Luděk Strašák

Epigenome and chromatin structure in human embryonic stem cells undergoing differentiation

Epigenetic histone (H3) modification patterns and the nuclear radial arrangement of select genetic elements were compared in human embryonic stem cells (hESCs) before and after differentiation. H3K9 acetylation, H3K9 trimethylation, and H3K79 monomethylation were reduced at the nuclear periphery of differentiated hESCs. Differentiation coincided with centromere redistribution, as evidenced by perinucleolar accumulation of the centromeric markers CENP‐A and H3K9me3, central repositioning of centromeres 1, 5, 19, and rearrangement of other centromeres at the nuclear periphery. The radial positions of PML, RARα genes, and human chromosomes 10, 12, 15, 17, and 19 remained relatively stable as hESCs differentiated. However, the female inactive H3K27‐trimethylated X chromosome occupied a more peripheral nuclear position in differentiated cells. Thus, pluripotent and differentiated hESCs have distinct nuclear patterns of heterochromatic structures (centromeres and inactive X chromosome) and epigenetic marks (H3K9me3, and H3K27me3), while relatively conserved gene density‐related radial chromatin distributions are already largely established in undifferentiated hES cells. Developmental Dynamics 237:3690–3702, 2008.

50 Hz magnetic field effect on the morphology of bacteria

Atomic force microscopy was used to distinguish changes in morphology of bacteria induced by 50 Hz 10 mT magnetic field exposure. It is known that alternating magnetic field exposure causes decrease of viability of different bacterial strains. Previously we found that the viability of rod-like bacteria exposed to magnetic field decreased twice more in comparison with the spherical ones. Motivated by this fact we carried out this study with bacterial cells of both shapes. We used Escherichia coli (rod-like) and Paracoccus denitrificans (spherical) bacteria. As a result we have not observed any change in bacterial morphology neither of rod-like nor of spherical bacteria after 1 h, 50 Hz and 10 mT magnetic field exposure.

H3K9 acetylation and radial chromatin positioning

Histone variants and their epigenetic modifications determine genome function, particularly transcription. However, whether regulation of gene expression can be influenced by nuclear organization or vice versa is not completely clear. Here, we analyzed the effect of epigenetic changes induced by a histone deacetylase inhibitor (HDACi) on the nuclear radial rearrangement of select genomic regions and chromosomes. The HDACi, sodium butyrate (NaBt), induced differentiation of human adenocarcinoma HT29 cells as well as a genome‐wide increase in H3K9 acetylation. Three‐dimensional analysis of nuclear radial distributions revealed that this increase in H3K9 acetylation was often associated with a repositioning of select loci and chromosomes toward the nuclear center. On the other hand, many centromeres resided sites more toward the nuclear periphery, similar to sites occupied by chromosome X. In more than two‐thirds of events analyzed, central nuclear positioning correlated with a high level of H3K9 acetylation, while more peripheral positioning within interphase nuclei correlated with a lower level of acetylation. This was observed for the gene‐rich chromosomes 17 and 19, TP53, and CCND1 genes as well as for gene‐poor chromosome 18, APC gene, regions of low transcriptional activity (anti‐RIDGEs), and the relatively transcriptionally less active chromosome X. These results are consistent with a role for epigenetic histone modifications in governing the nuclear radial positioning of genomic regions during differentiation. J. Cell. Physiol. 220: 91–101, 2009.

Effects of 50 Hz Magnetic Fields on the Viability of Different Bacterial Strains

The effect of a 50 Hz magnetic field on the growth of different bacterial strains was studied. We used bacteria with different cell shapes. We compared rod-shaped bacteria (E. coli, L. adecarboxylata) and spherical bacteria (S. aureus, P. denitrificans, S. paucimobilis, R. erythropolis). The growth curves of control and magnetic fields exposed samples were measured. The cylindrical coil induced magnetic fields with inductions up to 10 mT. Duration of exposure varied up to 24 min. Exposure took place at laboratory temperature (24–26°C) and the air ventilator maintained the temperature of sample. We observed the decrease of optical densities in exposed samples. The magnetic field effect was bigger for rod-like bacteria. We concluded that magnetic field effects depend on the shape of exposed cells.

Single-cell c-myc gene expression in relationship to nuclear domains.

Nuclear locations of the c-myc gene and its transcripts (c-mycT) have been investigated in relation to nuclear domains involved in RNA synthesis and processing. Transcription of the c-myc gene appears to be linked to the late G1- and preferentially to S-phases of the cell cycle. The c-myc gene and its transcripts were positioned non-randomly within the interphase nucleus; additionally, c-myc RNA signals accumulated at nucleoli. Using oligo-probes, designed to exon II and exon III of the c-myc gene, single c-mycT was preferentially observed in human carcinoma HT29 and A549 cells. Conversely, human embryonal teratocarcinoma NTERA cells were characterized by the presence of multiple c-myc RNA signals located in both the nucleoli and nucleoplasm. When accumulated at nucleoli, c-mycT occupied the periphery of this organelle, though not those associated with the cultivation surface. In HT29 cells, approximately 80% of c-mycT co-localized with the RNAP II positive regions, so-called transcription factories. However, in ∼20% of the cells with c-myc transcripts, the c-mycT was released from the site of synthesis, and was not associated with either transcription factories or SC35 domains. In ∼60% of nuclei with c-mycT, these signals were located in close proximity to the SC35 regions, but promyelocytic leukaemia bodies were associated with c-mycT only in ∼20% of the nuclei. Taken together, c-myc RNA signals were positioned in the most internal parts of the cell nuclei preferentially associated with the nucleoli. Specific nuclear and nucleolar positioning probably reflects the kinetics of c-myc RNA metabolism.

Extremely-Low Frequency Magnetic Field Effects on Sulfate Reducing Bacteria Viability

50 Hz magnetic fields effects on Sulfate Reducing Bacteria (SRB) viability were studied by colony forming units (CFU) counting. We found a 15% decrease of CFU number after magnetic field exposure (B=7.1 mT, f=50 Hz, t=24 min) compared to the control samples. These results are in good agreement with our previous work on other bacterial strains. The magnetic field effects on SRB are relatively large for small magnetic fields. The data correlations have been subjected to a simple physical chemical analysis, yielding surprisingly large estimates for the characteristic magnetic reaction susceptibility, even when the entire bacterium is assumed to be the direct target of interaction of the magnetic ac fields for the exposures in the time range from 3–24 min.

Fibrinogen and cellular adherability on differently treated titanium as implants

Adsorption of human plasma fibrinogen, osteoblasts, and fibroblasts on differently treated titanium samples as implants were examined in this study. Titanium samples were mechanically polished, chemically etched (with and without surface material loss), and grinded. The main goal of this study is to find the best surface treatment of titanium for its possible use as implants. Atomic force microscopy was used to evaluate the adsorption of human plasma fibrinogen onto the titanium samples. Cell counting was used to determine the adherability of osteoblasts and fibroblasts on the titanium samples. Our preliminary results show that the etched titanium surface with surface material loss is the best surface treatment used in our experiments.