Effect of tissue fixatives on telomere length determination by quantitative PCR

Telomere length is a well established marker of cellular senescence and thus biological age. Quantitative PCR allows the determination even from very low amounts of tissue by using telomere specific and single copy gene primers. Comparing a directly processed tissue sample to a 4% formaldehyde fixed one showed a significantly reduced efficiency of PCR reactions (mainly in single copy gene experiments) in a storage time-dependent manner resulting in an artificial increase in reported relative telomere length. This effect was not seen when the tissue was stored in RNA later solution. In summary, telomere length determination from formaldehyde fixed material by quantitative PCR is not a reliable method. Unfortunately therefore, many easily accessible tissue samples from pathology laboratories are unsuitable for this technique.     

一种新型的乙型肝炎病毒剪接变异体

目的 证实乙型肝炎病毒(hepatitis Bvirus，HBV)前基因组RNA在458nt～1308nt(nt，核苷酸)之间可发生剪接并产生相应的基因组剪接变异体。方法对1株分离自慢性乙型肝炎患者血清的亚基因组HBVDNA(2366bp)进行测序并以Vector NT16．0软件进行分析。将全基因组HBVDNA(3215bp)理论上可能的剪接供体及受体内的核苷酸进行定点突变并将突变株转染HepG2细胞，以位于HBVDNA458nt及1308nt两侧的特异引物检测转染后的细胞内HBV核心颗粒DNA，并以相同引物检测458nt～1308nt发生缺失的HBV DNA在不同病程的乙型肝炎患者血清中的存在情况。结果23661bp HBV DNA在458nt～1308nt之间发生缺失并符合GT-AG剪接模式。3215bp全基因组HBV DNA在459nt及1306nt分别发生G→A及A→C突变后均不能产生458nt～1308nt缺失。458nt～1308nt发生缺失的HBVDNA在慢性乙型肝炎、肝硬化及原发性肝细胞癌患者血清的检出率分别为80％、75％及70％，显著高于HBV无症状携带者10％的检出率。结论HBV前基因组RNA在458m～1308nt之间可发生剪接并产生长度为2366bp的基因组剪接变异体。该类型基因组剪接变异体基因结构特点及在HBV相关性肝病中广泛存在提示它与HBV致病性密切相关。     

Construction of a risk model of steroid-induced necrosis of the femoral head and prediction of potential Chinese medicine treatments北大核心

背景:随着疾病治疗模式的改变,人们已经意识到中医药在激素性股骨头坏死治疗过程中的重要性,因此利用生物信息学从分子水平分析激素性股骨头坏死的发病机制,构建疾病风险模型,并预测具有潜在治疗作用的中药,为后期中医药治疗激素性股骨头坏死提供一定的理论依据。目的:基于生物信息学挖掘激素性股骨头坏死的竞争性内源RNA(ceRNA)调控网络,分析其在激素性股骨头坏死中的分子调控机制,预测相关疾病靶点并构建疾病风险模型,同时预测具有潜在治疗作用的中药。方法:检索GEO数据库,下载激素性股骨头坏死的矩阵文件GSE123568和基因注释文件GPL15207。借助R语言等软件分析得到差异表达的长链非编码RNA与mRNA,并通过公共数据库预测与差异表达长链非编码RNA关联的miRNA-mRNA,再将预测到的mRNA与差异表达mRNA取交集,整合得到ceRNA网络。随后采用STRING数据库和Cytoscape软件筛选关键基因,利用R语言分析关键基因的功能与相关通路,并挖掘关键ceRNA网络。最后根据关键基因构建激素性股骨头坏死的风险模型,并进行中药预测。结果与结论:(1)与健康对照相比,激素性股骨头坏死患者共有7个长链非编码RNA和1763个mRNAs存在差异表达;(2)筛选出STAT3、KAT2B、AGO4、JAK2、JAK1、PTGS2共6个关键基因;(3)关键基因所富集的功能包括对肽激素的反应、白细胞介素6介导的信号通路、细胞对白细胞介素6的反应等生物学过程,涉及JAK-STAT、脂肪细胞因子、催乳素等信号通路;(4)4种mi RNAs(mi R-135a-5p、mi R-137、mi R-17-5p、miR-20b-5p)和2种长链非编码RNA(SNHG11、C20orf197)可能在导致激素性股骨头坏死发生发展过程中发挥关键作用;(5)KAT2B最有可能是激素性股骨头坏死发生发展的风险因子;(6)郁金、淫羊藿、黄芪具备治疗激素性股骨头坏死疾病靶点的可能。通过对激素性股骨头坏死相关长链非编码RNA介导的ceRNA网络进行分析,识别出潜在的疾病靶点、信号通路及潜在治疗中药,为进一步阐明其发病机制,并为后续的实验研究提供参考依据。     

Effect of chronic carbon tetrachloride poisoning on the turnover of RNA fractions in rat liver tissue

Changes in the content and incorporation of 5-³H-uridine after brief exposure to its labeled precursor were studied in the individual liver RNA fractions of rats during administration of carbon tetrachloride for 24 weeks. These fractions were obtained by preparative electrophoresis in 2.5% polyacrylamide gel from previously isolated nuclear and cytoplasmic RNA. Administration of CCl₄ to rats was shown to reduce the quantity of transfer and ribosomal RNA in the liver tissue. Chronic CCl₄ poisoning also disturbs the synchronization of the turnover of the individual components of fast-labeled RNA.Laboratory of Biochemistry, Professor N. N. Petrov Scientific-Research Institute of Oncology, Ministry of Health of the USSR, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR S. N. Golikov). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 423–425, April, 1977.     

Co-expression of Epstein-Barr virus latent membrane protein and vimentin in “aggressive” histological subtypes of Hodgkin's disease

Summary The presence of Epstein-Barr virus (EBV) genome in Hodgkin's and Reed-Sternberg (HRS) cells, as detected using in situ hybridization (ISH) with biotinylatedBamHI V probes, along with the expression of EBV-encoded latent membrane protein (LMP) and vimentin was examined in paraffin-embedded sections of 39 immunomorphologically characterized cases of Hodgkin's disease (HD). ISH demonstrated EBV in HRS cells in 15 of 39 cases, whereas LMP expression was detected in 11 of 39 cases, only in the presence of EBV genome detection. With the exception of 1 case, in which HRS cells expressed B-cell-associated antigens, the LMP-positive cases included specimens in which HRS cells were of non-B, non-T phenotype. LMP expression showed a stronger association with lymphocyte depletion (LD) (3/3) and mixed cellularity (MC) (6/11) than with lymphocyte predominance (0/5) or nodular sclerosis (2/20) subtypes. Vimentin expression on HRS cells was found in all the LMP-expressing cases and only in a fraction (13/28) of LMP-negative cases. This study supports the view that HD represents a heterogeneous group of diseases also in terms of EBV association, LMP expression being strongly related to the aggressive LD and MC histological subtypes. In light of the supposed interactions between vimentin and LMP, their coexpression on HRS cells, as detected in this study, provides further evidence for a significant role of EBV in the development of a proportion of HD cases.     

Mutations in activation-induced cytidine deaminase in patients with hyper IgM syndrome

Recent studies have shown that mutations in a newly described RNA editing enzyme, activation-induced cytidine deaminase (AID), can cause an autosomal recessive form of hyper IgM syndrome. To determine the relative frequency of mutations in AID, we evaluated a group of 27 patients with hyper IgM syndrome who did not have defects in CD40 ligand and 23 patients with common variable immunodeficiency. Three different mutations in AID were identified in 18 patients with hyper IgM syndrome, including 14 French Canadians, 2 Lumbee Indians, and a brother and sister from Okinawa. No mutations were found in the remaining 32 patients. In the group of patients with hyper IgM syndrome, the patients with mutations in AID were older at the age of diagnosis, were more likely to have positive isohemagglutinins, and were less likely to have anemia, neutropenia, or thrombocytopenia. Lymphoid hyperplasia was seen in patients with hyper IgM syndrome and normal AID as well as the patients with hyper IgM syndrome and defects in AID.     

High frequency of rotavirus viremia in children with acute gastroenteritis: Discordance of strains detected in stool and sera

Recently, rotavirus antigenemia and viremia have been identified in patients with acute gastroenteritis. This study examined rotavirus viremia in children hospitalized for acute gastroenteritis in order to establish its association with fecal shedding of rotavirus, infecting genotypes and antibody marker of acute infection. Thirty‐one pairs of stool–serum specimens were collected from November 2004 to February 2005 together with clinical information. All paired specimens were screened for rotavirus RNA by RT‐PCR using the VP6 gene primers. All stool and serum specimens were tested for rotavirus antigen and anti‐rotavirus IgM respectively by ELISA. Sixteen of 31 stool–serum pairs showed the presence of rotavirus RNA. Nine stool and two serum specimens were positive only by RT‐PCR. The total positivity in rotavirus RNA was significantly higher in both stools (80.6%) and sera (58.1%) than that of stool antigen (38.7%) and anti‐rotavirus IgM (25.8%) (P < 0.01). All PCR positive paired specimens were typed for the VP7 (G) and VP4 (P) genes. Five of sixteen pairs could be typed for both genes. Three of the five pairs showed concordance (G2P[4]/G2P[4]) while two showed discordance (G12P[8]/G2P[4], G8P[4]/G2P[4]) in the genotypes detected in stool and serum specimens respectively. The study documents a high frequency of rotavirus viremia in patients with acute diarrhea. The discordance of rotavirus strains at the genotypic level in the serum and stool of individual patients with diarrhea suggests the susceptibility of extra‐intestinal sites for rotavirus infection and the possibility of differential dissemination of rotavirus strains from the intestine. J. Med. Virol. 80:2169–2176, 2008. © 2008 Wiley‐Liss, Inc.     

Action of acute cardiac ischemia on activity of the protein-synthesizing system of the inner mitochondrial membranes of the myocardium

Acute cardiac ischemia caused by ligation of the descending branch of the left coronary artery leads to a sharp increase in the activity of the protein-synthesizing system of the inner mitochondrial membranes of the myocardium throughout the period of organization of the experimental infarct, as reflected in an incease in the synthesis of protein and RNA in the mitochondria. During development of the infarct considerable changes are observed in the ultrastructure of the inner mitochondrial membranes of the myocardium, the degree of which is directly dependent on the stage of development of the pathological process.Institute of General Pathology and Pathophysiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. N. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 9, pp. 295–297, September, 1978.     

Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

The genome of equine arteritis virus (EAV) produces a 3 coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5 end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) strands of the EAV leader RNA. Several cell proteins with molecular masses ranging from 74 to 31 kDa and 58 to 32 kDa were detected in UV-induced cross-linking assays with the EAV leader RNA (+) and (-) strands, respectively. In both cases, intense bands were observed at the 58–52 kDa molecular weight markers. Results from competition gel mobility shift assays using overlapping cold RNA probes spanning the leader RNA (+) strand indicated that nt 140–206 are not necessary for binding to cell proteins.