Even though amyloid aggregates were discovered many years ago the mechanism of their formation is still a mystery. Because of their connection to many of untreatable neurodegenerative diseases the motivation for finding a common aggregation path is high. We report a new high heat induced fibrillization path of a model protein β-lactoglobulin (BLG) when incubated in glycine instead of water at pH 2. By combining atomic force microscopy (AFM), transmission emission microscopy (TEM), dynamic light scattering (DLS) and circular dichroism (CD) we predict that the basic building blocks of fibrils made in glycine are not peptides, but rather spheroid oligomers of different height that form by stacking of ring-like structures. Spheroid oligomers linearly align to form fibrils by opening up and combining. We suspect that glycine acts as an hydrolysation inhibitor which consequently promotes a different fibrillization path. By combining the known data on fibrillization in water with our experimental conclusions we come up with a new fibrillization scheme for BLG. We show that by changing the fibrillization conditions just by small changes in buffer composition can dramatically change the aggregation pathway and the effect of buffer shouldn't be neglected. Fibrils seen in our study are also gaining more and more attention because of their pore-like structure and a possible cytotoxic mechanism by forming pernicious ion-channels. By preparing them in a simple model system as BLG we opened a new way to study their formation.
- MeSH
- amyloid * chemie MeSH
- glycin farmakologie MeSH
- laktoglobuliny * chemie MeSH
- mikroskopie atomárních sil metody MeSH
- voda MeSH
- Publikační typ
- časopisecké články MeSH
Traction force microscopy (TFM) has emerged as a versatile technique for the measurement of single-cell-generated forces. TFM has gained wide use among mechanobiology laboratories, and several variants of the original methodology have been proposed. However, issues related to the experimental setup and, most importantly, data analysis of cell traction datasets may restrain the adoption of TFM by a wider community. In this review, we summarize the state of the art in TFM-related research, with a focus on the analytical methods underlying data analysis. We aim to provide the reader with a friendly compendium underlying the potential of TFM and emphasizing the methodological framework required for a thorough understanding of experimental data. We also compile a list of data analytics tools freely available to the scientific community for the furtherance of knowledge on this powerful technique.
The design of efficient and safe gene delivery vehicles remains a major challenge for the application of gene therapy. Of the many reported gene delivery systems, metal complexes with high affinity for nucleic acids are emerging as an attractive option. We have discovered that certain metallohelices-optically pure, self-assembling triple-stranded arrays of fully encapsulated Fe-act as nonviral DNA delivery vectors capable of mediating efficient gene transfection. They induce formation of globular DNA particles which protect the DNA from degradation by various restriction endonucleases, are of suitable size and electrostatic potential for efficient membrane transport and are successfully processed by cells. The activity is highly structure-dependent-compact and shorter metallohelix enantiomers are far less efficient than less compact and longer enantiomers.
- MeSH
- buněčné linie MeSH
- DNA chemie ultrastruktura MeSH
- exprese genu MeSH
- fluorescenční protilátková technika MeSH
- genetické vektory * chemie ultrastruktura MeSH
- kationty chemie MeSH
- kovové nanočástice chemie ultrastruktura MeSH
- lidé MeSH
- mikroskopie atomárních sil metody MeSH
- molekulární struktura MeSH
- průtoková cytometrie MeSH
- reportérové geny MeSH
- technika přenosu genů * MeSH
- transfekce MeSH
- viabilita buněk MeSH
- železnaté sloučeniny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Zein is renewable plant protein with valuable film-forming properties that can be used as a packaging material. It is known that the addition of natural cross-linkers can enhance a film's tensile properties. In this study, we aimed to prepare antimicrobial zein-based films enriched with monolaurin, eugenol, oregano, and thyme essential oil. Films were prepared using the solvent casting technique from ethanol solution. Their physicochemical properties were investigated using structural, morphological, and thermal techniques. Polar and dispersive components were analyzed using two models to evaluate the effects on the surface free energy values. The antimicrobial activity was proven using a disk diffusion method and the suppression of bacterial growth was confirmed via a growth kinetics study with the Gompertz function. The films' morphological characteristics led to systems with uniform distribution of essential oils or eugenol droplets combined with a flat-plated structure of monolaurin. A unique combination of polyphenolic eugenol and amphiphilic monoglyceride provided highly stretchable films with enhanced barrier properties and efficiency against Gram-positive and Gram-negative bacteria, yeasts, and molds. The prepared zein-based films with tunable surface properties represent an alternative to non-renewable resources with a potential application as active packaging materials.
