The human body is constantly exposed to ionizing radiation of different qualities. Especially the exposure to high-LET (linear energy transfer) particles increases due to new tumor therapy methods using e.g. carbon ions. Furthermore, upon radiation accidents, a mixture of radiation of different quality is adding up to human radiation exposure. Finally, long-term space missions such as the mission to mars pose great challenges to the dose assessment an astronaut was exposed to. Currently, DSB counting using γH2AX foci is used as an exact dosimetric measure for individuals. Due to the size of the γH2AX IRIF of ~ 0.6 μm, it is only possible to count DSB when they are separated by this distance. For high-LET particle exposure, the distance of the DSB is too small to be separated and the dose will be underestimated. In this study, we developed a method where it is possible to count DSB which are separated by a distance of ~ 140 nm. We counted the number of ionizing radiation-induced pDNA-PKcs (DNA-PKcs phosphorylated at T2609) foci (size = 140 nm ± 20 nm) in human HeLa cells using STED super-resolution microscopy that has an intrinsic resolution of 100 nm. Irradiation was performed at the ion microprobe SNAKE using high-LET 20 MeV lithium (LET = 116 keV/μm) and 27 MeV carbon ions (LET = 500 keV/μm). pDNA-PKcs foci label all DSB as proven by counterstaining with 53BP1 after low-LET γ-irradiation where separation of individual DSB is in most cases larger than the 53BP1 gross size of about 0.6 μm. Lithium ions produce (1.5 ± 0.1) IRIF/μm track length, for carbon ions (2.2 ± 0.2) IRIF/μm are counted. These values are enhanced by a factor of 2-3 compared to conventional foci counting of high-LET tracks. Comparison of the measurements to PARTRAC simulation data proof the consistency of results. We used these data to develop a measure for dosimetry of high-LET or mixed particle radiation exposure directly in the biological sample. We show that proper dosimetry for radiation up to a LET of 240 keV/μm is possible.
- MeSH
- biologické markery MeSH
- dávka záření MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- fluorescenční mikroskopie metody MeSH
- fosforylace účinky záření MeSH
- HeLa buňky MeSH
- lidé MeSH
- lineární přenos energie MeSH
- lithium škodlivé účinky MeSH
- oprava DNA účinky záření MeSH
- proteinkinasy účinky záření MeSH
- radiační expozice MeSH
- radiometrie metody MeSH
- těžké ionty škodlivé účinky MeSH
- uhlík škodlivé účinky MeSH
- záření gama škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.
- MeSH
- fluorescenční mikroskopie * metody MeSH
- genetická transkripce * MeSH
- lidé MeSH
- regulace genové exprese * MeSH
- RNA-polymerasa II metabolismus MeSH
- transkripční faktory metabolismus MeSH
- vazba proteinů MeSH
- zobrazení jednotlivé molekuly metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza účinky léků MeSH
- bisbenzimidazol chemie MeSH
- buněčná smrt účinky léků MeSH
- buněčné jádro účinky léků metabolismus MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- cisplatina farmakologie MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie metody MeSH
- fragmentace DNA účinky léků MeSH
- kamptothecin farmakologie MeSH
- lidé MeSH
- průtoková cytometrie metody MeSH
- reprodukovatelnost výsledků MeSH
- staurosporin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.
- MeSH
- chromozomy rostlin chemie genetika metabolismus MeSH
- DNA-topoisomerasy typu II metabolismus MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční mikroskopie metody MeSH
- indoly chemie MeSH
- ječmen (rod) cytologie genetika MeSH
- konfokální mikroskopie metody MeSH
- metafáze genetika MeSH
- reprodukovatelnost výsledků MeSH
- zobrazení jednotlivé molekuly metody MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The tear film at the ocular surface is covered by a thin layer of lipids. This oily phase stabilizes the film by decreasing its surface tension and improving its viscoelastic properties. Clinically, destabilization and rupture of the tear film are related to dry eye disease and are accompanied by changes in the quality and quantity of tear film lipids. In dry eye, eye drops containing oil-in-water emulsions are used for the supplementation of lipids and surface-active components to the tear film. We explore in detail the biophysical aspects of interactions of specific surface-active compounds, cetalkonium chloride and poloxamer 188, which are present in oil-in-water emulsions, with tear lipids. The aim is to better understand the macroscopically observed eye drops-tear film interactions by rationalizing them at the molecular level. To this end, we employ a multi-scale approach combining experiments on human meibomian lipid extracts, measurements using synthetic lipid films, and in silico molecular dynamics simulations. By combining these methods, we demonstrate that the studied compounds specifically interact with the tear lipid film enhancing its structure, surfactant properties, and elasticity. The observed effects are cooperative and can be further modulated by material packing at the tear-air interface.
