ETHNOPHARMACOLOGICAL RELEVANCE: Salvia officinalis L., Sambucus nigra L., Matricaria chamomilla L., Agrimonia eupatoria L., Fragaria vesca L. and Malva sylvestris L. are plants that have a long tradition in European folk medicine. To this day, they are part of medicinal teas or creams that help with the healing of skin wounds and the treatment of respiratory or intestinal infections. However, so far these plants have not been investigated more deeply than in their direct antibacterial effect. AIM OF THE STUDY: Our research is focused on adjuvants that inhibit the mechanism of antibiotic resistance or modulate bacterial virulence. Based on a preliminary screening of 52 European herbs, which commonly appear as part of tea blends or poultice. Six of them were selected for their ability to revert the resistant phenotype of nosocomial bacterial strains. METHODS: Herbs selected for this study were obtained from commercially available sources. For the extraction of active compounds ethanol was used. Modulation of virulence was observed as an ability to inhibit bacterial cell-to-cell communication using two mutant sensor strains of Vibrio campbellii. Biofilm formation, and planktonic cell adhesion was measured using a static antibiofilm test. Ethidium bromide assay was used to checked the potential of inhibition bacterial efflux pumps. The antibacterial activities of the herbs were evaluated against resistant bacterial strains using macro dilution methods. RESULTS: Alcohol extracts had antibacterial properties mainly against Gram-positive bacteria. Of all of them, the highest antimicrobial activity demonstrated Malva sylvestris, killing both antibiotic resistant bacteria; Staphylococcus aureus with MIC of 0.8 g/L and Pseudomonas aeruginosa 0.7 g/L, respectively. Fragaria vesca extract (0.08 g/L) demonstrated strong synergism with colistin (4 mg/L) in modulating the resistant phenotype to colistin of Pseudomonas aeruginosa. Similarly, the extract of S. officinalis (0.21 g/L) reverted resistance to gentamicin (1 mg/L) in S. aureus. However, Sambucus nigra and Matricaria chamomilla seem to be a very promising source of bacterial efflux pump inhibitors. CONCLUSION: The extract of F. vesca was the most active. It was able to reduce biofilm formation probably due to the ability to decrease bacterial quorum sensing. On the other hand, the activity of S. nigra or M. chamomilla in reducing bacterial virulence may be explained by the ability to inhibit bacterial efflux systems. All these plants have potential as an adjuvant for the antibiotic treatment.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacteria MeSH
- biofilmy MeSH
- kolistin farmakologie MeSH
- léčivé rostliny * MeSH
- mikrobiální testy citlivosti MeSH
- Pseudomonas aeruginosa MeSH
- rostlinné extrakty farmakologie MeSH
- Staphylococcus aureus MeSH
- virulence MeSH
- Publikační typ
- časopisecké články MeSH
(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.
Antibiotic resistance is currently a serious health problem. Since the discovery of new antibiotics no longer seems to be a sufficient tool in the fight against multidrug-resistant infections, adjuvant (combination) therapy is gaining in importance as well as reducing bacterial virulence. Silymarin is a complex of flavonoids and flavonolignans known for its broad spectrum of biological activities, including its ability to modulate drug resistance in cancer. This work aimed to test eleven, optically pure silymarin flavonolignans for their ability to reverse the multidrug resistance phenotype of Staphylococcus aureus and reduce its virulence. Silybin A, 2,3-dehydrosilybin B, and 2,3-dehydrosilybin AB completely reversed antibiotic resistance at concentrations of 20 μM or less. Both 2,3-dehydrosilybin B and AB decreased the antibiotic-induced gene expression of representative efflux pumps belonging to the major facilitator (MFS), multidrug and toxic compound extrusion (MATE), and ATP-binding cassette (ABC) families. 2,3-Dehydrosilybin B also inhibited ethidium bromide accumulation and efflux in a clinical isolate whose NorA and MdeA overproduction was induced by antibiotics. Most of the tested flavonolignans reduced cell-to-cell communication on a tetrahydrofuran-borate (autoinducer-2) basis, with isosilychristin leading the way followed by 2,3-dehydrosilybin A and AB, which halved communication at 10 μM. Anhydrosilychristin was the only compound that reduced communication based on acyl-homoserine lactone (autoinducer 1), with an IC50 of 4.8 μM. Except for isosilychristin and anhydrosilychristin, all of the flavonolignans inhibited S. aureus surface colonization, with 2,3-dehydrosilybin A being the most active (IC50 10.6 μM). In conclusion, the selected flavonolignans, particularly derivatives of 2,3-dehydrosilybin B, 2,3-dehydrosilybin AB, and silybin A are non-toxic modulators of S. aureus multidrug resistance and can decrease the virulence of the bacterium, which deserves further detailed research.
