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Formalin-fixed, paraffin-embedded (FFPE) samples are generally used for histology-study, however, they also possess important molecular diagnostics information. While it has been reported that the N-glycan moieties of glycoproteins is not affected by the FFPE process, no information is available about the effect of the elapsed time between sampling and fixation on the resulting N-glycosylation profile. In this study, lung, brain, heart, spleen, liver, kidney, and intestine mouse tissue specimens were used for N-glycan profiling analysis and the elapsed sampling time effect was investigated with the lung tissue. N-glycan extraction from the tissue samples was performed by glycoprotein retrieval from the FFPE specimens using radioimmunoprecipitation assay (RIPA) buffer followed PNGase F digestion. The released oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis-laser induced fluorescent detection (CE-LIF). N-glycosylation profiles of freshly collected lung-tissue samples (zero time point), as well as 1 and 2 h after sampling were compared by carbohydrate profiling and exoglycosidase treatment based deep glycomic analysis. It was found that up to two hours of room temperature storage of tissue specimens apparently did not cause changes in the N-glycosylation profiles of complex carbohydrates, but resulted in considerable decrease in the amount of linear glucose oligomers and high mannose type glycans present in the samples., (© The Authors. Electrophoresis Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

In early S phase the newly replicated DNA is folded back to increasingly compact structures. The process of chromatin condensation inside the nucleus starts with the formation of a micronucleus observed in five established cell lines (K562, CHO, Indian muntjac, murine preB and SCC). Supercoiling of chromatin generates a polarized end-plate region extruded from the nucleus. The extruded chromatin is turned around itself forming the head portion (micronucleus) visible by fluorescence microscopy until the middle of S phase when chromatin structures are succeeded by distinguishable early forms of chromosomes. The generation of micronuclei upon apoptotic treatment was achieved by the methotrexate (MTX) treatment of cells. A close correlation was found between the frequency of micronucleus and MTX concentration, with low frequency at low (0.1 µM) and increasingly higher frequency between 1 and 100 µM concentrations. Characteristic deformation and shrinkage of nuclei indicated apoptosis. High MTX concentration (100 µM) caused the enlargement and necrotic disruption of nuclei. Inhibition of DNA synthesis during replicative DNA synthesis by biotinylated nucleotide prevented the formation of metaphase chromosomes and elevated the frequency of early intermediates of chromosome condensation including micronucleus formation. Based on these observations the micronucleus is regarded as: (a) a regularly occuring element of early chromatin condensation and (b) a typical sign of nuclear membrane damage under toxic conditions. Explanation is given why the micronucleus is hidden in nuclei under normal chromatin condensation and why chromatin motifs including micronuclei become visible upon cellular damage.

Project: Beside its useful functions at very low concentrations, selenium including supplementary Se sources pose a potential toxicological risk. The toxicity of selenium species was tested in HaCaT cell culture and related nephrotoxicity in mice., Procedure: The apoptotic shrinkage and necrotic expansion of cells were measured by time-lapse image microscopy. Acute nephrotoxicity was estimated upon administration of various selenium species to mice for two weeks. To confirm or to refute the accumulation of Se in the kidney and its potential chronic effect, Se concentration in kidney tissue and histopathlology were tested., Results: The comparison of selenium species showed that organic lactomicroSe did not affect cell growth at 5ppm, but inorganic nanoSe severely hampered it at lower concentration (1ppm). The in vivo Se treatment (0.5, 5, 50ppm, corresponding to 4, 40 and 400μg/kg) was misleading as it did neither affect the outward appearance nor the weight of the kidney. Se accumulation was observed after selenate, selenite, SelPlex, selenite and nanoSe administration, while lactomicroSe caused no traceable accumulation. In vivo, ex vivo and in vitro experiments reflected this order of selenium toxicity: selenate>selenite>SelPlex=nanoSe>lactomicroSe., Conclusion: Within the tested species lactomicroSe was the only non-nephrotoxic selenium source recommended for nutritional Se supplementation., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Stem cell line from human limbal area was established to study in vitro cell growth and response to the toxic effects of antibiotics used in ophthalmology in terms of cell migration rates and structure of interphase chromatin. Recovery from cellular damages caused by ophthalmologic antibiotics was mimicked by an in vitro scratch model and followed by time-lapse microscopy, scanning electronmicroscopy and chromatin image analysis. Experiments revealed that broad spectrum antibiotics, chloramphenicol (0.5-1.0mg/ml) and rifampicin (0.1-0.2mg/ml), corresponding to concentrations in common clinical practice, slowed down the regeneration process. Results show that nuclei of naturally occurring limbal cells contain the same intermediates of chromatin condensation as seen in mammalian tumor cells and follow the common pathway of chromosome condensation. These intermediates included decondensed veil-like chromatin, fibrillary chromatin, supercoiled ribbon, chromatin bodies, early linear forms and metaphase chromosomes. Upon chloramphenicol and rifampicin treatment characteristic distorsions took place in the intermediates of chromosome condensation. Damaging effects in limbal stem cells in the presence of chloramphenicol or rifampicin indicate that ophthalmologic treatment with antibiotics should be used cautiously., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Malignant transformation is likely to render cells hyperploid, primarily tetraploid. We have measured the frequency of division into three rather than two daughter cells as a function of ploidy. Such trivisions were followed in near-tetraploid uveal melanoma (UM), hypotetraploid HaCaT (<4 N), hypertriploid HeLa (>3 N), and in near-diploid (∼2 N) lung epithelial cell lines by time-lapse image analyses. A stepwise analysis of cytokinesis revealed higher frequency of cell trivisions relative to divisions in hyperploid HeLa (1:24, 4%), HaCaT (1:126, 8%), and UM (1:186, 0.5%) cells. The occurrence of trivision was significantly lower in near-diploid endothelial cells (1:1400, 0.07%). We have previously observed the phenomenon of trivision in HaCaT cells treated with heavy metal lead, and here we describe that trivision is a spontaneous process taking place without genotoxic treatment. Beside re-diploidization by trivision, the hyperploid state decreases the cell size of the daughter cells and is likely to increase the time of cytokinesis. On the basis of the results, it is hypothesized that among other cancer-related causes, hyperploidy could be related to cell trivision, could cause random aneuploidy, and could generate new cancer-specific karyotypes.