In the presented study, the cells of the glacial alga Ancylonema alaskanum collected in the Austrian Alps were analyzed. Algae were imaged both in their natural environment and in laboratory conditions using transmitted light and fluorescence microscopy. Using appropriate fluorochromes, the cell wall and cell organelles were studied. Oval nuclei located in the middle of the cell next to the chloroplasts and active mitochondria as well as lipid thylakoids of chloroplasts were imaged. Scanning electron microscopy showed that the surface of the algal cell wall was not significantly differentiated, and atomic force microscope imaging recorded little roughness. The SEM EDS analysis revealed that carbon, nitrogen, oxygen, and magnesium were the main components of the cells. It is worth emphasizing that the analyzed living algal cells were obtained directly from the glacier surface and demonstrated normal respiratory processes i.e. undisturbed physiological functions. Additionally, the mineral material accompanying the cells in their natural environment - fragments of the rock were imaged by Differential Interference Contrast microscopy and analyzed by Fourier Transform Infrared Spectroscopy. The study provides new data on the morphology and physicochemical characteristics of A. alaskanum, contributing to a more comprehensive characterization of their place in this harsh ecosystem.
- MeSH
- Ice Cover * MeSH
- Microscopy, Electron, Scanning MeSH
- Spectroscopy, Fourier Transform Infrared MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Austria MeSH
Many photosensitive substances suitable for photodynamic therapy (PDT) have limited applications due to their insufficient solubility in polar solvents. Our research overcomes this challenge by means of nanotechnology in order to transform hydrophobic compounds into stable aqueous solutions, enabling them to use their full potential and unique properties in cancer therapy. In this study, the novel nano-composite cGQDs-PEG-curcumin was developed to overcome the insolubility of curcumin in water and its extraordinary efficacy in PDT was evaluated. Complex characterization was performed using high-resolution transmission electron microscopy (HR-TEM), FTIR, and UV-Vis spectroscopy. Further analysis involved fluorescence lifetime imaging (FLIM), and its cellular localization was mapped with confocal microscopy. In order to evaluate PDT effectiveness, cells treated with cGQDs-PEG-curcumin were irradiated with 5 J/cm2 of 414 nm light. After irradiation, cell viability assay, scanning electron microscopy (SEM), reactive oxygen species (ROS) detection, comet assay, and γH2AX-based DNA double-strand breaks (DSBs) detection were assessed and revealed a remarkable ability of the nano-composite to induce DNA damage after irradiation without ROS production. Our findings highlight the potential of cGQDs-PEG-curcumin as a cutting-edge PDT agent, capable of disrupting cell membrane and nucleolar integrity and impairing ribosomal synthesis, which is crucial for proliferating tumour cells.
- MeSH
- Cell Nucleolus * drug effects metabolism MeSH
- DNA Breaks, Double-Stranded drug effects MeSH
- Photochemotherapy * methods MeSH
- Photosensitizing Agents * pharmacology MeSH
- Graphite * chemistry pharmacology MeSH
- Curcumin * pharmacology chemistry MeSH
- Quantum Dots * chemistry MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Neoplasms * drug therapy MeSH
- Polyethylene Glycols * chemistry pharmacology MeSH
- DNA Damage * drug effects MeSH
- Reactive Oxygen Species metabolism MeSH
- Cell Survival drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
... Hybridization methods for the study of nucleic acids 132 -- 9.2.1 Southern blotting 132 -- 9.2.2 Fluorescence ... ... Rotrekl) 194 -- 13.1 Introduction to developmental biology 194 -- 13.2 Polarization of the embryo 196 ...
First edition 227 stran : ilustrace ; 30 cm
- Conspectus
- Lékařské vědy. Lékařství
- Učební osnovy. Vyučovací předměty. Učebnice
- NML Fields
- biologie
- NML Publication type
- učebnice vysokých škol
Molecules of fluorescent proteins (FPs) exhibit distinct optical directionality. This optical directionality is characterized by transition dipole moments (TDMs), and their orientation with respect to the molecular structures. Although our recent observations of FP crystals allowed us to determine the mean TDM directions with respect to the framework of representative FP molecules, the dynamics of TDM orientations within FP molecules remain to be ascertained. Here we describe the results of our investigations of the dynamics of TDM directions in the fluorescent proteins eGFP, mTurquoise2 and mCherry, through time-resolved fluorescence polarization measurements and microsecond time scale all-atom molecular dynamics (MD) simulations. The investigated FPs exhibit initial fluorescence anisotropies (r0) consistent with significant differences in the orientation of the excitation and emission TDMs. However, based on MD data, we largely attribute this observation to rapid (sub-nanosecond) fluorophore motions within the FP molecular framework. Our results allow improved determinations of orientational distributions of FP molecules by polarization microscopy, as well as more accurate interpretations of fluorescence resonance energy transfer (FRET) observations.
The aim of the study is to investigate the differences in the interaction of three structurally diverse anthocyanidins, namely peonidin, petunidin, and delphinidin, as well as their glucosides with model biological membranes, human albumin, and plasmid DNA in order to look into their structure-activity relationships. Fluorimetric studies, as well as ATR-FTIR analyses, were jointly used in order to determine the changes observed in both the hydrophilic and hydrophobic layers of cell-mimic membranes (MM) which reflected the membrane lipid composition of tumour cells and red blood cell membranes (RBCM). Our results showed that anthocyanins and anthocyanidins can cause an increase in the packing order of the polar heads of lipids, as well as interact with their deeper layers by reducing the fluidity of lipid chains. The results presented here indicate that all compounds tested here possessed the ability to bind to human serum albumin (HSA) and the presence of a glucose molecule within the structures formed by anthocyanidin reduces their ability to bind to proteins. Using fluorescence correlation spectroscopy, it was demonstrated that the compounds tested here were capable of forming stable complexes with plasmid DNA and, particularly, strong DNA conformational changes were observed in the presence of petunidin and corresponding glucoside, as well as delphinidin. The results we obtained can be useful in comprehending the anthocyanins therapeutic action as molecular antioxidants and provide a valuable insight into their mechanism of action.
Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS.
- Publication type
- Journal Article MeSH
P2X receptors (P2X1-7) are trimeric ion channels activated by extracellular ATP. Each P2X subunit contains two transmembrane helices (TM1 and TM2). We substituted all residues in TM1 of rat P2X7 with alanine or leucine one by one, expressed mutants in HEK293T cells, and examined the pore permeability by recording both membrane currents and fluorescent dye uptake in response to agonist application. Alanine substitution of G27, K30, H34, Y40, F43, L45, M46, and D48 inhibited agonist-stimulated membrane current and dye uptake, and all but one substitution, D48A, prevented surface expression. Mutation V41A partially reduced both membrane current and dye uptake, while W31A and A44L showed reduced dye uptake not accompanied by reduced membrane current. Mutations T28A, I29A, and L33A showed small changes in agonist sensitivity, but they had no or small impact on dye uptake function. Replacing charged residues with residues of the same charge (K30R, H34K, and D48E) rescued receptor function, while replacement with residues of opposite charge inhibited (K30E and H34E) or potentiated (D48K) receptor function. Prolonged stimulation with agonist-induced current facilitation and a leftward shift in the dose-response curve in the P2X7 wild-type and most functional mutants, but sensitization was absent in the W31A, L33A, and A44L. Detailed analysis of the decay of responses revealed two kinetically distinct mechanisms of P2X7 deactivation: fast represents agonist unbinding, and slow might represent resetting of the receptor to the resting closed state. These results indicate that conserved and receptor-specific TM1 residues control surface expression of the P2X7 protein, non-polar residues control receptor sensitization, and D48 regulates intrinsic channel properties.
- MeSH
- Adenosine Triphosphate pharmacology metabolism MeSH
- Biological Transport MeSH
- HEK293 Cells MeSH
- Ion Channels * metabolism MeSH
- Rats MeSH
- Humans MeSH
- Mutation genetics MeSH
- Protein Domains MeSH
- Receptors, Purinergic P2X7 * genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The new ultra-high performance liquid chromatography method with tandem mass spectrometry and fluorescence detection allowing fast, selective, and high-throughput analysis of neopterin, kynurenine, tryptophan, and creatinine in gingival crevicular fluid (GCF) has been optimized. Defining the pathophysiology of periodontal disease and identification of potential diagnostic test for active periodontitis remains a significant challenge in the field of oral disease diagnosis. Analysis of GCF provides a non-invasive means of evaluating the role of the host response in periodontal disease. In addition, the analysis of GCF provides an information about current inflammation level of sampled site/tooth. Determination of GCF inflammatory biomarkers such as neopterin, kynurenine, and tryptophan can contribute to diagnosis, evaluation of treatment, and progression of periodontal diseases such as gingivitis and periodontitis. The separation of target analytes was carried out using a column KinetexTM Polar C18 100 Å, (100 × 3.0 mm) packed with 2.6 μm core-shell particles applying an elution with a gradient formed from 0.2% aqueous formic acid and 90% aqueous acetonitrile. Kynurenine, tryptophan, and creatinine were detected using mass spectrometry with electrospray ionization to improve the sensitivity while neopterin was detected using fluorescence detection. The separation of these four substances was achieved after using a very simple sample preparation technique convenient for small amount of biological sample. Only less than 20 μL sample was needed and the separation was completed in 4 min. MS/MS analysis was performed using multiple reaction monitoring (MRM) under a positive ionization mode. Deuterium labeled internal standard was used for the more precise quantification. The lower limits of quantification (LLOQ) for target analytes were 0.50 × 10-3 μmol/L for neopterin, 0.10 μmol/L for kynurenine, and 0.20 μmol/L for tryptophan and creatinine. The within-run and between-run accuracy were in a range of 96.67-114.77% for all quality controls and LLOQ of all analytes. Matrix effect, extraction recovery, and stability testing have all been investigated. The method was tested with real-life samples using GCF collected from patients suffering from periodontitis and from healthy controls. Neopterin levels in patients were significantly higher (P = 0.020) than in healthy subjects and indicate good potential of this method for using in evaluation of periodontal pathogenesis and healing outcomes following a treatment.
Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.
A novel method for semiautomated assessment of directions of collagen fibers in soft tissues using histological image analysis is presented. It is based on multiple rotated images obtained via polarized light microscopy without any additional components, i.e., with just two polarizers being either perpendicular or nonperpendicular (rotated). This arrangement breaks the limitation of 90° periodicity of polarized light intensity and evaluates the in-plane fiber orientation over the whole 180° range accurately and quickly. After having verified the method, we used histological specimens of porcine Achilles tendon and aorta to validate the proposed algorithm and to lower the number of rotated images needed for evaluation. Our algorithm is capable to analyze 5·105 pixels in one micrograph in a few seconds and is thus a powerful and cheap tool promising a broad application in detection of collagen fiber distribution in soft tissues.
- MeSH
- Achilles Tendon metabolism MeSH
- Algorithms MeSH
- Extracellular Matrix metabolism MeSH
- Collagen metabolism MeSH
- Microscopy methods MeSH
- Optical Imaging methods MeSH
- Image Processing, Computer-Assisted methods MeSH
- Microscopy, Polarization methods MeSH
- Swine MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
