Neuroglobin基因体内转染对水杨酸钠给药后小鼠下丘核神经元听反应的影响

实验以48只成年健康昆明小鼠为实验对象,研究GeneJamer转染试剂介导的neuroglobin(NGB)基因体内转染对水杨酸钠给药后小鼠下丘核区听反应的影响。实验分4组,每组12只。A1组:对照组1(阴性对照,将GeneJamer转染试剂6μl和pEGFP-C12μg混合后注入下丘核脑区);A2组:对照组2[阳性对照,将GeneJamer转染试剂(6μl)和pEGFP-NGB(质粒载体pEGFP-C1与NGB基因全编码序列构建的重组子2μg)混合后注入下丘核脑区];B组:水杨酸钠给药组(450mg/kg·d-1)+pEGFP-C1;C组:水杨酸钠(450mg/kg.d-1)+pEGFP-NGB组。以直接注射法将GeneJamer转染试剂和重组质粒pEGFP-NGB混合后注入小鼠下丘核区。采用RT-PCR和Westernblot技术检测小鼠下丘核区NGBmRNA和蛋白的表达;采用细胞外记录技术,研究小鼠下丘核区神经元在水杨酸钠给药后转染重组质粒pEGFP-NGB对强度-发放率函数(刺激声强与实验鼠下丘核区神经元在接受声刺激所产生的电发放的关系曲线)、强度-潜伏期函数(刺激声强与实验鼠下丘核区神经元在接受声刺激至产生电发放潜伏期之间的关系曲线)和频率调谐曲线(实验鼠下丘核区神经元在各个频率纯音刺激下起反应的阈值绘制的曲线)的影响。实验观察到:(1)经GeneJamer转染试剂介导NGB基因可有效地转染小鼠下丘核区脑组织并得到表达。(2)水杨酸钠给药后神经元的强度-发放率函数曲线升高。对照组A1、A2各项指标进行比较均无统计学意义。对照组A1、A2和水杨酸钠+pEGFP-NGB组神经元的强度-发放率函数以非单调型(随刺激强度增加时,发放率表现为先降后升呈“V”形或“U”形)为主,分别占74.6%、72.2%和59.3%,水杨酸钠给药组以不规则型强度-发放率函数为主,占47%,与对照组A1、A2和水杨酸钠+pEGFP-NGB组比较,有显著性差异(P<0.01、P<0.01、P<0.05)。(3)水杨酸钠给药后神经元的强度-潜伏期函数曲线降低。对照组A1、A2各项指标进行比较均无统计学意义。水杨酸钠给药组以非单调型强度-潜伏期率函数为主,与对照组A1、A2和水杨酸钠+pEGFP-NGB组比较,有显著性差异(P<0.01、P<0.05)。(4)A1和A2对照组听反应神经元的调谐曲线,Q-10dB值均大于5.00,其调谐曲线为狭窄型。记录水杨酸钠给药组72个听神经元的调谐曲线,有53个神经元的Q-10dB值小于5.00,Q-10dB值最小为2.12,其调谐曲线为宽阔型;其余19个神经元的Q-10dB值大于5.00,属于狭窄型调谐曲线。水杨酸钠+pEGFP-NGB组67个听神经元的调谐曲线,有12个神经元的Q-10dB值小于5.00,Q-10dB值最小为2.87,其调谐曲线为宽阔型,其它的神经元的值大于5.00。它们的调谐曲线均属狭窄型。以上结果提示外源性NGB基因在水杨酸钠给药后小鼠下丘核区局部高表达,提示GeneJamer转染试剂介导NGB体内转基因治疗水杨酸钠引起的下丘核区的损伤的方法是可行的。实验小鼠转染NGB基因后可逆转因水杨酸钠给药引起的强度-发放率函数曲线升高以及强度-潜伏期函数曲线降低,并可逆转水杨酸钠引起的部分听神经元对声刺激强度的编码类型。     

PTEN regulates RPA1 and protects DNA replication forks

Tumor suppressor PTEN regulates cellular activities and controls genome stability through multiple mechanisms. In this study, we report that PTEN is necessary for the protection of DNA replication forks against replication stress. We show that deletion of PTEN leads to replication fork collapse and chromosomal instability upon fork stalling following nucleotide depletion induced by hydroxyurea. PTEN is physically associated with replication protein A 1 (RPA1) via the RPA1 C-terminal domain. STORM and iPOND reveal that PTEN is localized at replication sites and promotes RPA1 accumulation on replication forks. PTEN recruits the deubiquitinase OTUB1 to mediate RPA1 deubiquitination. RPA1 deletion confers a phenotype like that observed in PTEN knockout cells with stalling of replication forks. Expression of PTEN and RPA1 shows strong correlation in colorectal cancer. Heterozygous disruption of RPA1 promotes tumorigenesis in mice. These results demonstrate that PTEN is essential for DNA replication fork protection. We propose that RPA1 is a target of PTEN function in fork protection and that PTEN maintains genome stability through regulation of DNA replication.     

Xinnaokang improves cecal microbiota and lipid metabolism to target atherosclerosis

This study aims to explore the potential mechanisms of Xinnaokang in atherosclerosis treatment. Firstly, the active components of Xinnaokang were analysed by HPLC, which contains ginsenoside Rg1, puerarin, tanshinone, notoginsenoside R1, ammonium glycyrrhizate and glycyrrhizin. Network pharmacology analysis showed there were 145 common targets of Xinnaokang, including the chemical stress, lipid metabolite, lipopolysaccharide, molecules of bacterial origin, nuclear receptor and fluid shear stress pathways. Then, the animal experiment showed that Xinnaokang reduced the body weight and blood lipid levels of atherosclerotic mice. Vascular plaque formation was increased in atherosclerotic mice, which was markedly reversed by Xinnaokang. In addition, Xinnaokang reduced CAV-1 expression and increased ABCA1, SREBP-1 and LXR expressions in the vasculature. Xinnaokang promoted SREBP-2 and LDLR expressions in the liver but decreased IDOL and PCSK9 expressions, indicating that Xinnaokang regulated lipid transport-related protein expression. Cecal microbiota diversity was reduced in atherosclerotic mice but increased after Xinnaokang treatment. Xinnaokang treatment also improved gut microbiota communities by enriching Actinobacteria, Bifidobacteriales and Bifidobacteriaceae abundances. Metabolic profile showed that Xinnaokang significantly reduced homogentisate, phenylacetylglycine, alanine and methionine expressions in the liver of atherosclerotic mice. Xinnaokang effectively alleviated atherosclerosis, and this effect might be linked with the altered features of the liver metabolite profiles and cecal microbiota.     

Structural basis of EZH2 recognition by EED

The WD-repeat domain is a highly conserved recognition module in eukaryotes involved in diverse cellular processes. It is still not well understood how the bottom of a WD-repeat domain recognizes its binding partners. The WD-repeat-containing protein EED is one component of the PRC2 complex that possesses histone methyltransferase activity required for gene repression. Here we report the crystal structure of EED in complex with a 30 residue peptide from EZH2. The structure reveals that the peptide binds to the bottom of the WD-repeat domain of EED. The structural determinants of EZH2-EED interaction are present not only in EZH2 and EZH1 but also in its Drosophila homolog E(Z), suggesting that the recognition of ESC by E(Z) in Drosophila employs similar structural motifs. Structure-based mutagenesis identified critical residues from both EED and EZH2 for their interaction. The structure presented here may provide a template for understanding of how WD-repeat proteins recognize their interacting proteins.     

Genome-wide DNA methylation indicates silencing of tumor suppressor genes in uterine leiomyoma

Background

Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown.

Principal Findings

We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels.

Conclusions

These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women.     

Levels of Hexachlorocyclohexanes and Dichloro-Dipheny-Trichloroethanes in Penguin Droppings Collected from Ardley Island,Maritime Antarctic

This article reports the concentrations of hexachlorocyclohexanes (HCHs) and dichloro-diphenyl-trichloroethanes (DDTs) in fresh Gentoo penguin (Pygoscelis Papua) droppings deposited on the Ardley Island (62°13′S, 58°56′W), West Antarctica, and those in the island's two lake cores. In the fresh penguin droppings, metabolites were the predominant constituents of the DDT group, most likely due to the ban on DDT use in agriculture and the higher stability of the metabolites in the environment. In contrast, γ-HCH comprised 66% of the total HCHs, apparently due to the continuous use of Lindane in the Southern Hemisphere. The concentrations of HCHs and DDTs in the fresh droppings are many times higher than those in penguin plume, the latter currently being used as biomaterial for monitoring coastal contaminations in Antarctica. Results of this study indicate that penguin droppings have a good potential for use as another biomonitoring material in detecting the organochlorine pesticides and other contaminants in the maritime Antarctic. Because penguin droppings can be well preserved in lake sediments, they also provide a good and continuous historical record of these contaminants. The slight increase of DDTs since the 1990s, as observed in the aged penguin droppings in the sediment cores, suggests there have been new inputs of DDT into the Antarctic coastal ecosystem, probably from the illegal use of DDT in the Southern Hemisphere.     

Hdac2 regulates the cardiac hypertrophic response by modulating Gsk3 beta activity

In the adult heart, a variety of stresses induce re-expression of a fetal gene program in association with myocyte hypertrophy and heart failure. Here we show that histone deacetylase-2 (Hdac2) regulates expression of many fetal cardiac isoforms. Hdac2 deficiency or chemical histone deacetylase (HDAC) inhibition prevented the re-expression of fetal genes and attenuated cardiac hypertrophy in hearts exposed to hypertrophic stimuli. Resistance to hypertrophy was associated with increased expression of the gene encoding inositol polyphosphate-5-phosphatase f (Inpp5f) resulting in constitutive activation of glycogen synthase kinase 3beta (Gsk3beta) via inactivation of thymoma viral proto-oncogene (Akt) and 3-phosphoinositide-dependent protein kinase-1 (Pdk1). In contrast, Hdac2 transgenic mice had augmented hypertrophy associated with inactivated Gsk3beta. Chemical inhibition of activated Gsk3beta allowed Hdac2-deficient adults to become sensitive to hypertrophic stimulation. These results suggest that Hdac2 is an important molecular target of HDAC inhibitors in the heart and that Hdac2 and Gsk3beta are components of a regulatory pathway providing an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure.