Danio rerio, commonly known as zebrafish, is an established model organism for the developmental and cell biology studies. Although significant progress has been made in the analysis of the D. rerio genome, cytogenetic studies face challenges due to the unclear identification of chromosomes. Here, we present a novel approach to the study of the D. rerio karyotype, focusing on the analysis of lampbrush chromosomes isolated from growing oocytes. Lampbrush chromosomes, existing during diplotene, serve as a powerful tool for high-resolution mapping and transcription analysis due to their profound decondensation and remarkable lateral loops decorated by RNA polymerases and ribonucleoprotein (RNP) matrix. In D. rerio, lampbrush chromosomes are about 20 times longer than corresponding metaphase chromosomes. We found that the lampbrush chromosome stage karyotype of D. rerio is generally undifferentiated, except for several bivalents bearing distinct marker structures, including loops with complex RNP matrix and locus-associated nuclear bodies. Locus-associated nuclear bodies were enriched for coilin and snRNAs; the loci where they formed presumably correspond to the histone gene clusters. Further, we observed the accumulation of splicing factors in giant terminal RNP aggregates on one bivalent. DAPI staining of Danio rerio lampbrush chromosomes revealed large and small chromomeres non-uniformly distributed along the axis. For example, D. rerio lampbrush chromosome 4, comprising the sex-determining region, is divided into two halves-with small chromomeres bearing long lateral loops and with large dense chromomeres bearing no or very tiny lateral loops. As centromeres were not distinguishable, we identified centromeric regions in all bivalents by FISH mapping of pericentromeric RFAL1, RFAL2, and RFAM tandem repeats. Through a combination of morphological analysis, immunostaining of marker structures, and centromere mapping, we developed cytological maps of D. rerio lampbrush chromosomes. Finally, by RNA FISH we revealed transcripts of pericentromeric and telomeric tandem repeats at the lampbrush chromosome stage.

• Klíčová slova
• Centromere, FISH-mapping, Germinal vesicle, Histone locus bodies, Karyotype analysis, Lampbrush chromosomes, Non-coding RNA, Oocyte nucleus, Tandem repeats, Telomere, Zebrafish,
• MeSH
• chromozomy * MeSH
• dánio pruhované * genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• karyotyp MeSH
• karyotypizace MeSH
• oocyty metabolismus   MeSH
• ribonukleoproteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ribonukleoproteiny MeSH

This study involved creating oligomeric conjugates of 3-hydroxy fatty acids and diclofenac, named Dic-oligo(3HAs). Advanced NMR techniques confirmed no free diclofenac in the mix. We tested diclofenac release under conditions resembling healthy and chronic wound skin. These oligomers were used to make P(3HO) blends, forming patches for drug delivery. Their preparation used the solvent casting/porogen leaching (SCPL) method. The patches' properties like porosity, roughness, and wettability were thoroughly analysed. Antimicrobial assays showed that Dic-oligo(3HAs) exhibited antimicrobial activity against reference (S. aureus, S. epidermis, S. faecalis) and clinical (Staphylococcus spp.) strains. Human keratinocytes (HaCaT) cell line tests, as per ISO 10993-5, showed no toxicity. A clear link between material roughness and HaCaT cell adhesion was found. Deep cell infiltration was verified using DAPI and phalloidin staining, observed under confocal microscopy. SEM also confirmed HaCaT cell growth on these scaffolds. The strong adhesion and proliferation of HaCaT cells on these materials indicate their potential as wound dressing layers. Additionally, the successful diclofenac release tests point to their applicability in treating both normal and chronic wounds.

