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
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 complexity of cancer biology and its clinical manifestation are driven by genetic, epigenetic, transcriptomic, proteomic and metabolomic alterations, supported by genomic instability as well as by environmental conditions and lifestyle factors. Although novel therapeutic modalities are being introduced, efficacious cancer therapy is not achieved due to the frequent emergence of distinct mechanisms of multidrug resistance (MDR). Advanced technologies with the potential to identify and characterize cancer MDR could aid in selecting the most efficacious therapeutic regimens and prevent inappropriate treatments of cancer patients. Herein, we aim to present technological tools that will enhance our ability to surmount drug resistance in cancer in the upcoming decade. Some of these tools are already in practice such as next-generation sequencing. Identification of genes and different types of RNAs contributing to the MDR phenotype, as well as their molecular targets, are of paramount importance for the development of new therapeutic strategies aimed to enhance drug response in resistant tumors. Other techniques known for many decades are in the process of adaptation and improvement to study cancer cells' characteristics and biological behavior including atomic force microscopy (AFM) and live-cell imaging. AFM can monitor in real-time single molecules or molecular complexes as well as structural alterations occurring in cancer cells induced upon treatment with various antitumor agents. Cell tracking methodologies and software tools recently progressed towards quantitative analysis of the spatio-temporal dynamics of heterogeneous cancer cell populations and enabled direct monitoring of cells and their descendants in 3D cultures. Besides, novel 3D systems with the advanced mimicking of the in vivo tumor microenvironment are applicable to study different cancer biology phenotypes, particularly drug-resistant and aggressive ones. They are also suitable for investigating new anticancer treatment modalities. The ultimate goal of using phenotype-driven 3D cultures for the investigation of patient biopsies as the most appropriate in vivo mimicking model, can be achieved in the near future.
- MeSH
- biotechnologie metody MeSH
- buněčné kultury metody MeSH
- chemorezistence genetika MeSH
- lidé MeSH
- mikroskopie atomárních sil metody MeSH
- mnohočetná léková rezistence genetika MeSH
- nádorové mikroprostředí genetika MeSH
- nádory genetika patologie MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Výskyt syndromu suchého oka je v oftalmologické praxi častý. Terapeutický postup závisí na příčinách choroby. Léčba je obvykle zahajována aplikací umělých slz. Autoři diskutují aktuální diagnostické a terapeutické možnosti.
Dry eye syndrome is a common ocular disease. Treatment depends on the underlaying cause. Arteficial tears are the usual first line treatment. New tools available to help diagnose the presence of this syndrome and innovative therapeutic trends are discussed.
- MeSH
- barvení a značení metody MeSH
- lidé MeSH
- mikroskopie atomárních sil metody MeSH
- nemoci slzného ústrojí chirurgie diagnostické zobrazování komplikace MeSH
- slzné ústrojí * anatomie a histologie patofyziologie účinky léků MeSH
- slzy fyziologie MeSH
- syndromy suchého oka * diagnóza patofyziologie terapie MeSH
- zvlhčující oční kapky farmakologie terapeutické užití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
A compromised detection of radiation-induced plasmid DNA fragments results in underestimation of calculated damage yields. Electrophoretic methods are easy and cheap, but they can only detect a part of the fragments, neglecting the shortest ones. These can be detected with atomic force microscopy, but at the expense of time and price. Both methods were used to investigate their capabilities to detect the DNA fragments induced by high-energetic heavy ions. The results were taken into account in calculations of radiation-induced yields of single and double strand breaks. It was estimated that the double strand break yield is twice as high when the fragments are at least partially detected with the agarose electrophoresis, compared to when they were completely omitted. Further increase by 13% was observed when the measured fragments were corrected for the fraction of the shortest fragments up to 300 base pairs, as detected with the atomic force microscopy. The effect of fragment detection on the single strand break yield was diminished.
The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.
- MeSH
- analýza dat MeSH
- elektrofyziologické jevy * MeSH
- kardiomyocyty cytologie fyziologie MeSH
- mechanické jevy * MeSH
- mikroskopie atomárních sil * přístrojové vybavení metody MeSH
- molekulární zobrazování MeSH
- spřažení excitace a kontrakce * MeSH
- vápníková signalizace MeSH
- Publikační typ
- časopisecké články MeSH
Atomic force microscopy (AFM) is not only a high-resolution imaging technique but also a sensitive tool able to study biomechanical properties of bio-samples (biomolecules, cells) in native conditions-i.e., in buffered solutions (culturing media) and stable temperature (mostly 37 °C). Micromechanical transducers (cantilevers) are often used to map surface stiffness distribution, adhesion forces, and viscoelastic parameters of living cells; however, they can also be used to monitor time course of cardiomyocytes contraction dynamics (e.g. beating rate, relaxation time), together with other biomechanical properties. Here we describe the construction of an AFM-based biosensor setup designed to study the biomechanical properties of cardiomyocyte clusters, through the use of standard uncoated silicon nitride cantilevers. Force-time curves (mechanocardiograms, MCG) are recorded continuously in real time and in the presence of cardiomyocyte-contraction affecting drugs (e.g., isoproterenol, metoprolol) in the medium, under physiological conditions. The average value of contraction force and the beat rate, as basic biomechanical parameters, represent pharmacological indicators of different phenotype features. Robustness, low computational requirements, and optimal spatial sensitivity (detection limit 200 pN, respectively 20 nm displacement) are the main advantages of the presented method.
- MeSH
- biomechanika * MeSH
- biosenzitivní techniky MeSH
- kardiomyocyty cytologie MeSH
- lidé MeSH
- mikroskopie atomárních sil * přístrojové vybavení metody MeSH
- pluripotentní kmenové buňky cytologie MeSH
- preklinické hodnocení léčiv MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Compromised detection of short DNA fragments can result in underestimation of radiation-induced clustered DNA damage. The fragments can be detected with atomic force microscopy (AFM), followed by image analysis to compute the length of plasmid molecules. Plasmid molecules imaged with AFM are represented by open or closed curves, possibly with crossings. For the analysis of such objects, a dedicated algorithm was developed, and its usability was demonstrated on the AFM images of plasmid pBR322 irradiated with 60Co gamma rays. The analysis of the set of the acquired AFM images revealed the presence of DNA fragments with lengths shorter than 300 base pairs that would have been neglected by a conventional detection method.
