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Metabolism of metals in microalgae and adaptation to metal excess are of significant environmental importance. We report a three-step mechanism that the green microalga Chlorella sorokiniana activates during the acquisition of and adaptation to manganese (Mn), which is both an essential trace metal and a pollutant of waters. In the early stage, Mn2+ was mainly bound to membrane phospholipids and phosphates in released mucilage. The outer cell wall was reorganized and lipids were accumulated, with a relative increase in lipid saturation. Intracellular redox settings were rapidly altered in the presence of Mn excess, with increased production of reactive oxygen species that resulted in lipid peroxidation and a decrease in the concentration of thiols. In the later stage, Mn2+ was chelated by polyphosphates and accumulated in the cells. The structure of the inner cell wall was modified and the redox milieu established a new balance. Polyphosphates serve as a transient Mn2+ storage ligand, as proposed previously. In the final stage, Mn was stored in multivalent Mn clusters that resemble the structure of the tetramanganese–calcium core of the oxygen-evolving complex. The present findings elucidate the bioinorganic chemistry and metabolism of Mn in microalgae, and may shed new light on water-splitting Mn clusters.

Introduction: Comprising of a unique combination of bioactive polyphenol compounds, grapes are shown to have a beneficial effect on the skin as well as the whole body. They exhibit high antioxidative and antiradical activity through mechanisms of heavy metals chelation, scavenging reactive oxygen species, inhibiting lipid peroxidation, and preserving the integrity of the cell membranes. In the winemaking industry, after crushing and pressing, grape pomace (stems, seeds, pulp, and skin) is removed as a by-product. This valuable source of bioactive compounds is often treated as animal food, compost, raw material in the brewery, but unfortunately mostly disposed of as unusable waste. Aim: The aim of this study was to compare redox properties of absolute EtOH, 50% EtOH, and H2 O grape wine skin extracts in a way of scavenging DPPH radicals, showing that biowaste obtained from grape wineskins could be used as an immense source of bioactive compounds with high antiradical activity. Material and Methods: Electron Paramagnetic Resonance (EPR) spectroscopy was used to detect the activity of grape skin extracts toward DPPH radicals and UHPLC-DAD MS/MS analysis to separate, identify and quantify their active components. Results: The results show that Cabernet Sauvignon and Pinot Blanc grape skin extracts have unexpectedly large potential to remove DPPH radicals from the system. Having in mind the concentration of redox-active components obtained by UHPLC-DAD MS/MS analysis and presented EPR results, there is strong evidence that primarily quercetin 3-O-glucoside, as well as kaempferol 3-O-glucoside, isorhamnetin 3-O-glucoside, kaempferol 7-O-glucoside supplemented with catechin and rutin are responsible for the antioxidative capacity of extracts. Conclusions: This study demonstrates that extracts obtained from grape skins, initially intended as biowaste, exhibit high antiradical activity. The largest quantity of the most redox-active components could be found in 50% EtOH extracts, which we propose to be the starting material for making potent redox-active cosmetic products and dietary supplements.

There is a significant interest in understanding coordination and photo-chemistry of tetracycline antibiotics, primarily in relation to the development of advanced oxidation processes for degradation of these pollutants in water processing. Herein we analyzed the pH-dependence of interactions of tetracycline with ferric iron and photosensitivity of tetracycline to UV-A and UV-B, using a set of methods – UV–vis, Raman and electron paramagnetic resonance spectroscopy, MS spectrometry, HPLC, and cyclic voltammetry. Tetracycline and Fe3+ mainly bind through amide and OH groups in tricarbonylamide moiety to form a stable complex with 1:1 stoichiometry at pH ≤ 5. The interaction is reversible and tetracycline is released from the complex with pH increase. Tetracycline in the complex is stabilized and less susceptible than free tetracycline to oxidation by hydroxyl radical that is produced by UV-induced photolysis of Fe3+–OH- complexes. Redox properties of tetracycline were altered with increasing pH and it showed increased susceptibility to UV-induced degradation. In close, the system composed of tetracycline and ferric iron shows coordination and photo-redox chemistry that is dependent of pH in relation to the solubility of Fe3+ species and protonation of tetracycline. The development and optimization of advanced oxidation processes should take into account that iron may bind and stabilize pollutants and that the redox landscape of water changes drastically with pH. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/5512]

