Homologous expression of Phanerochaete chrysosporium manganese peroxidase,using bialaphos resistance as a dominant selectable marker

Manganese peroxidase (MnP) is a major extracellular component of the lignin-degrading system of the white-rot fungus, Phanerochaete chrysosporium. Homologous expression of recombinant MnP isozyme 1 (rMnP1) in P. chrysosporium was achieved using a novel transformation system for this fungus, which utilizes the Streptomyces hygroscopicus bialaphos-resistant gene, bar, as the selectable marker. The transformation frequency for this system is approximately 100 bialaphos-resistant transformants per microgram of plasmid DNA. Transformed strains all contain plasmid DNA, ectopically integrated into the fungal genome. Using this transformation system, the promoter region of the P. chrysosporium translation elongation factor gene was used to drive expression of mnp1, encoding MnP1, in primary metabolic cultures of P. chrysosporium, where endogenous MnP was not expressed. Approximately 2–3 mg of active recombinant MnP1 per liter of extracellular medium was produced in agitated cultures of transformants.Communicated by U. Kück     

Electron microscopic radioautographic study on nucleic acid synthesis in amitotic hepatocytes of the aging mouse

Twenty groups of aging mice, each consisting of three individuals from fetal day 19 to postnatal month 24, were injected with either ³H-thymidine or ³H-uridine. The mice were killed 1h and the livers processed for light and electron microscopic radioautography. On radioautograms obtained from each animal, amitotic nuclear divisions and resulting binucleate hepatocytes were detected and compared to mononucleate hepatocytes. From the results, it was demonstrated that only a few hepatocytes showing amitotic nuclear division were found labeled with the two precursors demonstrating DNA and RNA synthesis. However, there were very few silver grains showing incorporation of labeled precursors in respective amitotic cells. It was clarified that the amitotic cells did not synthesize such macromolecules as mononucleate hepatocytes did. On the other hand, the binucleate cells were found more often than the amitotic cells. DNA synthesis of binucleate hepatocyte nuclei was observed at the perinatal stage but disappeared at the adult stage. RNA syntheses in karyoplasm and cytoplasm in both mononucleate and binucleate cells significantly increased from the perinatal stage, reaching the maxima at the adult stage, then significantly decreased to the senescent stage. Grain counts revealed that RNA was synthesized significantly more in binucleate cells than in mononucleate cells at the respective aging stages.     

Immune activation by nucleic acids: A role in pregnancy complications

Cell‐free self‐DNA or RNA may induce an immune response by activating specific sensing receptors. During pregnancy, placental nucleic acids present in the maternal circulation further activate these receptors due to the presence of unmethylated CpG islands. A higher concentration of cell‐free foetal DNA is associated with pregnancy complications and a higher risk for foetal rejection. Cell‐free foetal DNA originates from placental trophoblasts. It appears in different forms: free, bound to histones in nucleosomes, in neutrophil extracellular traps (NETs) and in extracellular vesicles (EVs). In several pregnancy complications, cell‐free foetal DNA triggers the production of proinflammatory cytokines, and this production results in a cellular and humoral immune response. This review discusses preeclampsia, systemic lupus erythematosus, foetal growth restriction, gestational diabetes, rheumatoid arthritis and obesity in pregnancy from an immunological point of view and closely examines the different pathways that result in maternal inflammation. Understanding the role of cell‐free nucleic acids, as well as the biogenesis of NETs and EVs, will help us to specify their functions or targets, which seem to be important in pregnancy complications. It is still not clear whether higher concentrations of cell‐free nucleic acids in the maternal circulation are the cause or consequence of various complications. Therefore, further clinical studies and, even more importantly, animal experiments that focus on the involved immunological pathways are needed.     

