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Fernandez J.L.,Complejo Hospitalario Universitario runa CHUAC | Fernandez J.L.,Centro Oncologico Of Galicia
Journal of Theoretical Biology

Two procedures for detection of Sister-Chromatid Exchanges (SCEs) in interphase cells within specific DNA sequence areas are delineated. Proliferating cells cultured with 5-bromodeoxyuridine (BrdU) for one cell cycle are subjected to cytokinesis-block to produce binucleated cells. Then, the BrdU-substituted DNA strands are removed as in the chromosome orientation-FISH (CO-FISH) procedure. In one case, the intact unsubstituted DNA strands may be hybridized with differentially labelled direct and reverse single-stranded DNA (ssDNA) probes from the target area. After formation of a SCE during the first cycle, specifically within the target area, three signals should be visualized in each nucleus of the binucleated cell. Two of them, of different color, must appear colocalized or adjacent simultaneously in both sister nuclei, corresponding to the SCE site, being accompanied by a signal from the other homologous locus showing a complementary color pattern in both sister nuclei. Other possibility is hybridizing two separated direct ssDNA probes flanking the target sequence area labelled with the same color and the two reverse complementary ssDNA probes labelled with other color. The presence of three signals with similar color and one of different color in each nucleus from the binucleated cell, following a complementary color pattern between both nuclei, would indicate the presence of a SCE within the DNA sequence area flanked by the DNA probes. The availability of complementary ssDNA probes specific for single locus should make the Interphase SCE-FISH a suitable procedure. © 2012 Elsevier Ltd. Source

Tamayo M.,Complejo Hospitalario Universitario runa CHUAC | Tamayo M.,Centro Oncologico Of Galicia | Santiso R.,Complejo Hospitalario Universitario runa CHUAC | Santiso R.,Centro Oncologico Of Galicia | And 5 more authors.
Archives of Microbiology

Lysostaphin digestion of peptidoglycan (PG) from Staphylococcus aureus resulted in chromosomal DNA fragmentation by released DNase, as directly visualized in situ on isolated nucleoids. Nevertheless, DNA digestion was partially prevented by previous incubation with antibiotics that inhibit PG synthesis. This inhibitory effect was much more remarkable with glycopeptides vancomycin and mainly teicoplanin than with beta-lactams cloxacillin and ceftazidime. Therefore, inhibition of PG chain elongation has a more significant inhibition of DNA degradation than inhibition of PG cross-linking, possibly due to a reduction in DNase storage at the cell wall. © Springer-Verlag 2012. Source

Santiso R.,Centro Oncologico Of Galicia | Santiso R.,Complejo Hospitalario Universitario runa CHUAC | Tamayo M.,Centro Oncologico Of Galicia | Tamayo M.,Complejo Hospitalario Universitario runa CHUAC | And 7 more authors.
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis

Sperm DNA fragmentation (SDF) is not a static seminal parameter, since the longevity of sperm DNA decreases progressively with time following ejaculation or thawing. While the dynamics of SDF is a species-specific characteristic, in the case of humans, there is still significant variation within patients. To evaluate the suitability of the dynamic SDF assay to assess the adverse effects of agents that cause genetic damage, fresh semen samples from different donors were exposed in vitro to (1) increasing acute doses of ionizing radiation, (2) elevated temperature (41°C and 45°C), (3) acidic pH (pH 4) and (4) the nitric oxide (NO) donor sodium nitroprusside (SNP). Sperm DNA fragmentation was analyzed after an incubation period of chronic (24. h), or acute (1. h) exposure to each treatment followed by incubation at 37°C over a period of 24. h. SDF was assessed using the sperm chromatin dispersion (SCD) test. Dynamic SDF for each treatment was analyzed using Kaplan-Meier survival curves. All agents, except for ionizing radiation, accelerated SDF kinetics following chronic exposure over a 24. h period. Transient exposure to NO and heat but not acidic pH increased the basal (T0) level of SDF. Despite the removal of the three toxicants, the remaining sperm following acute exposure showed a decrease in their expected DNA longevity. It is concluded that the assessment of sperm DNA fragmentation dynamics is an effective methodological approach for revealing latent damage associated with toxicants that is not initially expressed following a single initial observation of SDF. © 2012 Elsevier B.V.. Source

Bou G.,Complejo Hospitalario Universitario runa CHUAC | Otero F.M.,Complejo Hospitalario Universitario runa CHUAC | Otero F.M.,Centro Oncologico Of Galicia | Santiso R.,Complejo Hospitalario Universitario runa CHUAC | And 8 more authors.
Journal of Clinical Microbiology

Infections caused by multidrug-resistant Acinetobacter baumannii constitute a major life-threatening problem worldwide, and early adequate antibiotic therapy is decisive for success. For these reasons, rapid detection of antibiotic susceptibility in this pathogen is a clinical challenge. Two variants of the Micromax kit were evaluated for a rapid detection in situ of susceptibility or resistance to meropenem or ciprofloxacin, separately, in 322 clinical isolates. Release of the nucleoid is the criterion of susceptibility to the beta-lactams (carbapenems), whereas diffusion of DNA fragments emerging from the nucleoid characterizes the quinolone activity. All the susceptible and resistant strains were correctly categorized in 100 min according to the MIC results and CLSI criteria. Thus, our technology is a promising tool for rapid identification of carbapenem and quinolone resistance of A. baumannii strains in hospital settings. Copyright © 2012, American Society for Microbiology. All Rights Reserved. Source

Tamayo M.,Complejo Hospitalario Universitario runa CHUAC | Tamayo M.,Centro Oncologico Of Galicia | Mosquera A.,Complejo Hospitalario Universitario runa CHUAC | Rego I.,Complejo Hospitalario Universitario runa CHUAC | And 4 more authors.
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis

Length of telomeric DNA sequences and numerical chromosome aberrations from uncultured human osteoarthritic (OA) articular chondrocytes were compared with those from peripheral blood leukocytes (PBL) from the same individual and from chondrocytes and PBL from control subjects. Cells were both obtained from 39 OA patients (age range: 43-80 years) and from 20 control subjects (age range: 39-94 years). Mean length of telomeric DNA sequences was determined using a quantitative real-time polymerase chain reaction (qPCR) assay and numerical chromosome aberrations were identified in interphase nuclei by Fluorescence In Situ Hybridization (FISH) using cocktails of specific DNA probes for chromosomes 7, 8 and for 18, X and Y. Chondrocytes revealed higher telomere size than PBL, both in control subjects and in OA patients, being 2 and 1.6 times higher respectively, thus revealing cell type specific differences. However, chondrocytes from OA patients showed significantly shorter telomere size than chondrocytes from control subjects (T/S ratio 1.64 ± 0.41 vs. 1.99 ± 0.54; mean ± sd; p=0.008). Regarding the percentage of numerical chromosome aberrations, OA chondrocytes showed 1.7 times higher than chondrocytes from control subjects (19.80 ± 3.31 vs.11.48 ± 4.11; p<0.01) and 1.5 times average higher than that from PBL from the own OA patient (13.06 ± 1.45; p<0.001). Moreover, PBL from OA patients also showed 1.4 times more anomalies than PBL from controls (13.06 ± 1.45 vs. 9.54 ± 1.61; p<0.001). No significant differences were found between chondrocytes and PBL in control subjects. Chromosome loss was the more frequent aneuploidy, mainly monosomy 18. The decreased telomere size and increased chromosome instability in chondrocytes from OA affected joints may imply a local advanced senescence that could contribute to the pathogenesis or progression of the degenerative articular disease. Moreover, the increased chromosomal abnormalities in PBL from OA patients suggest a more general accelerated senescence phenotype that could promote the age-related degenerative joint pathology. © 2011 Elsevier B.V. Source

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