Oxidative stress is involved in the pathogenesis of colon cancer. We wanted to elucidate at which stage of the disease this phenomenon occurs. In the examined groups of patients with colorectal cancer (CRC, n = 89), benign adenoma (AD, n = 77) and healthy volunteers (controls, n = 99), we measured: vitamins A, C and E in blood plasma, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in leukocytes and urine, leukocyte 8-oxoGua excision activity, mRNA levels of APE1, OGG1, 8-oxo-7,8-dihydrodeoxyguanosine 5'-triphosphate pyrophosphohydrolase (MTH1) and OGG1 polymorphism. The vitamin levels decreased gradually in AD and CRC patients. 8-OxodG increased in leukocytes and urine of CRC and AD patients. 8-OxoGua was higher only in the urine of CRC patients. 8-OxoGua excision was higher in CRC patients than in controls, in spite of higher frequency of the OGG1 Cys326Cys genotype, encoding a glycosylase with decreased activity. mRNA levels of OGG1 and APE1 increased in CRC and AD patients, which could explain increased 8-oxoGua excision rate in CRC patients. MTH1 mRNA was also higher in CRC patients. The results suggest that oxidative stress occurs in CRC and AD individuals. This is accompanied by increased transcription of DNA repair genes, and increased 8-oxoGua excision rate in CRC patients, which is, however, insufficient to counteract the increased DNA damage.

We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 39-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 39-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 39-end cleavage and polyadenylation, that is, cotranscriptionally.

The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions. We have analyzed which lesions lead to these mutations. We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of [3H]MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of [3H]MNU alkylated poly(A) and poly(dA). After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.

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Cockayne syndrome complementation group B (CSB) protein is engaged in transcription-coupled repair (TCR) of UV induced DNA damage and its deficiency leads to progressive multisystem degeneration and premature aging. Here, we show that human CSB-deficient cells are hypersensitive to physiological concentrations (1–10 µM) of a lipid peroxidation product, trans-4-hydroxy-2-nonenal (HNE), and in response to HNE they develop a higher level of sister chromatid exchanges (SCEs) in comparison to the wild-type cells. HNE-DNA adducts block in vitro transcription by T7 RNA polymerase, as well as by HeLa cell-free extracts. Treatment of wild-type cells with 1–20 µM HNE causes dephosphorylation of the CSB protein, which stimulates its ATPase activity necessary for TCR. However, high HNE concentrations (100–200 µM) inhibit in vitro CSB ATPase activity as well as the transcription machinery in HeLa cell-free extracts. Cell lines expressing CSB protein mutated in different ATPase domains exhibit different sensitivities to HNE. The motif II mutant, which binds ATP, but is defective in ATP hydrolysis was as sensitive to HNE as CSB-null cells. In contrast, motif V mutant cells were as sensitive to HNE as were the cells bearing wild-type protein, while motif VI mutant cells showed intermediate sensitivity to HNE. These mutants exhibit decreased ATP binding, but retain residual ATPase activity. Homology modeling suggested that amino acids mutated in motifs II and VI are localized closer to the ATP binding site than amino acids mutated in ATPase motif V. These results suggest that HNE-DNA adducts are extremely toxic endogenous DNA lesion, and that their processing involves CSB. When these lesions are not removed from the transcribed DNA strand due to CSB gene mutation or CSB protein inactivation by high, pathological HNE concentrations, they may contribute to accelerated aging.

Background: ROS1 is the third specific driver gene after EGFR and ALK in NSCLC, and the occurrence frequency is about 1-2%. Compared with traditional FISH and ICH methods, NGS has significant advantages in detecting ROS1 variation. Not only fusion can be detected, but also ascertain fusion partners and fracture sites can be discovered by using NGS. Liquid biopsy based on ctDNA is more and more used in some type of cancers, the use of ctDNA to detect ROS1 variation has become a reliable method for precision treatment of NSCLC. However, the occurrence frequency, common fusion partners and common fracture sites of ROS1 malignant mutation (MUT) in Chinese NSCLC patients are still rarely reported, and the difference in positive rate between tissue and liquid biopsy is still unclear.Methods: The genetic testing data of Chinese NSCLC patients detected by 3D Medicines (Shanghai, China) with NGS method were retrospectively analyzed. Illumina Neseq-500 sequencing platform was used to detect tissue and peripheral blood samples. abstracts Annals of Oncology Volume 31 -Issue S4 -2020 S891

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Obesity is an increasing health problem in affluent societies that contributes to the prevalence of serious health diseases such as diabetes and heart disease. Current pharmacotherapies for obesity have side effects and are not effective in the long term, therefore, opportunities exist to develop novel therapeutics to treat this disorder. Peptides derived from the C-terminal domain of human growth hormone have shown to have anti-lipogenic and lipolytic activity. Growth hormone is a multifunctional protein (22-26 kDa) comprised of discrete bioactive domains responsible for several biological actions including the reduction and redistribution of body fat (Pullin et al., 1981) and the promotion of growth. Growth hormone is synthesized in the pituitary gland and stored in the cells of the anterior pituitary (Goodman et al., 1969). In mature animals it is released from these cells in response to insulin-induced hypoglycemia, starvation, amino acid infusion and neural stimuli including emotional stress. These responses suggest that growth hormone may play a role in the regulation of metabolism of energy in adult animals.AOD9401 is a cyclic 15 amino acid peptide containing a disulphide bond derived from human growth hormone. Biological investigations have revealed that AOD9401 has lipolytic and anti-lipogenic actions on rodent, porcine and human adipose tissues. Moreover, the biological activity of AOD9401 has been improved by substitution of key amino acids or functional side chains. These active mutant AOD9401 analogues show great promise for the development of new and more potent anti-obesity peptides. We are investigating whether the lipolytic and antilipogenic activity of AOD9401 and analogues are related to a specific molecular conformation. A conformational analysis of the peptides will be presented.Escherichia coli possesses two different N-methylpurine-D N A glycosylases. 3-methyladenine-DNA glycosylase I1 induced as a one of enzymes of adaptive response to alkylating agents with broad substrate specificity. 3-methyladenine-D N A glycosylase I (TagA protein) is expressed constitutively and excises only 3-methyladenine and 3-methylguanine from alkylated DNA. The structure of this enzyme has not yet been determined experimentally. We propose a tree-dimensional model of the TagA protein based on the threading algorithm. The model shows that TagA is mostly a a-helical protein, in agreement with circular dichroism measurements.None of the eight cysteines present in the TagA sequence forms a disulfide bridge in the model structure, which has also been experimentally verified with the use of Ellman method. Investigation of functional aspects of the N-terminal region ofEscherichia coli T-protein of the glycine cleavage system The glycine cleavage system is composed of four proteins named P-, H-, T-, and L-protein. T-protein is a folate-dependent enzyme and catalyzes the formation of NH) and CH2-THF from the aminomethyl intermediate attached to H-protein after the decarboxylation of glycine. A mutant with N-terminal7-r...