- MeSH
- film jako téma * MeSH
- fluorescenční mikroskopie metody MeSH
- kvartérní amoniové sloučeniny chemie MeSH
- lidé MeSH
- lipidy chemie MeSH
- mastné alkoholy chemie MeSH
- meibomské žlázky metabolismus MeSH
- poloxamer chemie MeSH
- simulace molekulární dynamiky * MeSH
- teoretické modely MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
V histologických vzorcích resekátu ledvin 104 pacientů se světiobuněčným karcinomem ledvin byla retrospektivně imunofluorescenčně vyšetřena frekvence výskytu primárních řasinek. Ostatní parametry byly vyšetřeny imunohistochemicky. Medián celkového přežití (overall survival, OS) byl signifikantně delší u pacientů s nižší frekvencí výskytu primárních řasinek (< 0,002) než u pacientů s vyšší frekvencí primárních řasinek (> 0,002) (p < 0,001). Medián OS byl signifikantně delší u pacientů s nižší expresí (< 25 %) CD8+ TIL (tumor infiltrující lymfocyty) v porovnání s pacienty s vyšší expresí (> 25 %) CD8+ TIL (p = 0,006). Medián OS byl signifikantně delší u pacientů s nižší expresí (< 25 %) PD-1 (receptor programované buněčné smrti 1) než u pacientů s vyšší expresí (> 25 %) PD-1 (p = 0,006). Tato studie poskytuje data o prognostickém významu primárních řasinek ve vztahu k vybraným parametrům nádorového mikroprostředí světlobuněčného karcinomu ledvin.
The presence of primary cilia, programmed cell death protein-1 receptor (PD-1) expression and intraepithelial CD8+ TIL (tumor infiltrating lymphocytes) expression were retrospectively evaluated in tumor tissue blocks of the resected specimens of the kidney in 104 patients with clear cell renal cell carcinoma. Median overall survival (OS) was significantly longer in patients with lower frequency of primary cilia (<0.002) than in patients with higher frequency of primary cilia (>0.002) (p<0.001). Median OS was significantly longer in patients with lower (<25%) CD8+ TIL expression than in patients with higher (>25%) CD8+ TIL expression (p=0.006). Median OS was significantly longer in patients with lower (<25%) PD-1 expression than in patients with higher (>25%) PD-1 expression (p=0.006). The present study provides the first data on the potential association and combined prognostic significance of frequency of primary cilia, CD8+ TIL expression and PD-1 expression in patients with clear cell renal cell carcinoma.
- MeSH
- analýza přežití MeSH
- antigeny CD279 MeSH
- cilie * patologie MeSH
- fluorescenční mikroskopie metody MeSH
- karcinom z renálních buněk * patologie ultrastruktura MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikrotubuly patologie MeSH
- nádorové mikroprostředí MeSH
- nefrektomie MeSH
- prognóza MeSH
- proporcionální rizikové modely MeSH
- proteiny hedgehog fyziologie MeSH
- senioři MeSH
- tumor infiltrující lymfocyty patologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- Publikační typ
- klinická studie MeSH
- práce podpořená grantem MeSH
Super -rozlišovací mikroskopie je schopna různými způsoby obejít difrakční limit a dosahovat izotropického rozlišení desítek nanometrů. V současné době se vyvíjí značné úsilí, aby stejná kvalita výsledků, které lze získat s fixovanými vzorky, byla dosažitelná i s buňkami živými. Vzhledem k četnosti dynamických vnitrobuněčných procesů je žádoucí, aby super -rozlišovací 3D mikroskopie byla co nejdříve běžným a dostupným způsobem, jak tyto procesy pozorovat a zkoumat. Mitochondrie, semiautonomní buněčné elektrárny, skrývají stále spoustu nezodpovězených otázek, na které by v budoucnu právě 3D super -rozlišovací mikroskopie živých buněk mohla nalézt odpovědi.
Super -resolution microscopy can variously circumvent the Abbe's diffraction limit and achieve an isotropic resolution of tens of nanometers. Considerable efforts are currently being made to ensure that the same quality of results that can be obtained using fixed samples are achievable using living cells. Since the numerous intracellular dynamic processes occur in cells, it is desirable to make super -resolution 3D microscopy an available method as soon as possible in order to investigate these processes. Mitochondria, semi -autonomous cellular power -plants, still hide a lot of unanswered questions, to which specifically 3D super -resolution microscopy of living cells could find answers in the future.
Fluid transport in the perivascular space by the glia-lymphatic (glymphatic) system is important for the removal of solutes from the brain parenchyma, including peptides such as amyloid-beta which are implicated in the pathogenesis of Alzheimer's disease. The glymphatic system is highly active in the sleep state and under the influence of certain of anaesthetics, while it is suppressed in the awake state and by other anaesthetics. Here we investigated whether light sheet fluorescence microscopy of whole optically cleared murine brains was capable of detecting glymphatic differences in sleep- and awake-mimicking anaesthesia, respectively. Using light-sheet imaging of whole brains, we found anaesthetic-dependent cerebrospinal fluid (CSF) influx differences, including reduced tracer influx along tertiary branches of the middle cerebral artery and reduced influx along dorsal and anterior penetrating arterioles, in the awake-mimicking anaesthesia. This study establishes that light sheet microscopy of optically cleared brains is feasible for quantitative analyses and can provide images of the entire glymphatic system in whole brains.
- MeSH
- anestezie MeSH
- arteria cerebri media fyziologie MeSH
- arterioly fyziologie MeSH
- fluorescenční mikroskopie metody MeSH
- glymfatický systém fyziologie MeSH
- mozek ultrastruktura MeSH
- mozkomíšní mok metabolismus MeSH
- mozkový krevní oběh fyziologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- neurozobrazování metody MeSH
- spánek fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. In addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.
- MeSH
- barva MeSH
- cholesterol chemie MeSH
- dimerizace MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie MeSH
- fúze membrán * MeSH
- konfokální mikroskopie MeSH
- lipidy chemie MeSH
- lipopeptidy chemie MeSH
- peptidy chemie MeSH
- polysorbáty chemie MeSH
- rezonanční přenos fluorescenční energie MeSH
- unilamelární lipozómy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
