The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes-termed herein as dancing-endosomes-which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth.

• Klíčová slova
• Arabidopsis thaliana, development, endosomes, interaction, root hair, spinning disc microscopy, structured illumination microscopy, trafficking,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Cytoskeleton can be observed in live plant cells in situ with high spatial and temporal resolution using a combination of specific fluorescent protein tag expression and advanced microscopy methods such as spinning disc confocal microscopy (SDCM) or variable angle epifluorescence microscopy (VAEM). Existing methods for quantifying cytoskeletal dynamics are often either based on laborious manual structure tracking, or depend on costly commercial software. Current automated methods also do not readily allow separate measurements of structure lifetime, lateral mobility, and spatial anisotropy of these parameters. RESULTS: We developed a new freeware-based, operational system-independent semi-manual technique for analyzing VAEM or SDCM data, QuACK (Quantitative Analysis of Cytoskeletal Kymograms), and validated it on data from Arabidopsis thaliana fh1 formin mutants, previously shown by conventional methods to exhibit altered actin and microtubule dynamics compared to the wild type. Besides of confirming the published mutant phenotype, QuACK was used to characterize surprising differential effects of various fluorescent protein tags fused to the Lifeact actin probe on actin dynamics in A. thaliana cotyledon epidermis. In particular, Lifeact-YFP slowed down actin dynamics compared to Lifeact-GFP at marker expression levels causing no macroscopically noticeable phenotypic alterations, although the two fluorophores are nearly identical. We could also demonstrate the expected, but previously undocumented, anisotropy of cytoskeletal dynamics in elongated epidermal cells of A. thaliana petioles and hypocotyls. CONCLUSIONS: Our new method for evaluating plant cytoskeletal dynamics has several advantages over existing techniques. It is intuitive, rapid compared to fully manual approaches, based on the free ImageJ software (including macros we provide here for download), and allows measurement of multiple parameters. Our approach was already used to document unexpected differences in actin mobility in transgenic A. thaliana expressing Lifeact fusion proteins with different fluorophores, highlighting the need for cautious interpretation of experimental results, as well as to reveal hitherto uncharacterized anisotropy of cytoskeletal mobility in elongated plant cells.

• Klíčová slova
• Actin, Anisotropy, FH1 (At3g25500), Kymogram, Lateral mobility, Lifeact, Microtubules, Spinning disc confocal microscopy, Structure stability, Variable angle fluorescence microscopy,
• Publikační typ
• časopisecké články MeSH

Microscopic filamentous fungi are ubiquitous microorganisms that adapt very easily to a variety of environmental conditions. Due to this adaptability, they can colonize a number of various surfaces where they are able to start forming biofilms. Life in the form of biofilms provides them with many benefits (increased resistance to desiccation, UV radiation, antimicrobial compounds, and host immune response). The aim of this study is to find a reliable and reproducible methodology to determine biofilm growth of selected microscopic filamentous fungi strains. Several methods (crystal violet staining, MTT assay, XTT assay, resazurin assay) for the determination of total biofilm biomass and its metabolic activity were tested on four fungi - Alternaria alternata, Aspergillus niger, Fusarium culmorum and Fusarium graminearum, and their biofilm was also imaged by spinning disc confocal microscopy using fluorescent dyes. A reproducible biofilm quantification method is essential for the subsequent testing of the biofilm growth suppression using antifungal agents or physical methods. Crystal violet staining was found to be a suitable method for the determination of total biofilm biomass of selected strains, and the MTT assay for the determination of metabolic activity of the biofilms. Calcofluor white and Nile red fluorescent stains successfully dyed the hyphae of microscopic fungi.

