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The mussels Mytilus galloprovincialis collected from a noncontaminated site (Chaib Rasso) were transplanted during one, three and six months at Ghazaouet harbor (GH), areas with a strong gradient of pollution. The micronucleus test (MN) was selected to monitor the impact of contamination, along with physiological indexes (condition index CI and organo-somatic indexes RI and GSI). The results show a negative correlation of MN variation in gill cells with CI but a positive correlation with transplantation duration. However, a significant correlation was found between the indexes. Moreover, the findings indicate that MN in the hemolymph and gills of transplanted mussels for one, three and six months at GH are significantly higher than those of the reference site. However, no significant differences were noted between the three transplants at the two organs. Monitoring the physiological status of mussels, in parallel with the biomarker measurements, is useful in assessing the impact of contaminants., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The DNA in eukaryotic cells is organized into loop domains that represent basic structural and functional units of chromatin packaging. The comet assay, a sensitive method for monitoring DNA damage and repair, involves electrophoresis of nucleoids comprising supercoiled DNA attached to the nuclear matrix. Breaks in the DNA relax the supercoiling and allow DNA loops to expand, and on electrophoresis to move towards the anode, giving the appearance of a comet tail. We use fluorescent in situ hybridization (FISH) to investigate the structure of the chromatin within comet preparations and to study specific DNA sequences within comets. In this chapter we describe our FISH comets protocols, deal with some technical questions and outline the theory. FISH with comets should be useful to researchers interested in the structural organization of DNA and chromatin, the localization of DNA damage, and the kinetics of repair of damage.

The comet assay is a sensitive and versatile method for assessing DNA damage in cells. In the traditional version of the assay, there are many manual steps involved and few samples can be treated in one experiment. High throughput (HT) modifications have been developed during recent years, and they are reviewed and discussed. These modifications include accelerated scoring of comets; other important elements that have been studied and adapted to HT are cultivation and manipulation of cells or tissues before and after exposure, and freezing of treated samples until comet analysis and scoring. HT methods save time and money but they are useful also for other reasons: large-scale experiments may be performed which are otherwise not practicable (e.g., analysis of many organs from exposed animals, and human biomonitoring studies), and automation gives more uniform sample treatment and less dependence on operator performance. The HT modifications now available vary largely in their versatility, capacity, complexity, and costs. The bottleneck for further increase of throughput appears to be the scoring.

Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period-for example, analysing samples of white blood cells from a large human biomonitoring trial-to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

Chondroitin sulfate proteoglycans (CSPGS) are extracellular matrix components that contain two structural parts with distinct functions: a protein core and glycosaminoglycan (GAG) side chains. CSPGs are known to be involved in important cell processes like cell adhesion and growth, receptor binding, or cell migration. It is recognized that the presence of CSPGs is critical in neuronal growth mechanisms including axon guidance following injury of nervous system components such as spinal cord and brain. CSPGs are upregulated in the central nervous system after injury and participate in the inhibition of axon regeneration mainly through their GAG side chains. Recently, it was shown that some CSPGs members like aggrecan, versican, and neurocan were strongly involved in brain disorders like bipolar disorder (BD), schizophrenia, and ADHD. In this paper, we present the chemical structure-biological functions relationship of CSPGs, both in health state and in genetic disorders, addressing methods represented by genome-wide and crystallographic data as well as molecular modeling and quantitative structure-activity relationship.

The retrospective research included 1983 patients with different abdominal surgical pathology. Parameters of homeostasis were estimated in preoperative period and early postoperative period. Frequency of occurrence and relevance of different clinical risk factors of thrombosis were analyzed. The rate of development of thromboembolic complications was investigated in studied subgroup of patients. It was revealed, that high risk groups of thrombosis progress were the patients with malignant disease of the pancreas, the esophagus, the large and straight intestine as well as obstructive jaundice of malignant genesis. The most significant clinical factors were the presence of malignant process, accompanied by cardiac pathology, dehydration and high number (3 and more) on ASA scale.

