3D FISH
Dotaz
Zobrazit nápovědu
During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl's configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl's orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery.
- MeSH
- buněčné jádro MeSH
- chromatin genetika MeSH
- chromozomy rostlin * MeSH
- hybridizace in situ fluorescenční * metody MeSH
- interfáze * genetika MeSH
- ječmen (rod) genetika MeSH
- počítačové zpracování obrazu MeSH
- průtoková cytometrie MeSH
- pšenice genetika MeSH
- žito genetika MeSH
- Publikační typ
- časopisecké články MeSH
The consideration of human and environmental exposure to dendrimers, including cytotoxicity, acute toxicity, and cell and tissue accumulation, is essential due to their significant potential for various biomedical applications. This study aimed to evaluate the biodistribution and toxicity of a novel methoxyphenyl phosphonium carbosilane dendrimer, a potential mitochondria-targeting vector for cancer therapeutics, in 2D and 3D cancer cell cultures and zebrafish embryos. We assessed its cytotoxicity (via MTT, ATP, and Spheroid growth inhibition assays) and cellular biodistribution. The dendrimer cytotoxicity was higher in cancer cells, likely due to its specific targeting to the mitochondrial compartment. In vivo studies using zebrafish demonstrated dendrimer distribution within the vascular and gastrointestinal systems, indicating a biodistribution profile that may be beneficial for systemic therapeutic delivery strategies. The methoxyphenyl phosphonium carbosilane dendrimer shows promise for applications in cancer cell delivery, but additional studies are required to confirm these findings using alternative labelling methods and more physiologically relevant models. Our results contribute to the growing body of evidence supporting the potential of carbosilane dendrimers as vectors for cancer therapeutics.
- MeSH
- dánio pruhované MeSH
- dendrimery * toxicita MeSH
- lidé MeSH
- nádory * farmakoterapie MeSH
- techniky 3D buněčné kultury MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We have explored the potential relationship between ploidy level, DNA content (pg DNA nucleus(-1)), and dimensional characteristics, such as volume (μm(3)), surface area (μm(2)), and 3-D structure of erythrocyte nuclei in a series of fish ploidy level models using Feulgen image analysis densitometry, flow cytometry, and confocal laser scanning microscopy. The species were diploid tench (Tinca tinca) (2n), Cuban gar (Atractosteus tristoechus) (2n), triploid tench (3n), evolutionary tetraploid sterlet (Acipenser ruthenus) (4n), evolutionary octaploid Siberian sturgeon (A. baerii) (8n), triploid Siberian sturgeon exhibiting dodecaploidy (12n), evolutionary 12n shortnose sturgeon (A. brevirostrum), and experimentally obtained sturgeon hybrids that were tetraploid, hexaploid (6n), heptaploid (7n), octaploid, decaploid (10n), dodecaploid and/or tetradecaploid (14n). Increase in ploidy was accompanied by growth of the nucleus and an increase in the number of flattened ellipsoid nuclei with increased transverse diameter. The volume (Vvoxel ) of erythrocyte nuclei, as the sum of voxels calculated from live cells, seems more accurate than volume (Vaxis ) calculated from measuring the major and minor axis, especially at higher and odd ploidy levels. Data of absolute and relative DNA content were in agreement with previously published reports. Species of the same ploidy level, but differing in DNA content, had a similar mean erythrocyte nuclear volume (Vvoxel ), as demonstrated in sterlet and a hybrid of sterlet and beluga (48.3 and 48.9 μm(3), respectively), with a respective mean DNA content of 3.74 and 3.10 pg DNA nucleus(-1). A similar relationship was found for the ploidy 6n, 10n, 12n. The surface-to-volume ratio decreased non-linearly with increasing ploidy. The DNA in erythrocyte nuclei appeared to be more densely packed with increase in ploidy level.
- MeSH
- buněčné jádro genetika fyziologie MeSH
- Cyprinidae genetika MeSH
- DNA analýza MeSH
- erytrocyty cytologie MeSH
- polyploidie MeSH
- zobrazování trojrozměrné MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This article compares two bioconcentration Quantitative Structure Activity Relationships (QSARs) for fish applied in human risk assessments with the mechanistic bioaccumulation model OMEGA and field data. It was found that all models are virtually similar up to a Kow of 10(6). For substances with a Kow higher than 10(6), the fish bioconcentration curve in the risk assessment model EUSES decreases parabolically. In contrast, OMEGA bioaccumulation outcomes approximately show a linear increase, based on mechanistic bioconcentration and biomagnification properties of chemicals. The OMEGA-outcomes are close to the fish bioconcentration outcomes of the risk assessment model CalTOX. For very hydrophobic substances, field accumulation data in freshwater and marine fish species are closer to OMEGA- and CalTOX-outcomes compared to EUSES. The results also show that it is important to include biomagnification in fish and lipid content of fish in human exposure models.