Biliverdin (BV) structure was analyzed by using NMR techniques and unrestricted density function theory simulations to explain the incapacity of BV to build coordination complex(es) with Cu2+ in dimethyl sulfoxide, which was confirmed by UV-Vis, EPR and NMR spectroscopy. NMR showed that N atoms are protonated in all four pyrrole rings. The structure is stabilized by two hydrogen bonds between NH moieties and carbonyl oxygens from opposite terminal pyrrole rings, and by the bending of propionyl chain with carboxyl group out of the plain toward central position of BV. The simulations of deprotonated BV, which builds copper complexes in water and chloroform as described previously, showed a different conformation and organization of hydrogen bonds. Taking into account that deprotonation represents a critical step in coordinate bonds formation, the protonation of an additional N atom may represent a key difference between the interactions of BV with copper in different solvents.

Biliverdin (BV), a product of heme catabolism, is known to interact with transition metals, but the details of such interactions under physiological conditions are scarce. Herein, we examined coordinate/redox interactions of BV with Cu2+ in phosphate buffer at pH 7.4, using spectrophotometry, HESI-MS, Raman spectroscopy, 1 H NMR, EPR, fluorimetry, and electrochemical methods. BV formed a stable coordination complex with copper in 1 : 1 stoichiometry. The structure of BV was more planar and energetically stable in the complex. The complex showed strong paramagnetic effects that were attributed to an unpaired delocalized e−. The delocalized electron may come from BV or Cu2+, so the complex is formally composed either of BV radical cation and Cu1+ or of BV radical anion and Cu3+. The complex underwent oxidation only in the presence of both O2 and an excess of Cu2+, or a strong oxidizing agent, and it was resistant to reducing agents. The biological effects of the stable BV metallocomplex containing a delocalized unpaired electron should be further examined, and may provide an answer to the long-standing question of high energy investment in the catabolism of BV, which represents a relatively harmless molecule per se. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3069] This is the peer-reviewed version of the following article: Dimitrijević, M. S.; Bogdanović Pristov, J.; Žižić, M.; Stanković, D. M.; Bajuk-Bogdanović, D.; Stanić, M.; Spasić, S.; Hagen, W.; Spasojević, I. Biliverdin-Copper Complex at Physiological PH. Dalton Transactions 2019, 48 (18), 6061–6070. [https://doi.org/10.1039/c8dt04724c] Published version: [https://vinar.vin.bg.ac.rs/handle/123456789/8200]

Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characterized by specialized metabolite profiles predominated by iridoids and phenolics, are differentiated according to the stereochemistry of major iridoid aglycone nepetalactone (NL). For the first time, the present study provides a comparative analysis of antimicrobial and immunomodulating activities of the two Nepeta species and their major iridoids isolated from natural sources—cis,trans-NL, trans,cis-NL, and 1,5,9-epideoxyloganic acid (1,5,9-eDLA), as well as of phenolic acid rosmarinic acid (RA). Methanol extracts and pure iridoids displayed excellent antimicrobial activity against eight strains of bacteria and seven strains of fungi. They were especially potent against food-borne pathogens such as L. monocytogenes, E. coli, S. aureus, Penicillium sp., and Aspergillus sp. Targeted iridoids were efficient agents in preventing biofilm formation of resistant P. aeruginosa strain, and they displayed additive antimicrobial interaction. Iridoids are, to a great extent, responsible for the prominent antimicrobial activities of the two Nepeta species, although are probably minor contributors to the moderate immunomodulatory effects. The analyzed iridoids and RA, individually or in mixtures, have the potential to be used in the pharmaceutical industry as potent antimicrobials, and in the food industry to increase the shelf life and safety of food products.

