12 research outputs found

    Hydroxyapatite nanoparticles-cell interaction: New approaches to disclose the fate of membrane-bound and internalised nanoparticles

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    Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.Peer ReviewedPostprint (published version

    Aufnahme, Transport und Toxizität von Mangan in zwei Varietäten der Douglasie (Pseudotsuga menziesii) unter dem Einfluß von Mykorrhizen: von der zellulären bis zur organismischen Ebene

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    Obwohl Mangan (Mn) ein essentielles Mikro-Nährelement in allen Organismen darstellt, ist es in höheren Konzentrationen toxisch. Es wurde in dieser Arbeit untersucht, wie sich zwei unterschiedlich Mn sensitive Varietäten der Douglasie (Pseudotsuga menziesii), Viridis und Glauca in ihrer Mn-Aufnahme, subzellulären Lokalisation, Transport und Verteilung von Mangan unterscheiden. Unter kontrollierten Bedingungen konnte in beiden Varietäten unter Mn-Überschuss vor allem eine unterschiedliche Entwicklung der Keimlinge und subzelluläre Lokalisation von Mn festgestellt werden. Während in der Varietät Glauca die Entwicklung der Wurzelbiomasse am stärksten durch Manganüberschuss gehemmt wurde, war in der Varietät viridis die Biomasseentwicklung des Sprosses am stärker betroffen. Durch Röntgen-Mikroanalyse konnte gezeigt werden, dass Mn in beiden Varietäten vor allem in der Epidermis und den Cortexzellen der Wurzel akkumulierte. Vermutlich stellt die Endodermis eine natürliche Barriere für Mn dar um das vaskuläre System vor Mn Überschuss zu schützen. In den Cortex- und Epidermiszellen der Wurzel wurden Vesikel sehr hohen Mn-Gehalten nachgewiesen. In diesen Vesikeln korrelierte der Gehalt an Mn mit Ca und P was auf eine Komplexierung von Mn durch diese Elemente hindeutet. In der Varietät glauca nicht aber in viridis wurde der P Gehalt auf zellulärer Ebene auch unter P Mangel konstant gehalten und P-Mangel führte zu höherer Toleranz gegenüber Mn. Es konnte nur ein begrenzter Einfluss von Mn auf mit Rhizopogon subareolatus mykorrhizierte Keimlinge beider Varietäten festgestellt werden. In mykorrhizierten Keimlingen waren im Vergleich zu nicht mykorrhizierten Pflanzen die Mn-Gehalte in Geweben nahe der Endodemis erhöht. Eine Mykorrhizierung mit Rhizopogon subareolatus hatte eher nachteilige Effekte auf die Biomasseentwicklung und Mn-Aufnahme der Douglasie. Eine molekulare Identifikation der Mykorrhiza von Douglasien beider Provinienzen, die zwei Jahre in natürlichen Böden zweier unterschiedlicher Standorte mit unterschiedlichen Mn-Konzentrationen gewachsen sind ergab, dass sich sowohl eine Boden spezifische- wie auch eine Varietäten spezifische Mykorrhiza Gesellschaft entwickelte. Die Netto Assimilation von Stickstoff, Phosphor und Kohlenstoff zeigte eine positive Korrelation mit der Intensität der Mykorrhizierung. Diese Ergebnisse deuten darauf hin, dass ein komplexer Zusammenhang zwischen Genotyp des pflanzlichen Partners, der Ausgangspopulation an potentiellen pilzlichen Partnern und der Bodenchemie in der Ausbildung einer spezifischen Mykorrhizapopulation besteht

    Oxalate oxidase and non-enzymatic compounds of the antioxidative system in young Serbian spruce plants exposed to cadmium stress

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    We studied changes in the concentrations of ascorbate and glutathione, composition of soluble phenolics, and activity of oxalate oxidase in 75-day-old Serbian spruce plants after exposure to 5 mu M and 50 mu M cadmium for 6-48 h. The presence of OxOx activity in a conifer species is here demonstrated for the first time. Both Cd concentrations induced a decrease of OxOx activity in treated plants in comparison with the control at all sampling dates. The concentrations of reduced glutathione, its oxidized form, and reduced ascorbate in the plants decreased during 48-h treatment with cadmium. Among simple phenolics, only catechin increased significantly during Cd treatment