Selaginella P. Beauv. is a group of vascular plants in the family Selaginellaceae Willk., found worldwide and numbering more than 700 species, with some used as foods and medicines. The aim of this paper was to compare methanolic (MeOH) and dichloromethane (DCM) extracts of eight Selaginella species on the basis of their composition and biological activities. Six of these Selaginella species are underinvestigated. Using ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis, we identified a total of 193 compounds among the tested Selaginella species, with flavonoids predominating. MeOH extracts recovered more constituents that were detected, including selaginellins, the occurrence of which is only typical for this plant genus. Of all the tested species, Selaginellaapoda contained the highest number of identified selaginellins. The majority of the compounds were identified in S. apoda, the fewest compounds in Selaginellacupressina. All the tested species demonstrated antioxidant activity using oxygen radical absorption capacity (ORAC) assay, which showed that MeOH extracts had higher antioxidant capacity, with the half maximal effective concentration (EC50) ranging from 12 ± 1 (Selaginellamyosuroides) to 124 ± 2 (Selaginellacupressina) mg/L. The antioxidant capacity was presumed to be correlated with the content of flavonoids, (neo)lignans, and selaginellins. Inhibition of acetylcholinesterase (AChE) was mostly discerned in DCM extracts and was only exhibited in S. myosuroides, S. cupressina, Selaginellabiformis, and S. apoda extracts with the half maximal inhibitory concentration (IC50) in the range of 19 ± 3 to 62 ± 1 mg/L. Substantial cytotoxicity against cancer cell lines was demonstrated by the MeOH extract of S. apoda, where the ratio of the IC50 HEK (human embryonic kidney) to IC50 HepG2 (hepatocellular carcinoma) was 7.9 ± 0.2. MeOH extracts inhibited the production of nitrate oxide and cytokines in a dose-dependent manner. Notably, S. biformis halved the production of NO, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 at the following concentrations: 105 ± 9, 11 ± 1, and 10 ± 1 mg/L, respectively. Our data confirmed that extracts from Selaginella species exhibited cytotoxicity against cancer cell lines and AChE inhibition. The activity observed in S. apoda was the most promising and is worth further exploration.
- Publikační typ
- časopisecké články MeSH
Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.
The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.
- MeSH
- AIDS opičí virologie MeSH
- buněčná membrána metabolismus virologie MeSH
- endozomy metabolismus virologie MeSH
- genové produkty env genetika metabolismus MeSH
- Masonův-Pfizerův opičí virus genetika fyziologie MeSH
- sestavení viru MeSH
- transport proteinů MeSH
- transportní vezikuly metabolismus virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Effect of processing by equal channel angular pressing (ECAP) on the degradation behaviour of extruded LAE442 magnesium alloy was investigated in a 0.1M NaCl solution, Kirkland's biocorrosion medium (KBM) and Minimum Essential Medium (MEM), both with and without 10% of foetal bovine serum (FBS). Uniform degradation of as extruded and ECAP processed samples in NaCl solution was observed, nevertheless higher corrosion resistance was found in the latter material. The increase of corrosion resistance due to ECAP was observed also after 14-days immersion in all media used. Higher compactness of the corrosion layer formed on the samples after ECAP was responsible for the observed decrease of corrosion resistance, which was proven by scanning electron microscope investigation. Lower corrosion rate in media with FBS was observed and was explained by additional effect of protein incorporation on the corrosion layer stability. A cytotoxicity test using L929 cells was carried out to investigate possible effect of processing on the cell viability. Sufficient cytocompatibility of the extruded samples was observed with no adverse effects of the subsequent ECAP processing. In conclusion, this in vitro study proved that the degradation behaviour of the LAE442 alloy could be improved by subsequent ECAP processing and this material is a good candidate for future in vivo investigation.