We used padlock probes to study the rate of gene specific repair of three genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) in human lymphocytes, in relation to the repair rate of Alu repeats and total genomic DNA. Padlock probes offer highly specific detection of short target sequences by combining detection by ligation and signal amplification. In this approach only genes in sequences containing strand breaks, which become single-stranded in the tail, are available for hybridisation. Thus the total number of signals from the padlock probes per comet gives a direct measure of the amount of damage (strand-breaks) present and allows the repair process to be monitored. This method could provide insights on the organisation of genomic DNA in the comet tail. Alu repeat containing DNA was repaired rapidly in comparison with total genomic DNA, and the studied genes were generally repaired more rapidly than the Alu repeats., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The comet assay is a simple and sensitive method for measuring DNA damage. Cells are embedded in agarose on a microscope slide, lysed, and electrophoresed; the presence of strand breaks allows the DNA to migrate, giving the appearance of a comet tail, the percentage of DNA in the tail reflecting the break frequency. Lesion-specific endonucleases extend the usefulness of the method to investigate different kinds of damage. DNA repair can be studied by treating cells with damaging agent and monitoring the damage remaining at intervals during incubation. An important feature of the assay is that damage is detected at the level of individual cells. By combining the comet assay with fluorescent in situ hybridization (FISH), using labeled probes to particular DNA sequences, we can examine DNA damage and repair at the level of single genes or DNA sequences. Here we provide protocols for the comet assay and the FISH modification, answer some technical questions, and give examples of applications of the technique.

The comet assay is widely used to measure DNA damage and repair in basic research, genotoxicity testing and human biomonitoring. The conventional format has 1 or 2 gels on a microscope slide, 1 sample per slide. To increase throughput, we have designed and tested a system with 12 smaller gels on one slide, allowing incubation of individual gels with different reagents or enzymes. Thus several times more samples can be analysed with one electrophoresis run, and fewer cells and smaller volumes of test solutions are required. Applications of the modified method include treatment with genotoxic agents at different concentrations; simultaneous analysis of different lesions using a range of enzymes; analysis of cell extracts for DNA repair activity; and fluorescent in situ hybridisation (FISH) to comet DNA with specific labelled probes., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

DNA oxidation, which can have potentially serious mutagenic consequences, commonly accompanies exposure to environmental mutagens. Oxidised bases can be measured chromatographically, but spurious oxidation during sample preparation leads to serious over-estimation of low levels of damage. A more reliable approach is to employ endonucleases specific for oxidised bases, to introduce breaks in cellular DNA that are then most commonly measured using the comet assay (alkaline single cell gel electrophoresis). The two enzymes in general use are formamidopyrimidine DNA glycosylase, which detects primarily 8-oxo-7,8-dihydroguanine (8-oxoGua), and endonuclease III which recognises oxidised pyrimidines. We give a brief account of the recommended experimental procedures, and then describe applications in various areas of environmental research. Cultured cell lines or white blood cells have been exposed to a range of environmental mutagens, including natural products, industrial chemicals, radiation and nanoparticles. In vivo exposure of animals and humans to pollutants is more challenging but can give particularly valuable information in relation to real life exposure. Possibly the most useful application is in biomonitoring of human population groups suffering actual exposure to environmental or occupational mutagens. Finally, the potential use of this technique to monitor effects of contaminants in the natural environment has yet to be fully exploited.