- MeSH
- biologické modely MeSH
- chemické látky znečišťující vodu metabolismus MeSH
- ekologie MeSH
- hodnocení rizik MeSH
- hydrofobní a hydrofilní interakce MeSH
- kvantitativní vztahy mezi strukturou a aktivitou MeSH
- lidé MeSH
- metabolismus lipidů MeSH
- oceány a moře MeSH
- ryby metabolismus MeSH
- sladká voda MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
- Geografické názvy
- oceány a moře MeSH
RNAscope® technology provided by Advanced Cell Diagnostics (ACD) allows the detection and evaluation of coinciding mRNA expression profiles in the same or adjacent cells in unprecedented quantitative detail using multicolor fluorescent in situ hybridization (FISH). While already extensively used in thinly sectioned material of various pathological tissues and, to a lesser extent, in some whole mounts, we provide here a detailed approach to use the fluorescent RNAscope method in the mouse inner ear and thick brain sections by modifying and adapting existing techniques of whole mount fluorescent in situ hybridization (WH-FISH). We show that RNAscope WH-FISH can be used to quantify local variation in overlaying mRNA expression intensity, such as neurotrophin receptors along the length of the mouse cochlea. We also show how RNAscope WH-FISH can be combined with immunofluorescence (IF) of some epitopes that remain after proteinase digestion and, to some extent, with fluorescent protein markers such as tdTomato. Our WH-FISH technique provides an approach to detect cell-specific quantitative differences in developing and mature adjacent cells, an emerging issue revealed by improved cellular expression profiling. Further, the presented technique may be useful in validating single-cell RNAseq data on expression profiles in a range of tissue known or suspected to have locally variable mRNA expression levels.
- MeSH
- fluorescenční protilátková technika metody MeSH
- hybridizace in situ fluorescenční MeSH
- kochlea metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- neurotrofin 3 metabolismus MeSH
- receptor trkB genetika metabolismus MeSH
- receptor trkC genetika metabolismus MeSH
- regulace genové exprese MeSH
- zobrazování trojrozměrné MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Chromosome pairing in meiosis usually starts in the vicinity of the telomere attachment to the nuclear membrane and congregation of telomeres in the leptotene bouquet is believed responsible for bringing homologue pairs together. In a heterozygote for an inversion of a rye (Secale cereale L.) chromosome arm in wheat, a distal segment of the normal homologue is capable of chiasmate pairing with its counterpart in the inverted arm, located near the centromere. Using 3D imaging confocal microscopy, we observed that some telomeres failed to be incorporated into the bouquet and occupied various positions throughout the entire volume of the nucleus, including the centromere pole. Rye telomeres appeared ca. 21 times more likely to fail to be included in the telomere bouquet than wheat telomeres. The frequency of the out-of-bouquet rye telomere position in leptotene was virtually identical to the frequency of telomeres deviating from Rabl's orientation in the nuclei of somatic cells, and was similar to the frequency of synapsis of the normal and inverted chromosome arms, but lower than the MI pairing frequency of segments of these two arms normally positioned across the volume of the nucleus. Out-of-position placement of the rye telomeres may be responsible for reduced MI pairing of rye chromosomes in hybrids with wheat and their disproportionate contribution to aneuploidy, but appears responsible for initiating chiasmate pairing of distantly positioned segments of homology in an inversion heterozygote.
- MeSH
- buněčné jádro genetika ultrastruktura MeSH
- centromera chemie ultrastruktura MeSH
- chiméra genetika MeSH
- chromozomální inverze * MeSH
- chromozomy rostlin chemie ultrastruktura MeSH
- druhová specificita MeSH
- heterozygot MeSH
- hybridizace in situ fluorescenční MeSH
- konfokální mikroskopie MeSH
- párování chromozomů MeSH
- počítačové zpracování obrazu statistika a číselné údaje MeSH
- profáze meiózy I * MeSH
- pšenice genetika ultrastruktura MeSH
- rostlinné buňky metabolismus ultrastruktura MeSH
- telomery chemie ultrastruktura MeSH
- žito genetika ultrastruktura MeSH
- zobrazování trojrozměrné metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. 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.
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
Přeruš. str. : il., tab. ; 30 cm
Pomocí technologie DNA-mikročipů budou stanoveny transkripční mapy u několika populací zdravých a nádorových buněk s různým stupněm progrese. Porovnáním transkripčních map se budeme snažit o nalezení kombinací genů, pomiňujících kancerogenezi. Dále budeopužita FISH u 3D fixovaných buněk a cytometrie s vysokým rozlišením ke zjištění struktury chromosomů v interfázním jádře. Změny ve struktuře oproti normální tkáni budou porovnány se změnami exprese. Bude učiněn závěr o míře souvislosti mezi strukturou aexpresí v průběhu karcinogeneze. Tím můžeme získat možnost použití nové, levné metody k detekci onkogenní transformace specifické tkáně. Projekt přinese také zavedení techniky DNA-mikročípů na druhém pracovišti v ČR, což bude mít význam pro regionální rozvoj progresivních technologií.; Using DNA-microarray technology, the transcirptome maps will be determined for several normal and tumor cell populations in different stages of progression. Comparison ot maps should allow us to find out combinations of genes related to cancerogenesis. The structure of chromosomes will be inbestigated in interphase 3D-fixed nuclei by means of FISH and high-resolution cytometry. Changes of the structure re latively to the normal tissue xill be compared with changes of gene expression. Conclusion will bemade on the relationship between the structure of chromosomes and expression of genes during cancerogenesis. It may give us new and relatively cheap method for the detection of oncogenic transformation in the specific tissue. The project will bring DNA-microarrays to the second laboratory in the Czech Republic, which will be important for the regional development of progressive tehnologies.
- MeSH
- buněčná diferenciace MeSH
- cytofotometrie MeSH
- epigeneze genetická MeSH
- genetická transkripce MeSH
- hybridizace in situ fluorescenční MeSH
- lékařská onkologie MeSH
- mikročipová analýza MeSH
- nádory MeSH
- struktury chromozomu MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biologie
- genetika, lékařská genetika
- onkologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