• Klíčová slova
• Artificial skin substitute, bioresorbable, porous layer of dressing material, Poly(3-hydroxyoctanoate), Polyhydroxyalkanoates, Targeted delivery of diclofenac,
• MeSH
• biokompatibilní materiály chemie   farmakologie   MeSH
• buněčné linie keratinocytů HaCaT MeSH
• buněčné linie MeSH
• chemické jevy MeSH
• diklofenak * farmakologie   chemie   MeSH
• hojení ran účinky léků   MeSH
• keratinocyty účinky léků   cytologie   MeSH
• kůže * účinky léků   MeSH
• lidé MeSH
• polymery chemie   MeSH
• poréznost MeSH
• proliferace buněk účinky léků   MeSH
• regenerace účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Hybrid taxa from the genus Pelophylax can propagate themselves in a modified way of sexual reproduction called hybridogenesis ensuring the formation of clonal gametes containing the genome of only one parental (host) species. Pelophylax grafi from South-Western Europe is a hybrid composed of P. ridibundus and P. perezi genomes and it lives with a host species P. perezi (P-G system). Yet it is unknown, whether non-Mendelian inheritance is fully maintained in such populations. In this study, we characterize P. perezi and P. grafi somatic karyotypes by using comparative genomic hybridization, genomic in situ hybridization, fluorescent in situ hybridization, and actinomycin D-DAPI. Here, we show the homeology of P. perezi and P. grafi somatic karyotypes to other Pelophylax taxa with 2n = 26 and equal contribution of ridibundus and perezi chromosomes in P. grafi which supports F1 hybrid genome constitution as well as a hemiclonal genome inheritance. We show that ridibundus chromosomes have larger regions of interstitial (TTAGGG)n repeats flanking the nucleolus organizing region on chromosome no. 10 and a high quantity of AT pairs in the centromeric regions. In P. perezi, we found species-specific sequences in metaphase chromosomes and marker structures in lampbrush chromosomes. Pericentromeric RrS1 repeat sequence was present in perezi and ridibundus chromosomes, but the blocks were stronger in ridibundus. Various cytogenetic techniques applied to the P-G system provide genome discrimination between ridibundus and perezi chromosomal sets. They could be used in studies of germ-line cells to explain patterns of clonal gametogenesis in P. grafi and broaden the knowledge about reproductive strategies in hybrid animals.

• Klíčová slova
• Pelophylax grafi, Pelophylax perezi, comparative genomic hybridization, fluorescent in situ hybridization, hybridogenesis, karyotype,
• MeSH
• centromera * genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• karyotypizace MeSH
• Ranidae * genetika   MeSH
• srovnávací genomová hybridizace MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided.

• Klíčová slova
• Best practices, DAPI, DNA-ploidy level, Desiccated tissues, Flow cytometry, Genome size, Plant nuclei isolation, Plant tissues, Propidium iodide, Seeds,
• MeSH
• buněčné jádro * genetika   chemie   MeSH
• délka genomu MeSH
• DNA rostlinná genetika   analýza   MeSH
• genom rostlinný MeSH
• ploidie MeSH
• průtoková cytometrie metody   MeSH
• rostliny * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH

We explored the mechanism of human osteosarcoma MG-63 cell apoptosis induced by asta-xanthin. The MTT assay was used to detect the effect of astaxanthin on cell viability. Morphological changes associated with apoptosis were observed after DAPI staining. Early and late stages of apoptosis were detected by flow cytometry with annexin V-FITC/PI staining. Activation of caspases-8, -9 and -3 was detected by enzyme activity in vitro. Changes in the mitochondrial membrane potential were detected by MitoCapture staining. Western blot was used to detect the cleavage of PARP, which is a caspase-3 substrate, the release of cytochrome c and Smac into the cytosol, the translocation of pro-apoptotic proteins Bax and Bak, and the expression of mitochondrial pathway-related proteins. The translocation of Bax was also detected by immunofluorescence assay. Astaxanthin significantly inhibited the viability of human osteosarcoma MG-63 cells with an IC50 value of 12.36 μg/ml. The DAPI-stained cells showed characteristic apoptotic morphological changes - cell shrinkage, cell membrane blebbing, nuclear condensation, and apoptotic body formation. Cytochrome c and Smac were released from mitochondria to the cytosol. Pro-apoptotic proteins Bax and Bak were rapidly translocated to mitochondria after six hours of astaxanthin action. Caspases-9 and -3 were activated and PARP was cleaved. The expression of anti-apoptotic proteins Bcl-2, Bcl-xL and XIAP was significantly decreased. Astaxanthin induced human osteosarcoma MG-63 cell apoptosis through the mitochondria-mediated endogenous apoptosis pathway.