- MeSH
- antibakteriální látky farmakologie MeSH
- antifungální látky farmakologie MeSH
- biomechanika účinky léků MeSH
- diferenciální skenovací kalorimetrie MeSH
- Escherichia coli účinky léků MeSH
- eugenol farmakologie MeSH
- laurany farmakologie MeSH
- mikroskopie atomárních sil MeSH
- monoglyceridy farmakologie MeSH
- obaly potravin * MeSH
- oleje prchavé farmakologie MeSH
- pára MeSH
- permeabilita MeSH
- povrchové vlastnosti MeSH
- smáčivost MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- Staphylococcus aureus účinky léků MeSH
- zein farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
The conditions determining network-forming and aggregation properties of hyaluronan on the mica surface were studied. The hyaluronan was deposited on the surface from aqueous and saline solutions and attached by a bivalent cation. The morphology of the immobilized assemblies was characterized by atomic force microscopy. The experimental results show that the morphology and size of the aggregates as well as the density of the interconnecting fibrillar network, both made of hyaluronan, at the liquid-solid phase interface are determined not only by its molecular weight or concentration in solution, but also by the dissolution conditions and storage time. These findings extend the current state of knowledge about the conformational variability of this biologically important polymer. Understanding the conformational variability is of great importance, as it governs the physiological functions of hyaluronan, as well as its processability and formulations. That in turn determines its usability in different pharmacological and biomaterial applications.
- MeSH
- hydrofobní a hydrofilní interakce MeSH
- hypertonický solný roztok chemie MeSH
- kyselina hyaluronová chemie MeSH
- mikroskopie atomárních sil metody MeSH
- molekulární struktura MeSH
- molekulová hmotnost MeSH
- polymery chemie MeSH
- povrchové vlastnosti MeSH
- rozpustnost MeSH
- silikáty hliníku chemie MeSH
- skladování léků MeSH
- voda chemie MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage-bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force-distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy.
- MeSH
- bakteriofágy * MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- povrchová plasmonová rezonance MeSH
- stafylokokové infekce * MeSH
- Staphylococcus aureus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Spatial light modulators have become an essential tool for advanced microscopy, enabling breakthroughs in 3D, phase, and super-resolution imaging. However, continuous spatial-light modulation that is capable of capturing sub-millisecond microscopic motion without diffraction artifacts and polarization dependence is challenging. Here we present a photothermal spatial light modulator (PT-SLM) enabling fast phase imaging for nanoscopic 3D reconstruction. The PT-SLM can generate a step-like wavefront change, free of diffraction artifacts, with a high transmittance and a modulation efficiency independent of light polarization. We achieve a phase-shift > π and a response time as short as 70 µs with a theoretical limit in the sub microsecond range. We used the PT-SLM to perform quantitative phase imaging of sub-diffractional species to decipher the 3D nanoscopic displacement of microtubules and study the trajectory of a diffusive microtubule-associated protein, providing insights into the mechanism of protein navigation through a complex microtubule network.
- MeSH
- časové faktory MeSH
- interferenční mikroskopie metody statistika a číselné údaje MeSH
- kovové nanočástice ultrastruktura MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- mikroskopie fázově kontrastní metody statistika a číselné údaje MeSH
- mikrotubuly metabolismus ultrastruktura MeSH
- nanotechnologie MeSH
- nanotrubičky ultrastruktura MeSH
- optické jevy MeSH
- počítačová simulace MeSH
- proteiny asociované s mikrotubuly metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- Schizosaccharomyces pombe - proteiny metabolismus MeSH
- světlo MeSH
- tubulin metabolismus MeSH
- zlato MeSH
- zobrazování trojrozměrné metody statistika a číselné údaje MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Photodynamic inactivation (PDI) is a promising approach for the efficient killing of pathogenic microbes. In this study, the photodynamic effect of sulfonated polystyrene nanoparticles with encapsulated hydrophobic 5,10,15,20-tetraphenylporphyrin (TPP-NP) photosensitizers on Gram-positive (including multi-resistant) and Gram-negative bacterial strains was investigated. The cell viability was determined by the colony forming unit method. The results showed no dark cytotoxicity but high phototoxicity within the tested conditions. Gram-positive bacteria were more sensitive to TPP-NPs than Gram-negative bacteria. Atomic force microscopy was used to detect changes in the morphological properties of bacteria before and after the PDI treatment.
- MeSH
- Bacteria účinky léků účinky záření MeSH
- fotochemické procesy * MeSH
- fotochemoterapie metody MeSH
- mikroskopie atomárních sil MeSH
- nanočástice * chemie MeSH
- polystyreny * chemie MeSH
- porfyriny aplikace a dávkování chemie MeSH
- příprava léků * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lipid membrane remodeling belongs to the most fundamental processes in the body. The skin barrier lipids, which are ceramide dominant and highly rigid, must attain an unusual multilamellar nanostructure with long periodicity to restrict water loss and prevent the entry of potentially harmful environmental factors. Our data suggest that the skin acid mantle, apart from regulating enzyme activities and keeping away pathogens, may also be a prerequisite for the multilamellar assembly of the skin barrier lipids. Atomic force microscopy on monolayers composed of synthetic or human stratum corneum lipids showed multilayer formation (approximately 10-nm step height) in an acidic but not in a neutral environment. X-ray diffraction, Fourier transform infrared spectroscopy, and permeability studies showed markedly altered lipid nanostructure and increased water loss at neutral pH compared with that at acidic pH. These findings are consistent with the data on the altered organization of skin lipids and increased transepidermal water loss under conditions such as inadequate skin acidification, for example, in neonates, the elderly, and patients with atopic dermatitis.
- MeSH
- atopická dermatitida patologie MeSH
- ceramidy chemie metabolismus MeSH
- cholesterol chemie metabolismus MeSH
- difrakce rentgenového záření MeSH
- epidermis chemie metabolismus patologie MeSH
- koncentrace vodíkových iontů MeSH
- kyseliny mastné neesterifikované chemie metabolismus MeSH
- lidé MeSH
- mastné kyseliny MeSH
- mikroskopie atomárních sil MeSH
- novorozenec MeSH
- permeabilita MeSH
- perspiratio insensibilis * MeSH
- senioři MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- věkové faktory MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of our research was to study the behaviour of adipose tissue-derived stem cells (ADSCs) and vascular smooth muscle cells (VSMCs) on variously modified poly(L-lactide) (PLLA) foils, namely on pristine PLLA, plasma-treated PLLA, PLLA grafted with polyethylene glycol (PEG), PLLA grafted with dextran (Dex), and the tissue culture polystyrene (PS) control. On these materials, the ADSCs were biochemically differentiated towards VSMCs by a medium supplemented with TGFβ1, BMP4 and ascorbic acid (i.e. differentiation medium). ADSCs cultured in a non-differentiation medium were used as a negative control. Mature VSMCs cultured in both types of medium were used as a positive control. The impact of the variously modified PLLA foils and/or differences in the composition of the medium were studied with reference to cell adhesion, growth and differentiation. We observed similar adhesion and growth of ADSCs on all PLLA samples when they were cultured in the non-differentiation medium. The differentiation medium supported the expression of specific early, mid-term and/or late markers of differentiation (i.e. type I collagen, αSMA, calponin, smoothelin, and smooth muscle myosin heavy chain) in ADSCs on all tested samples. Moreover, ADSCs cultured in the differentiation medium revealed significant differences in cell growth among the samples that were similar to the differences observed in the cultures of VSMCs. The round morphology of the VSMCs indicated worse adhesion to pristine PLLA, and this sample was also characterized by the lowest cell proliferation. Culturing VSMCs in the differentiation medium inhibited their metabolic activity and reduced the cell numbers. Both cell types formed the most stable monolayer on plasma-treated PLLA and on the PS control. The behaviour of ADSCs and VSMCs on the tested PLLA foils differed according to the specific cell type and culture conditions. The suitable biocompatibility of both cell types on the tested PLLA foils seems to be favourable for vascular tissue engineering purposes.
- MeSH
- aorta metabolismus MeSH
- biokompatibilní materiály MeSH
- biopolymery chemie MeSH
- buněčná adheze MeSH
- buněčná diferenciace účinky léků MeSH
- kmenové buňky cytologie MeSH
- mikroskopie atomárních sil MeSH
- myocyty hladké svaloviny cytologie MeSH
- oxaziny chemie MeSH
- polyestery chemie MeSH
- polymery chemie MeSH
- polysacharidy chemie MeSH
- polystyreny chemie MeSH
- povrchové vlastnosti MeSH
- prasata MeSH
- proliferace buněk MeSH
- svaly hladké cévní cytologie MeSH
- testování materiálů MeSH
- tkáňové inženýrství metody MeSH
- tuková tkáň metabolismus MeSH
- xantheny chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