Validation of Gelbond® high‐throughput alkaline and Fpg‐modified comet assay using a linear mixed model

Even if the comet assay has been widely used for decades, there is still a need for controlled studies and good mathematical models to assess the variability of the different versions of this assay and in particular to assess potential intra‐experimental variability of the high‐throughput comet assay. To address this point, we further validate a high‐throughput comet assay that uses hydrophilic polyester film (Gelbond®). Experiments were performed using human peripheral blood mononuclear cells (PBMC) either untreated or treated with different concentration of MMS (methyl methanesulfonate). A positive control for the Fpg (Formamidopyrimidine DNA glycosylase)‐modified comet assay (Ro 19‐8022 with light) was also included. To quantify the sources of variability of the assay, including intradeposit variability, instead of summarizing DNA damage on 50 cells from a deposit by the mean or median of their percentage DNA tail, we analyzed all logit‐transformed data with a linear mixed model. The main source of variation in our experimental data is between cells within the same deposit, suggesting genuine variability in the response of the cells rather than variation caused by technical treatment of cell samples. The second source of variation is the inter‐experimental variation (day‐to‐day experiment); the coefficient of this variation for the control was 13.6%. The variation between deposits in the same experiment is negligible. Moreover, there is no systematic bias because of the position of samples on the Gelbond^® film nor the position of the films in the electrophoresis tank. This high‐throughput comet assay is thus reliable for various applications. Environ. Mol. Mutagen. 59:595–602, 2018. © 2018 Wiley Periodicals, Inc.     

DNA定量分析检测对胸腔积液的诊断价值研究

目的 探讨细胞DNA定量分析在不明原因胸水诊断中的应用价值。方法 收集本院2015年9月~2016年3月122例胸腔积液患者抽取胸水,标本制片后分别经巴氏染色行脱落细胞学检查,Feulgen染色后行细胞DNA定量分析。结果 122例胸腔积液中有56例恶性胸腔积液,66例良性胸腔积液。细胞学检测恶性胸水敏感度为82.14%,特异性为98.48%,阳性预测值97.87%,阴性预测值86.67%。细胞DNA定量分析检测恶性胸水敏感度为80.36%,特异度为90.91%,阳性预测值88.24%,阴性预测值84.51%。两种方法诊断结果一致性一般（Kappa=0.473,P＜0.001）,差异无统计学意义（P=1.0）。结论 DNA定量分析检测是一种较敏感而特异的肺癌的筛查技术,DNA异倍体有望作为良恶性胸水诊断有价值的标志物。     

Proteins of the retinoblastoma pathway,FEN1 and MGMT are novel potential prognostic biomarkers in pancreatic adenocarcinoma

Background

We studied the expression of some major proteins involved in cell-cycle regulation and DNA repair, the roles of which are not well known in pancreatic ductal adenocarcinoma (PDAC), but which have a significant impact on carcinogenesis of many other cancers.

Diagnostic value of RASSF1A hypermethylation in colorectal cancer: a meta-analysis

Purpose

Ras association domain family 1 isoform A (RASSF1A), a member of Ras association domain family, plays an important role in tumorigenesis. The goal of our meta-analysis was to assess the diagnostic value of RASSF1A hypermethylation in colorectal cancer (CRC).

Novel non‐neutral mitochondrial DNA mutations found in childhood acute lymphoblastic leukemia

Mitochondria produce adenosine triphosphate (ATP) for energy requirements via the mitochondrial oxidative phosphorylation (OXPHOS) system. One of the hallmarks of cancer is the energy shift toward glycolysis. Low OXPHOS activity and increased glycolysis are associated with aggressive types of cancer. Mitochondria have their own genome (mitochondrial DNA [mtDNA]) encoding for 13 essential subunits of the OXPHOS enzyme complexes. We studied mtDNA in childhood acute lymphoblastic leukemia (ALL) to detect potential pathogenic mutations in OXPHOS complexes. The whole mtDNA from blood and bone marrow samples at diagnosis and follow‐up from 36 ALL patients were analyzed. Novel or previously described pathogenic mtDNA mutations were identified in 8 out of 36 patients. Six out of these 8 patients had died from ALL. Five out of 36 patients had an identified poor prognosis genetic marker, and 4 of these patients had mtDNA mutations. Missense or nonsense mtDNA mutations were detected in the genes encoding subunits of OXPHOS complexes, as follows: MT‐ND1, MT‐ND2, MT‐ND4L and MT‐ND6 of complex I; MT‐CO3 of complex IV; and MT‐ATP6 and MT‐ATP8 of complex V. We discovered mtDNA mutations in childhood ALL supporting the hypothesis that non‐neutral variants in mtDNA affecting the OXPHOS function may be related to leukemic clones.