Synthesis and living cell imaging of a novel fluorescent sensor for selective cupric detection.

Copper is an important element indispensable for human life and health. Many copper-determining probes have been created for exploring its functional behavior in various cell types but few of them contains both fluorescent and colorimetric characters. In the present study, we developed a set of copper probes by synthesizing several novel thiophene-based Schiff bases in order to make a suitable sensor for quantifying and imaging copper in living cells. We find that the ligand FS-1 has a splendid selectivity and affinity toward Cu2+ among the common divalent metal ions. Living cell imaging show that FS-1 has a robust and repetitive fluorescence response in the presence of Cu2+ only in the cytosolic space of Hepg2 cell and not in the other cells examined. These data suggest that we have developed a new copper probe that can be used as a Cu2+ fluorescent and colorimetric sensor for in vivo and in vitro copper studies.

Interleukin-33 Protects Ischemic Brain Injury by Regulating Specific Microglial Activities.

Interleukin-33 (IL-33), a novel member of the IL-1 family, expressed in many tissue and cell types, is involved in inflammation and immune functions. Previous studies suggest that IL-33 may play a role in ischemic stroke. Here, we evaluated the effect of IL-33 in cerebral ischemia-reperfusion-induced injury and investigated its underlying mechanism. Our data indicated that IL-33 deficiency exacerbated the neurological dysfunction caused by cerebral ischemia-reperfusion injury in mice and led to the formation of larger cerebral infarct volume as shown by 2,3,5-triphenyltetrazolium chloride staining and magnetic resonance imaging. Furthermore, the M1 and M2 macrophage-like microglial immune responses with decreased expression of the corresponding cytokines were seen in IL-33-deficient mice. IL-33 deficiency led to more biased to M2-like activities. The aggravated cerebral ischemia-reperfusion injury in IL-33-deficient mice is partially restored by intracerebroventricular injection of IL-33. These data suggest that IL-33 promotes the amplification of macrophage polarization and cytokine production associated with M2 macrophage-like microglial immune phenotype, which may contribute to the protective effects in the ischemic stroke, and that IL-33 may be a potential therapeutic target for ischemic stroke.

Mechanism of salvianolic acid B neuroprotection against ischemia/reperfusion induced cerebral injury.

The purpose of this study was to evaluate the cerebral protection of salvianolic acid B (Sal B) against cerebral I/R injury and investigate the underlying mechanism. As shown by 2,3,5-Triphenyltetrazolium chloride (TTC) staining and magnetic resonance imaging (MRI) analyses, Sal B significantly reduced cerebral infarct size, and accompanied with improved neurobehavioral functions as indicated by the modified Bederson score and Longa five-point scale. Sal B decreased the production of reactive oxygen species (p < .05, n = 10). The data of Western blotting and reverse transcription quantitative real time polymerase chain reaction (qRT-PCR) analyses showed that the expression of GFAP, Iba1, IL-1ß, IL-6, TNF-α and Cleaved-caspase 3 was significantly reduced by Sal B in I/R injured brain tissues as compared to corresponding controls (p < .05, n = 10). Over activation of astrocytes and microglia were inhibited by Sal B as shown by immunostaining of GFAP and Iba 1. These data suggest that Sal B has neural protective effects against I/R-induced cerebral injury and could be an effective candidate for further development of clinical therapy.

A modified bisulfite conversion method for the detection of DNA methylation.

AIM: Our purpose is to improve the conventional procedures for bisulfite conversion used to detect 5-methylcytosine in DNA. METHODS: Impacts of different bisulfite salts, bisulfite conversion temperature, antioxidants and denaturants on DNA conversion and degradation were assessed by methylation-sensitive melt curve analysis. The modified method was tested on different genes and the conversion efficiency was analyzed by bisulfite sequencing. RESULTS: We developed a modified bisulfite conversion method that completes this process within 2 h. We demonstrate that high temperature denaturation is the major cause for DNA degradation, and the addition of ethylene glycol dimethyl ether is an effective way to accelerate the bisulfite conversion. The conversion efficiency is comparable to many other commercial kits. CONCLUSION: Our modified bisulfite conversion method is simple, cost efficient and less time consuming and is compatible with different genes and samples, thus has a great potential for the future research and clinical applications.

The relationship between DNA methylation and Reprimo gene expression in gastric cancer cells.

Reprimo (RPRM) is a tumor suppressor involved in the development of a number of malignant tumors including gastric cancer which is highly related to its gene hypermethylation. However, the regulation of RPRM gene expression by DNA methylation in gastric cancer is not well understood. We examined the RPRM gene methylation in gastric cancer tissues or plasma samples by bisulfite sequencing, and investigated the relationship between DNA methylation and the RPRM gene expression by quantitative reverse transcription-PCR and Western blotting. We found that the RPRM gene promoter region is hypermethylated in gastric cancer tissues (75%, 45/60), plasma samples (86.3%, 44/51) and various cancer cell lines (75%, 3/4), which is correlated with the decrease of RPRM gene expression. The hypermethylation-induced RPRM reduction can be recovered by treating with zebularine, a demethylating agent, and by inhibition of the DNA methyltransferases via RNA interference and CRISPR/Cas9-mediated gene knockout. In addition, we generated RPRM gene-knockout cells and studied the effects of the RPRM deficiency on tumor formation by inoculating these cells in mice. The data show that the loss of RPRM can promote tumorigenesis. These data suggest that the RPRM expression is inhibited by DNA methyltransferases and the RPRM normal function can be restored by treating with DNA methylation inhibitors. The study provides important information regarding the role of RPRM and its methylation related to gastric cancer development.

Methylation-Sensitive Melt Curve Analysis of the Reprimo Gene Methylation in Gastric Cancer.

Reprimo (RPRM) is a p53-induced tumor suppressor gene. Its aberrant DNA methylation is correlated with carcinogenesis and may be used as a surrogate marker for the early detection of gastric cancer. However, the detail information regarding its DNA methylation has not been revealed. Here, we investigated the RPRM gene methylation in gastric cancer tumor and plasma samples by methylation-sensitive melt curve analysis (MS-MCA) and bisulfite sequencing in depth. We developed a semi-quantitative method based on MS-MCA for detecting DNA methylation and unraveled the RPRM gene methylation pattern in gastric cancer. This study provides a solid foundation for the future application of detecting RPRM gene methylation in human plasma or serum samples to help diagnose gastric cancer or for prognosis evaluation.

Regulatory effect of hypoxia-inducible factor-1α on hCG-stimulated endothelin-2 expression in granulosa cells from the PMSG-treated rat ovary.

Endothelin (ET)-2 plays a crucial role in ovarian ovulation in mammals. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated transcriptional activation contributes to the increased expression of ET-2 gene in response to hCG in rat ovarian granulosa cells (GCs) during gonadotropin-induced superovulation. By real-time RT-PCR analysis, ET-2 mRNA expression was found to significantly increase in cultured ovarian GCs after treatment with hCG, or even N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal (MG-132), while this increased ET-2 mRNA expression could also be blocked by ferrous ammonium sulfate (FAS) under human chorionic gonadotropin (hCG) treatment. Further analysis also found that these changes of ET-2 mRNA were consistent with HIF-1α expression or HIF-1 activity, and HIF-1α inhibitor echinomycin inhibited ovulation in rats. Taken together, these results indicate that ET-2 is transcriptionally activated by hCG through HIF-1α-mediated mechanism in GCs. This HIF-1α-induced transcriptional activation may be one of the important mechanisms mediating the increase of ET-2 expression in GCs during the gonadotropin-induced mammalian ovulatory process in vivo.

Effects of echinomycin on endothelin-2 expression and ovulation in immature rats primed with gonadotropins.

Echinomycin is a small-molecule inhibitor of hypoxia- inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1α mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1α and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1α and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1α directly mediated the transcriptional activation of ET-2 during gonadotropin- induced superuvulation. Taken together, these results demonstrated that HIF-1α-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.