    Phosphorus homeostasis in Populus alba L. under excess phosphate conditions, assessed by 31P nuclear magnetic resonance spectroscopy and X-ray microfluorescence

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    The phosphates (Pi) are nowadays recognized as pollutants. We studied the effect of Pi (0.625-12.500 mM KH2PO4) in the culture medium on in vitro grown 2-month-old Populus alba trees. The levels of sugar phosphates and vacuolar and cytoplasmic Pi in cell compartments of roots and stems were determined using P-31 NMR, while tissue-specific micro- and macroelements mapping on stem cross-sections were performed using synchrotron-based X-ray microfluorescence. Plants grown on 0.625 mM Pi (MS/2 medium) showed a survival rate of 70%. With the increase in Pi concentrations up to 6.250 mM, plant growth and survival increased, without changes in total P content per mass or in the levels of cytoplasmic and vacuolar phosphates, in both stems and roots, while the levels of Fe, Cu, Zn, Ca and Mn in stems increased. Further increase in Pi to 9.375 and 12.500 mM in the medium resulted in inhibited growth comparable with plants grown on MS/2, with the increase in total P content per mass up to 50%, in both stems and roots, but with no changes in cytoplasmic and vacuolar phosphates; 12.500 mM Pi affected even plant survival (70%) and thus might be considered as mildly toxic. P-31 NMR results indicate that the high tolerance of P. alba to increased Pi could result from its ability to maintain an intracellular P homeostasis, despite P accumulation up to 50%, in both stems and roots, indicating P. alba as a promising wood species for dendroremediation

    Variability of antioxidant enzyme activity and isoenzyme profile in needles of Serbian spruce (Picea omorika (Panc.) Purkinye)

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    Variations were studied of the activity and isoenzyme patterns of soluble peroxidase, catalase, catechol oxidase and superoxide dismutase, in needles of the Balkan endemic conifer Serbian spruce, Picea omorika (Panc.) Purkinye. The samples were collected from the natural habitat of the species, Mt. Tara. Seasonal changes were found to affect enzymatic activities and isoenzyme profiles. Total protein content was significantly lower in the summer than in other seasons. Several isoforms of peroxidase, catechol oxidase and superoxide dismutase (SOD), as well as two catalase isoenzymes were detected. The number of peroxidase isoenzymes was greatest during the vegetative season. Catalase and catechol oxidase peaked in summer and spring, respectively. Total SOD and Mn-SOD activities were significantly higher in the winter samples than the summer ones. (C) 2006 Elsevier Ltd. All rights reserved

    Lipid status of A2780 ovarian cancer cells after treatment with ruthenium complex modified with carbon dot nanocarriers: a multimodal SR-FTIR spectroscopy and MALDI TOF Mass Spectrometry Study MALDI TOF Mass Spectrometry Study

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    In the last decade, targeting membrane lipids in cancer cells has been a promising approach that deserves attention in the field of anticancer drug development. To get a comprehensive understanding of the effect of the drug [Ru(¿5-Cp)(PPh3)2CN] (RuCN) on cell lipidic components, we combine complementary analytical approaches, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI TOF MS) and synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectroscopy. Techniques are used for screening the effect of potential metallodrug, RuCN, without and with drug carriers (carbon dots (CDs) and nitrogen-doped carbon dots (N-CDs)) on the lipids of the human ovarian cancer cell line A2780. MALDI TOF MS results revealed that the lysis of ovarian cancer membrane lipids is promoted by RuCN and not by drug carriers (CDs and N-CDs). Furthermore, SR-FTIR results strongly suggested that the phospholipids of cancer cells undergo oxidative stress after the treatment with RuCN that was accompanied by the disordering of the fatty acid chains. On the other hand, using (N-)CDs as RuCN nanocarriers prevented the oxidative stress caused by RuCN but did not prevent the disordering of the fatty acid chain packing. Finally, we demonstrated that RuCN and RuCN/(N-)CDs alter the hydration of the membrane surface in the membrane—water interface region.ALBA Synchrotron, MIRAS Beamline (experiment No. 2019093770). This work was also supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant 451-03-68/2020-14/200017); FCT(CQM Base Fund—UIDB/00674/2020, Programmatic Fund—UIDP/00674/2020); Madeira 14-20 Program (project Reforço do Investimentoem Equipamentos e Infrastructures Científcasna RAM (PROEQUIPRAM) M1420-01-0145-FEDER-000008); and Agência Regional para o Desenvolvimento da Investigação, Tecnologia e Inovação (ARDITI) through the ARDITI-CQM/2018/007-PDG (Fellowship Grant to M.G.), project M1420-01-0145-FEDER-000005-CQM+ (Madeira 14—20)