- MeSH
- buněčná smrt účinky léků MeSH
- buněčné linie MeSH
- chlorid sodný farmakologie MeSH
- fibroblasty cytologie účinky léků metabolismus MeSH
- hořčík chemie MeSH
- ionty MeSH
- koroze MeSH
- myši MeSH
- roztoky MeSH
- slitiny chemie MeSH
- testování materiálů metody MeSH
- vodík analýza MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The aim of this work was to compare water and organic extracts, infusions and tinctures from flowers and leaves of Calendula officinalis in terms of their biological activity and composition. The purpose of work was investigation whether the leaves and stems are really the waste or they contain interesting substances which could be utilized. Antimicrobial, antifungal, antioxidant and anti-inflammatory activities were studied. Then, the ability to inhibit collagenase was studied as well. Cytotoxicity was tested for all the samples on mammalian cell lines. METHODS: To determine the composition of extracts, infusions and tinctures phytochemical analysis (the set of colour reactions for the detection of groups of biologically active compounds) was carried out and showed that samples from flowers and leaves contain the same groups of biologically active substances (proteins and amino acids, reducing sugars, flavonoids, saponins, phenolics, terpenoids, steroids, glycosides). The antimicrobial activity of tested samples was proved, where the most sensitive bacterium was Micrococcus luteus and the most sensitive yeast was Geotrichum candidum. RESULTS: The study of anti-collagenase activity has shown that the enzymatic reaction of collagenase was affected by all tested samples and their effect was concentration dependent. Cytotoxicity of water and methanol extracts at cell lines HEK 293T and HepG2 was observed. CONCLUSION: Cells HepG2 were more sensitive than cells HEK 293T. Using cell line RAW 264.7, antiinflammatory activity of all samples was observed. Tincture of leaves was the most effective.
- MeSH
- antiflogistika izolace a purifikace toxicita MeSH
- antiinfekční látky izolace a purifikace toxicita MeSH
- antioxidancia izolace a purifikace toxicita MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- HEK293 buňky MeSH
- květy chemie MeSH
- lidé MeSH
- listy rostlin chemie MeSH
- měsíček chemie MeSH
- rostlinné extrakty izolace a purifikace toxicita MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Recently, iron-based materials have been considered as candidates for the fabrication of biodegradable load-bearing implants. Alloying with palladium has been found to be a suitable approach to enhance the insufficient corrosion rate of iron-based alloys. In this work, we have extensively compared the microstructure, the mechanical and corrosion properties, and the cytotoxicity of an FePd2 (wt%) alloy prepared by three different routes - casting, mechanical alloying and spark plasma sintering (SPS), and mechanical alloying and the space holder technique (SHT). The properties of the FePd2 (wt%) were compared with pure Fe prepared in the same processes. The preparation route significantly influenced the material properties. Materials prepared by SPS possessed the highest values of mechanical properties (CYS~750-850MPa) and higher corrosion rates than the casted materials. Materials prepared by SHT contained approximately 60% porosity; therefore, their mechanical properties reached the lowest values, and they had the highest corrosion rates, approximately 0.7-1.2mm/a. Highly porous FePd2 was tested in vitro according to the ISO 10993-5 standard using L929 cells, and two-fold diluted extracts showed acceptable cytocompatibility. In general, alloying with Pd enhanced both mechanical properties and corrosion rates and did not decrease the cytocompatibility of the studied materials.
New materials with appropriate mechanical properties and an antibacterial effect are constantly being sought for orthopedic and dental applications. The aim of this study was to investigate newly developed TiSi alloys coated with titania sol-gel containing silver. Titanium alloys with 5 or 10wt% of silicon were prepared by vacuum arc remelting and dip-coated with titania sol containing either AgNO3or Ag3PO4in two concentrations. The size and distribution of the particles in the layer were evaluated, as well as layer compactness (SEM). The antibacterial effect (against E. coli and S. epidermidis) and cytotoxicity (towards L929 and U-2 OS cell lines) of these materials were then tested. Despite cracking of the coatings after firing, the coatings demonstrated very good antibacterial effects against both E. coli and S. epidermidis after 24h of interaction. None of the tested materials were toxic to both cell lines. Collectively, our results suggest that these materials are promising candidates for orthopedic applications.
