expression of the Tat-EGFP fusion protein was verified by matrix-assisted laser desorption/ionization-time of flight analysis, and the protein was transduced into cell cytoplasm as observed under a fluorescence microscope. Electrophoresis under reducing conditions demonstrated the covalent linkage between Tat-EGFP and AsAMs. Under a laser confocal microscope and a transmission electron microscope, TEAsAMs surrounded by green fluorescence were shown to enter the cells faster than EGFP-As2O3-AMs, with an increase in the intracellular arsenic content being observed in cells treated with TEAsAMs compared with those treated with EGFP-As2O3-AMs. These results suggest that Tat peptide promotes the cellular uptake of large albumin microspheres with encapsulated arsenicals.

[Mechanism of the effect of LPSp on the production of anti-HBs in mice].

AIM: To investigate the mechanism of the effect of avirulent Pantoea agglomerans lipopolysaccharide (LPSp) as the adjuvant of HBsAg on the production of anti-HBs in mice. METHODS: The peritoneal macrophages of BALB/c mice were pulsed in vitro by HBsAg+LPSp, HBsAg, LPSp, and NS respectively. The levels of anti-HBs IgG in serum were determined by ELISA. The variation of phagocytosis of the macrophages was detected. The culture supernatants were harvested and assayed for TNF-alpha and NO. HBsAg-pulsed peritoneal macrophages in the presence or absence of LPSp were injected in the hind footpads of syngeneic mice. Lymph node cells were harvested and cultured by HBsAg. IL-4 and IFN-gamma in the supernatants were quantitated by ELISA. RESULTS: The titer of anti-HBs IgG from the mice immunized with HBsAg-pulsed peritoneal macrophages treated with LPSp was higher than that of the mice immunized in the absence of LPSp. The phagocytosis rate of the LPSp-treated peritoneal macrophages was higher than that of the untreated macrophages. The level of TNF-alpha and NO of the LPSp-treated peritoneal macrophages was higher than that of the untreated macrophages. HBsAg-pulsed peritoneal cells in the presence of LPSp can induced higher production of IL-4 than those in the absence of LPSp. CONCLUSION: LPSp can act as the adjuvant of HBsAg and effectively increase the anti-HBS in the mice immunized by HBsAg. LPSp can activate macrophages and stimulate HBsAg-pulsed macrophages to induce the production of IL-4 of lymph node cells.