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.

Upon release in response to stress, epinephrine (Epi) may interact with labile iron pool in human plasma with potentially important (patho)physiological consequences. We have shown that Epi and Fe3+ build stable 1:1 high-spin bidentate complex at physiological pH, and that Epi does not undergo degradation in the presence of iron. However, the interactions of Epi with the more soluble Fe2+, and the impact of iron on biological activity of Epi are still not known. Herein we showed that Epi and Fe2+ build colorless complex which is stable under anaerobic conditions. In the presence of O2, Epi promoted the oxidation of Fe2+ and the formation of Epi-Fe3+ complex. Cyclic voltammetry showed that mid-point potential of Epi-Fe2+ complex is very low (−582 mV vs. standard hydrogen electrode), which explains catalyzed oxidation of Fe2+. Next, we examined the impact of iron binding on biological performance of Epi using patch clamping in cell culture with constitutive expression of adrenergic receptors. Epi alone evoked an increase of outward currents, whereas Epi in the complex with Fe3+ did not. This implies that the binding of Epi to adrenergic receptors and their activation is prevented by the formation of complex with iron. Pro-oxidative activity of Epi-Fe2+ complex may represent a link between chronic stress and cardiovascular problems. On the other hand, labile iron could serve as a modulator of biological activity of ligands. Such interactions may be important in human pathologies that are related to iron overload or deficiency.

Introduction. We tested whether the microclimate under grey hail protection nets (HPN) had an influence on the phenology, vegetative and reproductive potential, and fruit quality traits of ’Duke’ highbush blueberry (Vaccinium corymbosum L.) in two consecutive years (2013, 2014). Materials and methods. Light intensity, air temperature and relative humidity were evaluated together with biological properties (flowering and ripening time, bush height and volume, number of flowers and fruits per bush, fruit set percentage, and yield per bush), and fruit quality including biometrical (fruit weight, index of fruit shape and number of seeds per fruit) and nutritional (soluble solid content – SSC, titratable acidity – TA, total anthocyanins content – TACY, total phenolic content – TPC and total antioxidant capacity – TAC) parameters. Results and discussion. In both seasons, light was reduced by 5–20% under HPN, whereas daily maximum temperature was 2.4 °C higher in the open field (OF). This caused two days earlier ripening time in OF compared to the HPN. The increased yield per bush under HPN was the result of larger fruit weight. SSC were unaffected by the net in 2013 while in 2014 significantly higher value of SSC was registered under HPN compared to OF. Although apparent differences in TACY and TPC were not observed between the treatments, TAC significantly increased under HPN in 2014. Conclusion. The use of grey HPN could be considered as a suitable alternative for the hail protection of highbush blueberry ensuring an increased fruit production without negative effects on their quality.

We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation-emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide.

The aim of the present study was to synthesize a composite of raw interstratified montmorillonite-kaolinite clay (Mt-K) with TiO2 as an additive by mechanochemical activation and investigate its usability as low-cost adsorbent for simultaneous removal of heavy metals from acidic aqueous solutions. The composite synthesized with amorphous TiO2 (TiO(2,)a) showed a significantly better removal ability of heavy metals from highly acidic solutions (pH = 2.0-4.0) compared with the Mt-K milled for an optimum period of time of 19 h (Mt-K,m) and composite synthesized with crystalline TiO2 (TiO(2,)c). As both of the composites showed the same morphologies, the same distribution of TiO2 particles on the clay matrix, the same particle size distribution (PSD) dependencies and point of zero charge (pH(PZC)) values, it has to be noticed that the difference in the adsorption behavior lies only in the different crystalline forms of the additive, i.e. TiO2 that was used for the composite preparation. It has been shown that 20% of TiO(2,)a was the optimal amount of an additive in the composite to achieve improved adsorption ability. (C) 2014 Elsevier B.V. All rights reserved.