DNA repair is one of the important determinants of susceptibility to cancer. It is therefore useful to be able to measure DNA repair capacity in samples from population studies. Our aim was, first, to develop a simple comet-based in vitro assay for nucleotide excision repair (NER), similar to that already in use for base excision repair (BER), and then to apply these in vitro assays to lymphocyte samples collected on several occasions from healthy subjects, to gain an impression of the degree of intra- and inter-individual variability. The in vitro assay consists of an incubation of lymphocyte extract with substrate nucleoid DNA from cells pretreated with specific damaging agent; either photosensitiser plus light to induce 8-oxoguanine, for BER, or short wavelength ultraviolet light irradiation for NER. In the new NER assay, which requires magnesium but not adenosine triphosphate, there was significant accumulation of UV-dependent incisions during a 30-min incubation of extract with DNA. We found significant correlations between individual repair rates from samples taken on different occasions; i.e. individuals have a characteristic repair capacity. There was also significant variation between individuals, to the extent of about fourfold for BER and tenfold for NER. There was no correlation between BER and NER rates. The BER and NER assays are simple to perform and can provide valuable information in molecular epidemiological studies in which DNA instability is an endpoint.

The comet assay is a sensitive method for measuring DNA strand breaks in eukaryotic cells. After embedding in agarose, cells are lysed and electrophoresed at high pH. DNA loops containing breaks (in which supercoiling is relaxed) escape from the nucleoid comet head to form a tail. Oligonucleotide probes were designed for 5' and 3' regions of the genes for dihydrofolate reductase (DHFR) and O6-methylguanine DNA methyltransferase (MGMT), both from the Chinese hamster, and the human tumour suppressor p53 gene. Alternate ends were labelled with either biotin or fluorescein. These probes were hybridized to the DNA of comets from Chinese hamster ovary (CHO) cells or human lymphocytes treated with H2O2 or photosensitizer plus light to induce oxidative damage. Amplification with Texas red- and fluorescein-tagged antibodies led, in the case of p53 in human cells, to red and green signals located in the comet tail (as well as in the head), indicating the presence of breaks in the vicinity of the gene. However, only one end of the MGMT gene appeared in the tail and almost no signals from the DHFR gene, either red or green, were in the tail of comets from CHO cells. Restriction on movement from the head to tail may result from the presence of a 'matrix-associated region' in the gene. The kinetics of repair of oxidative damage were followed; strand breaks in the p53 gene were repaired more rapidly than total DNA. Thus, fluorescent in situ hybridization in combination with the comet assay provides a powerful method for studying repair of specific genes in relation to chromatin structure.

24,564 autopsy protocols from the central city hospital and oncology dispensary were analyzed in order to evaluate the rate and dynamics of postoperative thrombotic complications (PTC). The PTC rate was 14.89% and went up two-fold within the recent 20 years. The rate of pulmonary thromboembolism (PTE) was 10.13%; it increased during the recent 20 years from 6.75% to 17.30%. No significant difference was registered between the PTE rates in oncology and other patients (16.01% and 14.10%, respectively). Pelvic vein thrombosis was defined as a source of PTE in 50.11% of cases; deep vein thrombosis was found only in 28.54% of cases. The rate of the in-life PTE diagnosis was 42.8%. Despite refinements and standardization related with the use of anticoagulants, prevention and PTC management, the discussed phenomenon still remains a crucial issue for practitioners.

Loss of heterozygosity (LOH) on the long arm of human chromosome 16 is a common genetic alteration observed in both invasive ductal and invasive lobular breast carcinomas. We have generated a high-resolution integrated map encompassing the smallest region of LOH overlap within chromosome 16q22.1 (SRO2). Southern hybridization experiments using more than 140 probes resulted in the assembly of 152 bacterial large-insert clones into a 2.8-Mb contig covering SRO2. The structure of the contig was verified by long-range mapping using total human genomic DNA, and the contig orientation was determined by fluorescence in situ hybridization. A total of 68 transcripts have been identified in the map. One of the genes residing within SRO2 is the E-cadherin gene, CDH1, which has previously been shown to be mutated in lobular breast carcinomas, resulting in loss of E-cadherin expression. In most cases of ductal carcinoma, which is the major mammary cancer type, E-cadherin is normally expressed, suggesting that other genes within 16q22.1 are involved in the development of this tumor subtype. The high-resolution map presented in this study provides a valuable resource for identification of tumor suppressor genes expected to be involved in the etiology of breast carcinomas.