• Klíčová slova
• Biofilm, Crystal violet, Filamentous fungi, Spinning disc confocal microscopy, Tetrazolium salt assay,
• MeSH
• antifungální látky farmakologie   metabolismus   MeSH
• barvicí látky metabolismus   MeSH
• biofilmy MeSH
• genciánová violeť * metabolismus   MeSH
• houby * metabolismus   MeSH
• hyfy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antifungální látky MeSH
• barvicí látky MeSH
• genciánová violeť * MeSH

Current models of plasma membrane (PM) postulate its organization in various nano- and micro-domains with distinct protein and lipid composition. While metazoan PM nanodomains usually display high lateral mobility, the dynamics of plant nanodomains is often highly spatially restricted. Here we have focused on the determination of the PM distribution in nanodomains for Arabidopsis thaliana flotillin (AtFLOT) and hypersensitive induced reaction proteins (AtHIR), previously shown to be involved in response to extracellular stimuli. Using in vivo laser scanning and spinning disc confocal microscopy in Arabidopsis thaliana we present here their nanodomain localization in various epidermal cell types. Fluorescence recovery after photobleaching (FRAP) and kymographic analysis revealed that PM-associated AtFLOTs contain significantly higher immobile fraction than AtHIRs. In addition, much lower immobile fractions have been found in tonoplast pool of AtHIR3. Although members of both groups of proteins were spatially restricted in their PM distribution by corrals co-aligning with microtubules (MTs), pharmacological treatments showed no or very low role of actin and microtubular cytoskeleton for clustering of AtFLOT and AtHIR into nanodomains. Finally, pharmacological alteration of cell wall (CW) synthesis and structure resulted in changes in lateral mobility of AtFLOT2 and AtHIR1. Accordingly, partial enzymatic CW removal increased the overall dynamics as well as individual nanodomain mobility of these two proteins. Such structural links to CW could play an important role in their correct positioning during PM communication with extracellular environment.

• Klíčová slova
• Arabidopsis thaliana, cell wall, cytoskeleton, flotillin, hypersensitive induced reaction protein, lateral mobility, membrane nanodomains, plasma membrane, tonoplast,
• MeSH
• aktiny metabolismus   MeSH
• Arabidopsis genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• buněčná stěna metabolismus   MeSH
• cytoskelet metabolismus   MeSH
• konfokální mikroskopie MeSH
• membránové mikrodomény metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• flotillins MeSH Prohlížeč
• membránové proteiny MeSH
• proteiny huseníčku MeSH

The increasing risk of antibiotic failure in the treatment of Pseudomonas aeruginosa infections is largely related to the production of a wide range of virulence factors. The use of non-thermal plasma (NTP) is a promising alternative to antimicrobial treatment. Nevertheless, there is still a lack of knowledge about the effects of NTP on the virulence factors production. We evaluated the ability of four NTP-affected P. aeruginosa strains to re-form biofilm and produce Las-B elastase, proteases, lipases, haemolysins, gelatinase or pyocyanin. Highly strains-dependent inhibitory activity of NTP against extracellular virulence factors production was observed. Las-B elastase activity was reduced up to 82% after 15-min NTP treatment, protease activity and pyocyanin production by biofilm cells was completely inhibited after 60 min, in contrast to lipases and gelatinase production, which remained unchanged. However, for all strains tested, a notable reduction in biofilm re-development ability was depicted using spinning disc confocal microscopy. In addition, NTP exposure of mature biofilms caused disruption of biofilm cells and their dispersion into the environment, as shown by transmission electron microscopy. This appears to be a key step that could help overcome the high resistance of P. aeruginosa and its eventual elimination, for example in combination with antibiotics still highly effective against planktonic cells.