    X-ray absorption near-edge structure micro-spectroscopy study of vanadium speciation in Phycomyces blakesleeanus mycelium

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    Vanadium speciation in the fungus Phycomyces blakesleeanus was examined by X-ray absorption near-edge structure (XANES) spectroscopy, enabling assessment of oxidation states and related molecular symmetries of this transition element in the fungus. The exposure of P. blakesleeanus to two physiologically important vanadium species (V5+ and V4+) resulted in the accumulation of this metal in central compartments of 24 h old mycelia, most probably in vacuoles. Tetrahedral V5+, octahedral V4+, and proposed intracellular complexes of V5+ were detected simultaneously after addition of a physiologically relevant concentration of V5+ to the mycelium. A substantial fraction of the externally added V4+ remained mostly in its original form. However, observable variations in the pre-edge-peak intensities in the XANES spectra indicated intracellular complexation and corresponding changes in the molecular coordination symmetry. Vanadate complexation was confirmed by V-51 NMR and Raman spectroscopy, and potential binding compounds including cell-wall constituents (chitosan and/or chitin), (poly)phosphates, DNA, and proteins are proposed. The evidenced vanadate complexation and reduction could also explain the resistance of P. blakesleeanus to high extracellular concentrations of vanadium

    Estimation of carbon dots amelioration of copper toxicity in maize studied by synchrotron radiation-FTIR

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    Carbon dots are biocompatible and non-toxic nanoparticles with chemical affinity to some heavy metals. Human activities increase soil pollution with copper. Cu is an essential microelement in plants, but excess can induce a harmful effects. In plant response to Cu, the cell wall plays an important role. This study aims to estimate possible amelioration effects of folic acid based CDs on Cu toxicity by studying the intracellular and cell wall compounds in maize (Zea mays L.) roots and leaves after 7 day-treatment in hydroponics. The sub-cellular compartmentalization and bio-macromolecular changes induced by 5 mu M Cu applied alone or with CDs (167 and 500 mg/L) were studied using the Synchrotron-based Fourier transformmicro-spectroscopy (SR-FTIR) combined with X-Ray photoelectron spectroscopy (XPS). Cu induced changes in content of cell wall polysaccharides, proteins, and lipids. The XPS detected CDs transport throughout the plants. The Cu/167CDs treatment reduced Cu concentration in the roots, possibly by complexation/trapping between the functional groups on CDs surface and Cu2+. Principal component analysis of FTIR spectra confirmed that Cu/500CDs treatment increased Cu adverse effects in most tissues but alleviated adverse Cu effects on cell wall polysaccharides in the root xylem, and on polysaccharides and proteins in leaf phloem and mesophyll

    Mechanisms of detoxification of high copper concentrations by the microalga Chlorella sorokiniana

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    Microalgae have evolved mechanisms to respond to changes in copper ion availability, which are very important for normal cellular function, to tolerate metal pollution of aquatic ecosystems, and for modulation of copper bioavailability and toxicity to other organisms. Knowledge and application of these mechanisms will benefit the use of microalgae in wastewater processing and biomass production, and the use of copper compounds in the suppression of harmful algal blooms. Here, using electron microscopy, synchrotron radiation-based Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, and X-ray absorption fine structure spectroscopy, we show that the microalga Chlorella sorokiniana responds promptly to Cu2+ at high non-toxic concentration, by mucilage release, alterations in the architecture of the outer cell wall layer and lipid structures, and polyphosphate accumulation within mucilage matrix. The main route of copper detoxification is by Cu2+ coordination to polyphosphates in penta-coordinated geometry. The sequestrated Cu2+ was accessible and could be released by extracellular chelating agents. Finally, the reduction in Cu2+ to Cu1+ appears also to take place. These findings reveal the biochemical basis of the capacity of microalgae to adapt to high external copper concentrations and to serve as both, sinks and pools of environmental copper
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