Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus

Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent long distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. Both N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.     

Genomic hypomethylation and CpG island hypermethylation in prostatic intraepithelial neoplasm

Altered DNA methylation in cancer cells is characterized by focal CpG island hypermethylation and diffuse genomic hypomethylation. Both types of aberrant methylation are frequently found in human prostate adenocarcinoma (PCa). Prostatic intraepithelial neoplasm (PIN), a precursor lesion of PCa, has been demonstrated to contain CpG island hypermethylation, but little is known about the role of DNA hypomethylation. We analyzed the methylation status at 12 CpG island loci and at two repetitive DNA elements (LINE-1 and SAT2) from normal prostate (n = 20), PIN (n = 25), and PCa (n = 35) tissues using MethyLight assay or combined bisulfite restriction analysis. The methylation levels in LINE-1 and SAT2 decreased with progression of lesion types from normal prostate to PIN to PCa (P < 0.05), whereas promoter CpG island loci displayed increased methylation. Ten genes were found to be hypermethylated in a cancer-specific manner and were further analyzed in another set of PCa tissues (n = 64). The number of methylated genes was closely associated with TNM stage, Gleason sum, and preoperative serum PSA levels (P = 0.020, 0.073, 0.033, respectively). These results suggest that genomic hypomethylation and CpG island hypermethylation, common among PCas, are early events in prostate carcinogenesis and may be implicated in the development of PIN. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Methylated DNA sequences for early cancer detection,molecular classification and chemotherapy response prediction

Molecular studies of many types of cancer have revealed that clinically evident tumours carry multiple genetic and epigenetic abnormalities, including DNA sequence alterations, chromosome copy number changes and aberrant promoter hypermethylation. Together, these aberrant changes result in the activation of oncogenes and inactivation of tumour-suppressor genes (TSG). In many cases these abnormalities can be found in premalignant lesions and even in histological normal adjacent cells. Many tumour types are difficult to detect early and are frequently resistant to available chemotherapy and radiotherapy. Therefore, the early detection, chemoprevention and the design of new therapeutic strategies based on the increased understanding of cancer molecular changes are one of the great challenges nowadays. Insertions of a methyl group at the fifth carbon of cytosines within the dinucleotide 5'- CpG-3' is the best studied epigenetic mechanism. DNA methylation acts together with others mechanisms like histone modification, chromatin remodelling and microRNAs to mould the DNA structure according to the functional state required. The aberrant methylation of the CpG islands located at the promoter region of specific genes is a common and early event involved in cancer development. Thus, hypermethylated DNA sequences from tumours are one of the most promising markers for early detection screenings as well as tumour classification and chemotherapy response in many types of cancer.     

改良外踝上动脉岛状皮瓣在足部皮肤缺损中的应用

目的了解应用改良外踝上动脉岛状皮瓣修复足踝部软组织缺损的临床效果。方法应用上述方法修复各种原因所致的足踝部软组织缺损共28例。结果27例皮瓣全部成活,1例远端皮肤1～2cm皮肤坏死,皮瓣最大面积16×9cm,最小6×4cm,随访6m～2y年,效果满意。结论应用此种皮瓣手术简便,不损伤小腿主要血管,是修复足踝、足背部软组织缺损理想方法。     

颞浅动脉岛状皮瓣联合口腔黏膜移植修复眼睑全层缺损

目的探讨以颞浅动脉岛状皮瓣联合口腔黏膜移植修复眼睑重度全层缺损的适应证和手术方式,评价其安全性和有效性。方法利用颞浅动脉岛状皮瓣联合口腔黏膜移植修复眼睑重度全层缺损患者13例(13眼),眼睑缺损范围为(33.67±8.35)mm×(16.44±4.45)mm。颞浅动脉岛状皮瓣大小为(38.56±8.99)mm×(18.44±4.00)mm,皮瓣内层覆盖游离唇黏膜8例,硬腭黏膜5例,术后随访6~24月。结果皮瓣完全存活10例;皮瓣部分存活2例;1例皮瓣因严重静脉淤滞坏死,术后3月行游离植皮术,植皮存活。移植的口腔黏膜均存活。5例患者因皮瓣臃肿,行Ⅱ期修整术,其中3例同时行Ⅱ期上睑下垂矫正额肌瓣悬吊术。存活的皮瓣色泽近似邻近皮肤,质地柔软,有效保护眼球。结论以颞浅动脉岛状皮瓣联合口腔黏膜移植可有效修复眼睑重度全层缺损,重建眼睑,保护眼球。     

Hypermethylation of promoter 5＇CpG island of p16 gene in glioma tissue and plasma

ObjectiveTo explore the clinical significance of methylation status of promoter CpG island of p16 gene in glioma tissue and plasma.MethodsMethylation specific polymerase chain reaction (MSP) was used to determine the methylation status of the promoter for p16 gene within glioma tissue and plasma.Immunohistochemicel method (SP) was used to analyze the expressions of p16 and Ki-67 proteins.ResultsHypermethylation was found in 17/40 (42.5%) of brain gliomas,in comparison with 11/40 (27.5%) plasma specimens (x2 = 1.9780,P = 0.1596).Loss of p16 expression was associated (P = 0.0229) with hypermethylation of CpG island of promoter regions.Hypermethylation of p16 gene CpG island was significantly related to the increase of malignant grade of brain glioma (TissueX2 = 11.4288,P = 0.0007;PlasmaX2 = 8.9439,P = 0.0028).The Ki-67 index increased significantly (P＜0.05) in brain gliomas methylated in contrast to those unmethylated.ConclusionP16 hypermethylation may be one of the major mechanisms of tumorigenesis of gliomas.Methylated tumor-specific DNA may be as a plasma biomarker for prognosis in patients with glioma.