• Klíčová slova
• antivirulence factors, biofilm disruption, cold atmospheric plasma (CAP), combined therapy, haemolytic activity,
• MeSH
• antibakteriální látky farmakologie   MeSH
• biofilmy MeSH
• endopeptidasy farmakologie   MeSH
• faktory virulence MeSH
• hemolyziny farmakologie   MeSH
• lidé MeSH
• pankreatická elastasa MeSH
• plankton MeSH
• plazmové plyny * farmakologie   MeSH
• proteasy MeSH
• pseudomonádové infekce * MeSH
• Pseudomonas aeruginosa MeSH
• pyokyanin MeSH
• quorum sensing MeSH
• želatinasy farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• endopeptidasy MeSH
• faktory virulence MeSH
• hemolyziny MeSH
• pankreatická elastasa MeSH
• plazmové plyny * MeSH
• proteasy MeSH
• pyokyanin MeSH
• želatinasy MeSH

The biofilms of filamentous-forming fungi are a novel and still insufficiently understood research topic. We have studied Aspergillus fumigatus, an ubiquitous opportunistic pathogenic fungus, as a representative model for a study of biofilm formation by filamentous fungi and for assessing the potential anti-biofilm activity of natural substances. The activity of antibiotic amphotericin B and selected natural substances: baicalein, chitosan and rhamnolipid was studied. The minimum suspension inhibitory concentrations (MIC) were determined and the biofilm susceptibility was investigated by determining the metabolic activity of sessile cells (XTT assay) and total biofilm biomass (crystal violet staining). Significant time-dependent differences in substances' anti-biofilm activity were observed. Images of A. fumigatus biofilm were obtained by Cellavista automatic light microscope and spinning disc confocal microscopy. Baicalein and rhamnolipid were not found as suitable substances for inhibition of the A. fumigatus biofilm formation, as neither of the substances inhibited the sessile cells metabolic activity or the total biofilm biomass even at tenfold MIC after 48 h. In contrast, chitosan at 10 × MIC (25 µg mL-1), suppressed the biofilm metabolic activity by 90 % and the total biofilm biomass by 80 % even after 72 h of cultivation. Amphotericin B inhibited only 14 % of total biofilm biomass (crystal violet staining) and 35 % of metabolic activity (XTT assay) of adherent cells under the same conditions. Our results therefore suggest chitosan as potential alternative for treating A. fumigatus biofilm-associated infections.

• Klíčová slova
• Amphotericin B, Aspergillus fumigatus, Biofilm, Chitosan, Rhamnolipid,
• MeSH
• amfotericin B farmakologie   MeSH
• antifungální látky farmakologie   MeSH
• Aspergillus fumigatus účinky léků   fyziologie   MeSH
• biofilmy účinky léků   MeSH
• chitosan farmakologie   MeSH
• flavanony farmakologie   MeSH
• glykolipidy farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amfotericin B MeSH
• antifungální látky MeSH
• baicalein MeSH Prohlížeč
• chitosan MeSH
• flavanony MeSH
• glykolipidy MeSH
• rhamnolipid MeSH Prohlížeč

Pterostilbene (PTE), a dimethylated analogue of resveratrol, mostly contained in Vitis vinifera leaves or in other plant sources is well-known for its antioxidant activity. Due to its bioavailability, low hydrophilicity and thus ability to penetrate hydrophobic biological membranes it was found to be an antimicrobial agent. These properties of PTE offer the possibility of its use in the treatment of microbial infections. The emergence of antibiotic resistance of microorganisms is often caused by their ability to form biofilm; new substances with antibiofilm activity are therefore sought. The representatives of opportunistic pathogenic gram-positive and gram-negative bacteria as well as fungi were used for the determination of minimum inhibitory concentrations (MIC50 and MIC80), minimum biofilm inhibitory concentrations (MBIC50 and MBIC80) and minimum biofilm eradication concentrations (MBEC50 and MBEC80) of PTE and commonly used antibiotics erythromycin, polymyxin B or antimycotic amphotericin B. Total biofilm biomass was investigated by crystal violet staining, and the results were confirmed using microscopic techniques. The most significant antibiofilm action was proved for gram-positive cocci, e.g., MBEC50 of PTE for all strains of Staphylococcus epidermidis tested was 25 mg/L. By contrast, the antibiotic ERM did not exhibit antibiofilm activity in most cases. The permeabilization of cell membranes of gram-positive cocci biofilm by MBIC50 and MBEC50 of PTE was confirmed by LIVE/DEAD staining using spinning disc confocal microscopy. PTE significantly influenced the ability of gram-positive cocci to form biofilm and it effectively eradicated pre-formed biofilm in vitro; its potential for the treatment of biofilm-associated infections of Staphylococcus spp. or Enterococcus faecalis is thus apparent.

• Klíčová slova
• Antibiofilm, Biofilm inhibition, Eradication, Gram-positive cocci, Pterostilbene,
• MeSH
• antibakteriální látky farmakologie   MeSH
• antioxidancia farmakologie   MeSH
• biofilmy účinky léků   růst a vývoj   MeSH
• Enterococcus faecalis účinky léků   MeSH
• gramnegativní bakterie účinky léků   MeSH
• grampozitivní bakterie účinky léků   MeSH
• grampozitivní koky účinky léků   MeSH
• listy rostlin chemie   MeSH
• mikrobiální testy citlivosti MeSH
• Pseudomonas aeruginosa účinky léků   MeSH
• rostlinné extrakty farmakologie   MeSH
• Staphylococcus epidermidis účinky léků   MeSH
• stilbeny farmakologie   MeSH
• Vitis chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• antioxidancia MeSH
• pterostilbene MeSH Prohlížeč
• rostlinné extrakty MeSH
• stilbeny MeSH

To understand the impact of binary doping in ZnO, nanosized Zn(Ag, Ni)O systems were synthesized by the sol-gel method. The amount of Ag was fixed at 2 at%, and that of Ni was varied from 1 to 15 at%. Ni incorporation equal to or beyond 3 at% gave rise to the development of the NiO phase. The presence of Ag and Ni did not have much influence on the lattice constants of ZnO. However, a larger addition of Ni impacted the unit cell of NiO, as indicated by the reduction of the lattice constant of NiO. The increase in NiO and Ag contents in ZnO reduced the second and third harmonic intensities under non-linear investigations. X-ray photoelectron spectroscopy analysis indicated that initial Ni addition varied randomly along with Ag, and it stabilized itself at higher concentration. Field emission scanning electron microscopy revealed that interlinked particles and chains with tamarind shapes were formed, closely matching the rod-like structures under high resolution. Ag and Ni addition altered the structures slightly and randomly till 5 at% Ni; thereafter they deviated from the particle shape to flat disc-shapes. Interestingly, the magnetic response of the sample was determined by the NiO phase, and the effect of Ni and Ag substitution in the ZnO host matrix was almost irrelevant at low temperatures toward magnetic contribution. Weak ferromagnetism at low temperatures (≤50 K) with superparamagnetic-like behavior (cusp in ZFC magnetization) was observed in all the samples. This could be attributed to the finite nano-size effect and uncompensated spins at the surface of the particle.

• Publikační typ
• časopisecké články MeSH

Focused ion beam (FIB) milling has been used to fabricate magnetic nanostructures (wires, squares, discs) from single magnetic layers (Co, permalloy) and spin-valve (permalloy/Cu/Co) multilayers (thicknesses 5-50 nm) prepared by ion beam sputtering deposition. Milled surfaces of metallic thin films typically exhibit residual roughness, which is also transferred onto the edges of the milled patterns. This can lead to domain wall pinning and influence the magnetization behaviour of the nanostructures. We have investigated the milling process and the influence of the FIB parameters (incidence angle, dwell time, overlap and ion beam current) on the roughness of the milled surface. It has been found that the main reasons for increased roughness are different sputter yields for various crystallographic orientations of the grains in polycrystalline magnetic thin films. We have found that the oblique ion beam angle, long dwell time and overlap < 1 are favourable parameters for suppression of this intrinsic roughness. Finally, we have shown how to determine the ion dose necessary to mill through the whole thin film up to the silicon substrate from scanning electron microscopy (SEM) images only.

• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH