The combination of aminophylline and salbutamol is frequently used in clinical practice in the treatment of obstructive lung diseases. While the side effects (including arrhythmias) of the individual bronchodilator drugs were well described previously, the side effects of combined treatment are almost unknown. We aimed to study the arrhythmogenic potential of combined aminophylline and salbutamol treatment in vitro. For this purpose, we used the established atomic force microscopy (AFM) model coupled with cardiac organoids derived from human pluripotent stem cells (hPSC-CMs). We focused on the chronotropic, inotropic, and arrhythmogenic effects of salbutamol alone and aminophylline and salbutamol combined treatment. We used a method based on heart rate/beat rate variability (HRV/BRV) analysis to detect arrhythmic events in the hPSC-CM based AFM recordings. Salbutamol and aminophylline had a synergistic chronotropic and inotropic effect compared to the effects of monotherapy. Our main finding was that salbutamol reduced the arrhythmogenic effect of aminophylline, most likely mediated by endothelial nitric oxide synthase activated by beta-2 adrenergic receptors. These findings were replicated and confirmed using hPSC-CM derived from two cell lines (CCTL4 and CCTL12). Data suggest that salbutamol as an add-on therapy may not only deliver a bronchodilator effect but also increase the cardiovascular safety of aminophylline, as salbutamol reduces its arrhythmogenic potential.
- MeSH
- albuterol * farmakologie MeSH
- aminofylin * farmakologie MeSH
- bronchodilatancia farmakologie MeSH
- buněčné linie MeSH
- kardiomyocyty účinky léků metabolismus MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- pluripotentní kmenové buňky účinky léků cytologie MeSH
- srdeční arytmie * farmakoterapie MeSH
- srdeční frekvence účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A comparative study of interaction between chicken egg white lysozyme (Lyz) with two hexavalent chromate ions; chromate and dichromate; which are prevalently known for their toxicity, was investigated using different spectroscopic techniques along with a molecular docking study. Both steady-state and time-resolved studies revealed that the addition of chromate/dichromate is responsible for strong quenching of intrinsic fluorescence in Lyz and the quenching is caused by both static and dynamic quenching mechanisms. Different binding and thermodynamic parameters were also calculated at different temperatures from the intrinsic fluorescence of Lyz. The conformational change in Lyz and thermodynamic parameters obtained during the course of interaction with chromate/dichromate were well-supported by the molecular docking results.
Drug repurposing requires a limited resource, cost-effective and faster method to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, this in silico studies attempts to identify the drug-likeness properties of ravidasvir, an II/III phase clinical trial chronic hepatitis C drug against 3-Chymotrypsin-like protease (3CLpro) of SARS-CoV-2 to combat the ongoing coronavirus disease 2019 (COVID-19) pandemic. This protease is predominantly involved in virus replication cycle; hence it is considered as a potent drug target. The molecular docking results showed that ravidasvir was found to be potent inhibitors of 3CLpro with scoring function based binding energy is -26.7 kJ/mol. Further dynamic behaviour of apo form and complex form of ravidasvir with 3CLpro were studied using molecular dynamics (MD) simulations over 500 ns each, total 2 μs time scale. The motion of the protein was studied using principal component analysis of the MD simulation trajectories. The binding free energy calculated using MM/PBSA method from the MD simulation trajectory was -190.3 ± 70.2 kJ/mol and -106.0 ± 26.7 kJ/mol for GROMOS96 54A7 and AMBER99SB-ILDN force field, respectively. This in silico studies suggesting ravidasvir might be a potential lead molecule against SARS-CoV-2 for further optimization and drug development to combat the life-threatening COVID-19 pandemic.Communicated by Ramaswamy H. Sarma.
- MeSH
- adipáty MeSH
- benzimidazoly MeSH
- COVID-19 * MeSH
- cysteinové endopeptidasy chemie MeSH
- farmakoterapie COVID-19 MeSH
- inhibitory proteas chemie MeSH
- koronavirové proteasy 3C MeSH
- lidé MeSH
- pandemie MeSH
- SARS-CoV-2 * MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- sukcináty MeSH
- valin analogy a deriváty MeSH
- virové nestrukturální proteiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 weeks, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochemistry takes 8-10 h. The procedures can be performed after standard laboratory training in nanomaterials research.
- MeSH
- imunosorbenty MeSH
- lidé MeSH
- nádorové biomarkery MeSH
- nádory * diagnóza MeSH
- nanočástice * chemie MeSH
- oxid křemičitý chemie MeSH
- polyethylenglykoly chemie MeSH
- streptavidin MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.
- MeSH
- buněčné linie MeSH
- cirkulární dichroismus MeSH
- cystein genetika MeSH
- dánio pruhované MeSH
- down regulace MeSH
- isoindoly farmakologie MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- mozek metabolismus MeSH
- mutageneze cílená MeSH
- organoselenové sloučeniny farmakologie MeSH
- proteiny 14-3-3 chemie genetika metabolismus MeSH
- proteiny dánia pruhovaného chemie metabolismus MeSH
- stabilita proteinů účinky léků MeSH
- tyrosin-3-monooxygenasa metabolismus MeSH
- vazba proteinů účinky léků MeSH
- vazebná místa účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata 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
Use of chemically inducible systems for transgene expression is a crucial requirement for modern plant biology research, as it allows (1) expression of transgenes that compromise plant viability or fertility when constitutively expressed and (2) spatiotemporal control of transgene expression levels. We describe the stringently regulated and highly responsive dexamethasone-inducible gene expression system pOp6/LhGR, which comprises the chimeric transcription activator LhGR and the corresponding pOp6 promoter. Upon induction, the LhGR activator binds to the pOp6 promoter and induces expression of the target gene of interest. We provide detailed protocols for inducing transgene expression at different developmental stages and in different plant species and discuss dexamethasone stability and use of its analogs. We also introduce new, versatile, GATEWAY-compatible binary vectors that are now available for the pOp6/LhGR system. © 2019 by John Wiley & Sons, Inc.
- MeSH
- aktivace transkripce * účinky léků MeSH
- Arabidopsis genetika MeSH
- dexamethason farmakologie MeSH
- DNA vazebné proteiny genetika MeSH
- genetické techniky MeSH
- geneticky modifikované rostliny genetika MeSH
- lac represory genetika MeSH
- promotorové oblasti (genetika) MeSH
- proteiny z Escherichia coli genetika MeSH
- receptory glukokortikoidů genetika MeSH
- Saccharomyces cerevisiae - proteiny genetika MeSH
- transgeny * MeSH
- transkripční faktory genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The 14-3-3 protein family performs regulatory functions in eukaryotic organisms by binding to a large number of phosphorylated protein partners. Whilst the binding mode of the phosphopeptides within the primary 14-3-3 binding site is well established based on the crystal structures of their complexes, little is known about the binding process itself. We present a computational study of the process by which phosphopeptides bind to the 14-3-3ζ protein. Applying a novel scheme combining Hamiltonian replica exchange molecular dynamics and distancefield restraints allowed us to map and compare the most likely phosphopeptide-binding pathways to the 14-3-3ζ protein. The most important structural changes to the protein and peptides involved in the binding process were identified. In order to bind phosphopeptides to the primary interaction site, the 14-3-3ζ adopted a newly found wide-opened conformation. Based on our findings we additionally propose a secondary interaction site on the inner surface of the 14-3-3ζ dimer, and a direct interference on the binding process by the flexible C-terminal tail. A minimalistic model was designed to allow for the efficient calculation of absolute binding affinities. Binding affinities calculated from the potential of mean force along the binding pathway are in line with the available experimental estimates for two of the studied systems.