• Klíčová slova
• MG-63 cells, apoptosis, astaxanthin, cytochrome c, mitochondria,
• MeSH
• apoptóza MeSH
• cytochromy c * metabolismus   MeSH
• lidé MeSH
• osteosarkom * farmakoterapie   metabolismus   MeSH
• PARP inhibitory farmakologie   terapeutické užití   MeSH
• protein X asociovaný s bcl-2 metabolismus   MeSH
• proteiny regulující apoptózu farmakologie   terapeutické užití   MeSH
• protoonkogenní proteiny c-bcl-2 metabolismus   MeSH
• xanthofyly MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• astaxanthine MeSH Prohlížeč
• cytochromy c * MeSH
• PARP inhibitory MeSH
• protein X asociovaný s bcl-2 MeSH
• proteiny regulující apoptózu MeSH
• protoonkogenní proteiny c-bcl-2 MeSH
• xanthofyly MeSH

(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.

• Klíčová slova
• ImageJ, bioimage analysis, genotoxicity, high-throughput, in vitro testing,
• MeSH
• biologické markery analýza   MeSH
• HeLa buňky MeSH
• lidé MeSH
• mikroskopie * MeSH
• pilotní projekty MeSH
• poškození DNA * MeSH
• reprodukovatelnost výsledků MeSH
• valinomycin toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• valinomycin MeSH

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

• Klíčová slova
• 3D organization, Nucleus, RNA Pol II, SIM, STED imaging, chromatin, chromosome, crossovers, image analysis, meiosis, metaphase, mitosis, nuclear bodies, nuclear speckles, oligo FISH, pachytene, quantification, segmentation, spatial distribution, transcription factories,
• MeSH
• buněčné jádro * MeSH
• chromatin * MeSH
• fluorescenční mikroskopie MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• průběh práce MeSH
• zelené fluorescenční proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin * MeSH
• zelené fluorescenční proteiny MeSH

Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.

• Klíčová slova
• Aegilops biuncialis, Aegilops geniculata, chromosome flow sorting, flow karyotyping, genome dissecting,
• Publikační typ
• časopisecké články MeSH

• MeSH
• barvení a značení MeSH
• buněčné jádro * MeSH
• fluorescenční barviva MeSH
• fluorescenční protilátková technika MeSH
• indoly * MeSH
• lidé MeSH
• propidium MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DAPI MeSH Prohlížeč
• fluorescenční barviva MeSH
• indoly * MeSH
• propidium MeSH

BACKGROUND AND AIMS: In eukaryotes, the total kinetochore size (defined as a chromosomal region containing CENH3-positive nucleosomes) per nucleus strongly correlates with genome size, a relationship that has been hypothesized to stem from general intracellular scaling principles. However, if larger chromosomes within a karyotype required larger kinetochores to move properly, it could also be derived from the mechanics of cell division. METHODS: We selected seven species of the plant subfamily Agavoideae whose karyotypes are characterized by the presence of small and very large chromosomes. We visualized the kinetochore regions and chromosomes by immunolabelling with an anti-CENH3 antibody and DAPI (6'-diamidino-2-phenylindole) staining. We then employed 2D widefield and 3D super-resolution microscopy to measure chromosome and kinetochore areas and volumes, respectively. To assess the scaling relationship of kinetochore size to chromosome size inside a karyotype, we log-transformed the data and analysed them with linear mixed models which allowed us to control for the inherent hierarchical structure of the dataset (metaphases within slides and species). KEY RESULTS: We found a positive intra-karyotype relationship between kinetochore and chromosome size. The slope of the regression line of the observed relationship (0.277 for areas, 0.247 for volumes) was very close to the theoretical slope of 0.25 for chromosome width based on the expected physics of chromosome passage through the cytoplasm during cell division. We obtained similar results by reanalysing available data from human and maize. CONCLUSIONS: Our findings suggest that the total kinetochore size to genome size scaling observed across eukaryotes may also originate from the mechanics of cell division. Moreover, the potential causal link between kinetochore and chromosome size indicates that evolutionary mechanisms capable of leading kinetochore size changes to fixation, such as centromere drive, could promote the size evolution of entire chromosomes and genomes.

• Klíčová slova
• Asparagaceae, cell division, centromere, chromosome size evolution, genome size evolution, intracellular scaling, linear mixed models, structured illumination microscopy,
• MeSH
• buněčné dělení MeSH
• centromera * genetika   MeSH
• karyotyp MeSH
• karyotypizace MeSH
• kinetochory * MeSH
• rostliny * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH