UV-induced fingerprint spectroscopy (UV-IFS), a new tool in a toolbox of analytical methods, is a powerful technique registering molecule-specific changes of fluorescence induced by UV irradiation. Analysis of fluorescence spectra of a sample prior and after UV irradiation enables an identification of a sample of a drug or pharmaceutics based on a comparison with signals of known standards. Moreover, UV-IFS uncovers the presence of undesired contaminations or intentional changes of the composition. Herein, we employ UV-IFS for qualitative as well as quantitative analysis of common medicines including analgesic/antipyretic (Acetaminophen), antihistamines (Loratadine and Desloratadine), and phosphodiesterase type 5 inhibitors (Tadalafil and Sildenafil citrate). UV irradiation (λem = 254 nm) for 2 - 10 min induced significant changes of fluorescence of the studied samples and according to the unique patterns, the quality and quantity were evaluated. Limits of detection for individual active ingredients were calculated as follows: Acetaminophen = 0.1 μg·mL-1, Loratadine = 0.1 μg·mL-1, Desloratadine = 0.01 μg·mL-1, Tadalafil = 0.04 μg·mL-1 and Sildenafil = 0.2 μg·mL-1. Moreover, genuine and fake CIALIS, VIAGRA and KAMAGRA tablets were reliably identified.
- MeSH
- loratadin * MeSH
- paracetamol * MeSH
- sildenafil citrát MeSH
- spektrální analýza MeSH
- tablety MeSH
- tadalafil MeSH
- Publikační typ
- časopisecké články MeSH
Crosslinked 3D porous collagen-polysaccharide scaffolds, prepared by freeze-drying, were modified with bovine platelet lysate (BPL) and evaluated in terms of chemical, physical and biological properties. Natural antibacterial polysaccharides like chitosan, chitin/chitosan-glucan complex and calcium salt of oxidized cellulose (CaOC) incorporated in collagen scaffolds affected not only chemo-physical properties of the composite scaffolds but also improved their biological properties, especially when BPL was presented. Lipophilic BPL formed microspheres in porous scaffolds while reduced by half their swelling ratio. The resistance of collagen sponges to hydrolytic degradation in water depended strongly on chemical crosslinking varying from 60 min to more than one year. According to in-vitro tests, chemically crosslinked scaffolds exhibited a good cellular response, cell-matrix interactions, and biocompatibility of the material. The combination of collagen with natural polysaccharides confirmed a significant positive synergistic effect on cultivation of cells as determined by MTS assay and PicoGreen method, as well as on angiogenesis evaluated by ex ovo Chick Chorioallantoic Membrane (CAM) assay. Contrary, modification only by BLP of pure collagen scaffolds exhibited decreased biocompatibility in comparison to unmodified pure collagen scaffold. We propose that the newly developed crosslinked collagen sponges involving bioactive additives could be used as scaffold for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration.
- MeSH
- buněčná adheze účinky léků MeSH
- buňky 3T3 MeSH
- fibroblasty cytologie účinky léků MeSH
- fyziologická neovaskularizace účinky léků MeSH
- hydrolýza MeSH
- kolagen farmakologie MeSH
- kur domácí MeSH
- myši MeSH
- polysacharidy farmakologie MeSH
- proliferace buněk účinky léků MeSH
- reagencia zkříženě vázaná chemie MeSH
- skot MeSH
- teplota MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- trombocyty metabolismus MeSH
- voda chemie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Suitable fluorophores are the core of fluorescence imaging. Among the most exciting, yet controversial, labels are quantum dots (QDs) with their unique optical and chemical properties, but also considerable toxicity. This hinders QDs applicability in living systems. Surface chemistry has a profound impact on biological behavior of QDs. This study describes a two-step synthesis of QDs formed by CdTe core doped with Schiff base ligand for lanthanides [Ln (Yb3+, Tb3+ and Gd3+)] as novel cytocompatible fluorophores. RESULTS: Microwave-assisted synthesis resulted in water-soluble nanocrystals with high colloidal and fluorescence stability with quantum yields of 40.9-58.0%. Despite induction of endocytosis and cytoplasm accumulation of Yb- and TbQDs, surface doping resulted in significant enhancement in cytocompatibility when compared to the un-doped CdTe QDs. Furthermore, only negligible antimigratory properties without triggering formation of reactive oxygen species were found, particularly for TbQDs. Ln-doped QDs did not cause observable hemolysis, adsorbed only a low degree of plasma proteins onto their surface and did not possess significant genotoxicity. To validate the applicability of Ln-doped QDs for in vitro visualization of receptor status of living cells, we performed a site-directed conjugation of antibodies towards immuno-labeling of clinically relevant target-human norepinephrine transporter (hNET), over-expressed in neuroendocrine tumors like neuroblastoma. Immuno-performance of modified TbQDs was successfully tested in distinct types of cells varying in hNET expression and also in neuroblastoma cells with hNET expression up-regulated by vorinostat. CONCLUSION: For the first time we show that Ln-doping of CdTe QDs can significantly alleviate their cytotoxic effects. The obtained results imply great potential of Ln-doped QDs as cytocompatible and stable fluorophores for various bio-labeling applications.
- MeSH
- analýza jednotlivých buněk metody MeSH
- fluorescenční barviva toxicita MeSH
- kvantové tečky toxicita MeSH
- lanthanoidy chemie MeSH
- lidé MeSH
- mikrovlny MeSH
- nádorové buněčné linie MeSH
- optické zobrazování metody MeSH
- povrchové vlastnosti MeSH
- Schiffovy báze chemie MeSH
- sloučeniny kadmia toxicita MeSH
- telur toxicita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Poly(3-hydroxybutyrate) (PHB) is a common carbon- and energy-storage compound simultaneously produced and degraded into its monomer 3-hydroxybutyrate (3HB) by numerous bacteria and Archae in a metabolic pathway called the PHB cycle. We investigated 3HB as a chemical chaperone capable of protecting model enzymes, namely lipase and lysozyme, from adverse effects of high temperature and oxidation. Heat-mediated denaturation of lipase in the presence or absence of 3HB was monitored by dynamic light scattering (DLS) revealing a significant protective effect of 3HB which increased as its concentration rose. Furthermore, when compared at the same molar concentration, 3HB showed a greater protective effect than the well-known chemical chaperones trehalose and hydroxyectoine. The higher protective effect of 3HB was also confirmed when employing differential scanning calorimetry (DSC) and lysozyme as a model enzyme. Furthermore, 3HB was capable of protecting lipase not only against thermal-mediated denaturation but also against oxidative damage by Cu(2+) and H2O2; its protection was higher than that of trehalose and comparable to that of hydroxyectoine. Taking into account that the PHB-producing strain Cupriavidus necator H16 reveals a 16.5-fold higher intracellular concentration than the PHB non-producing mutant C. necator PHB(-4), it might be expected that the functional PHB cycle might be responsible for maintaining a higher intracellular level of 3HB which, aside from other positive aspects of functional PHB metabolism, enhances stress resistance of bacterial strains capable of simultaneous PHB synthesis and mobilization. In addition, 3HB can be used in various applications and formulations as an efficient enzyme-stabilizing and enzyme-protecting additive.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- Cupriavidus necator chemie enzymologie metabolismus MeSH
- hydroxybutyráty metabolismus MeSH
- kyselina 3-hydroxymáselná chemie metabolismus MeSH
- muramidasa chemie metabolismus MeSH
- ochranné látky chemie metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres MeSH
- polyestery metabolismus MeSH
- stabilita enzymů MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen-thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences.
Coffee is one of the world's most popular beverages and has been growing steadily in commercial importance. Nowadays, coffee is the second largest traded commodity in the world, after petroleum. Hence, coffee industry is responsible for the generation of large amounts of waste, especially spent coffee grounds (SCG). Various attempts to valorize this waste stream of coffee industry were made. This article summarizes our research and publications aiming at the conversion of SCG into valuable products - polyhydroxyalkanoates (PHAs) and carotenoids. At first, oil extracted from SCG (approx. 15 wt% oil in SCG) can be efficiently (YP/S=0.82 g/g) converted into PHA employing Cupriavidus necator H16. Further, the solid residues after oil extraction can be hydrolyzed (by the combination of chemical and enzymatic hydrolysis) yielding fermentable sugars, which can be further used as a substrate for the production of PHAs employing Bacillus megaterium (YP/S=0.04 g/g) or Burkholderia cepacia (YP/S=0.24 g/g). Alternatively, SCG hydrolysate can be used as a substrate for biotechnological production of carotenoids by carotenogenic yeast Sporobolomyces roseus. Solid residues after either oil extraction or hydrolysis can be used as fuel in industrial boilers to generate heat and energy. Therefore, entire biomass of SCG can be used for sustainable production of PHAs and/or carotenoids employing bio-refinery approach.
- MeSH
- biopaliva mikrobiologie MeSH
- biotechnologie metody MeSH
- extrakce kapalina-kapalina metody MeSH
- karotenoidy biosyntéza chemie izolace a purifikace MeSH
- káva chemie mikrobiologie MeSH
- odpadky - odstraňování metody MeSH
- polyhydroxyalkanoáty biosyntéza chemie izolace a purifikace MeSH
- zachování přírodních zdrojů metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Using random chemical mutagenesis we obtained the mutant of Cupriavidus necator H16 which was capable of improved (about 35 %) production of poly(3-hydroxybuytrate) (PHB) compared to the wild-type strain. The mutant exhibited significantly enhanced specific activities of enzymes involved in oxidative stress response such as malic enzyme, NADP-dependent isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. Probably, due to the activation of these enzymes, we also observed an increase of NADPH/NADP⁺ ratio. It is likely that as a side effect of the increase of NADPH/NADP⁺ ratio the activity of PHB biosynthetic pathway was enhanced, which supported the accumulation of PHB. Furthermore, the mutant was also able to incorporate propionate into copolymer poly(3-hydroxybuytyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] more efficiently than the wild-type strain (Y3HV/prec = 0.17 and 0.29 for the wild-type strain and the mutant, respectively)). We assume that it may be caused by lower availability of oxaloacetate for the utilization of propionyl-CoA in 2-methylcitrate cycle due to increased action of malic enzyme. Therefore, propionyl-CoA was incorporated into copolymer rather than transformed to pyruvate via 2-methylcitrate cycle. Thus, the mutant was capable of the utilization of waste frying oils and the production of P(3HB-co-3HV) with better yields and improved content of 3HV resulting in better mechanical properties of copolymer than the wild-type strain. The results of this work may be used for the development of innovative fermentation strategies for the production of PHA and also it might help to define novel targets for the genetic manipulations of PHA producing bacteria.
- MeSH
- acylkoenzym A metabolismus MeSH
- bakteriální proteiny genetika metabolismus MeSH
- bioreaktory MeSH
- biosyntetické dráhy MeSH
- Brassica rapa MeSH
- Cupriavidus necator účinky léků genetika metabolismus MeSH
- fermentace MeSH
- hydroxybutyráty metabolismus MeSH
- mutageneze * MeSH
- oleje rostlin chemie metabolismus MeSH
- oxidační stres MeSH
- polyestery metabolismus MeSH
- polyhydroxyalkanoáty metabolismus MeSH
- průmyslový odpad MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
