Non-thermal plasma induces changes in aflatoxin production, devitalization, and surface chemistry of Aspergillus parasiticus.

Non-thermal plasma (NTP) represents the fourth state of matter composed of neutral molecules, atoms, ions, radicals, and electrons. It has been used by various industries for several decades, but only recently NTPs have emerged in fields such as medicine, agriculture, and the food industry. In this work, we studied the effect of NTP exposure on aflatoxin production, conidial germination and mycelial vitality, morphological and surface changes of conidia and mycelium. When compared with colonies grown from untreated conidia, the colonies from NTP-treated conidia produced significantly higher levels of aflatoxins much earlier during development than colonies from untreated conidia. However, at the end of cultivation, both types of cultures yielded similar aflatoxin concentrations. The increase in the accumulation of aflatoxins was supported by high transcription levels of aflatoxin biosynthetic genes, which indicated a possibility that NTP treatment of conidia was having a longer-lasting effect on colony development and aflatoxins accumulation. NTP generated in the air at atmospheric pressure effectively devitalized Aspergillus parasiticus in conidia and hyphae within a few minutes of treatment. To describe devitalization kinetics, we applied Weibull and Hill models on sets of data collected at different exposure times during NTP treatment. The damage caused by NTP to hyphal cell wall structures was displayed by raptures visualized by scanning electron microscopy. Fourier transform infrared spectroscopy demonstrated that changes in cell envelope correlated with shifts in characteristic chemical bonds indicating dehydration, oxidation of lipids, proteins, and polysaccharides. Key points • Non-thermal plasma increases aflatoxin production shortly after treatment. • Non-thermal plasma rapidly devitalizes Aspergillus parasiticus. • Non-thermal plasma disrupts the cell surface and oxidizes biological components.

In vitro biological evaluation and consideration about structure-activity relationship of silver(I) aminoacidate complexes.

Two silver(I) aminoacidate complexes {[Ag4(L-HAla)4(NO3)3]NO3}n (AgAla, complex 1, Ala = alanine) and {[Ag(L-Phe)]}n (AgPhe, complex 2, Phe = phenylalanine) were prepared and characterized by elemental, spectral analysis (FT-IR, NMR techniques) and single crystal X-ray analysis in solid state and their solution stability was measured in biological testing time-scale by 1H NMR. The bridging coordination modes of the zwitterionic Ala and deprotonated Phe ligands led to the formation of 1D polymeric chains of the complexes. The significant argentophilic interactions are presented in the structure of AgAla. Antimicrobial testing of prepared Ag(I) complexes was evaluated by IC50 and MIC values and were compared with AgGly, silver(I) sulfadiazine and AgNO3 samples. Moreover, MTS test was used to the testing of broad range antiproliferative activity of studied compounds against different cancer cell lines and also to the investigation of calf thymus DNA interactions by absorption spectroscopy, fluorescence spectroscopy, Ethidium bromide/Hoechst 33258 displacement experiments and circular dichroism spectroscopy. To evaluate the pUC19 DNA fragmentation by silver(I) complexes, the agarose gel electrophoresis was used. In addition to biological evaluation we used lipophilicity measurement results in the discussion about structure-activity relationship (SAR).

Silver pyridine-2-sulfonate complex - its characterization, DNA binding, topoisomerase I inhibition, antimicrobial and anticancer response.

In the current study the ability of silver pyridine-2-sulfonate complex to exert multiple biological activities is compared with the pharmacological action of silver sulfadiazine (AgSD). Polymeric form of {[Ag(py-2-SO3)]}n (AgPS) was synthesized and characterized by analytical techniques (IR, CHN, TG/DTA, MS) and its molecular formula was established. The crystal structure was determined by X-ray diffraction method and the polymeric complex crystallizes in the triclinic P-1 space group. The stability of Ag(I) complex was verified by 1H and 13C NMR measurements and the interaction with calf thymus DNA through UV-VIS and fluorescence quenching experiments was studied. The Ag(I) complex was able to interact with DNA by dual binding mode: partial intercalation along groove binding. The binding constants were calculated to be in the order of 103 M-1. Topoisomerase I inhibition study have shown that silver complex is inhibiting its activity at concentration of 30 µM. The cytotoxic activity of AgPS and AgSD against mouse leukaemia L1210 S, R and T cell line was also evaluated. AgPS showed higher cytotoxicity than AgSD after 48 h incubation. The results suggest that mechanism of cell death is necrosis with a contribution of late apoptosis. Antimicrobial testing indicates higher growth inhibition effect of AgPS with comparison to commercially available AgSD.

Cold plasma treatment triggers antioxidative defense system and induces changes in hyphal surface and subcellular structures of Aspergillus flavus.

The cold atmospheric-pressure plasma (CAPP) has become one of the recent effective decontamination technologies, but CAPP interactions with biological material remain the subject of many studies. The CAPP generates numerous types of particles and radiations that synergistically affect cells and tissues differently depending on their structure. In this study, we investigated the effect of CAPP generated by diffuse coplanar surface barrier discharge on hyphae of Aspergillus flavus. Hyphae underwent massive structural changes after plasma treatment. Scanning electron microscopy showed drying hyphae that were forming creases on the hyphal surface. ATR-FTIR analysis demonstrated an increase of signal intensity for C=O and C-O stretching vibrations indicating chemical changes in molecular structures located on hyphal surface. The increase in membrane permeability was detected by the fluorescent dye, propidium iodide. Biomass dry weight determination and increase in permeability indicated leakage of cell content and subsequent death. Disintegration of nuclei and DNA degradation confirmed cell death after plasma treatment. Damage of plasma membrane was related to lipoperoxidation that was determined by higher levels of thiobarbituric acid reactive species after plasma treatment. The CAPP treatment led to rise of intracellular ROS levels detected by fluorescent microscopy using 2',7'-dichlorodihydrofluorescein diacetate. At the same time, antioxidant enzyme activities increased, and level of reduced glutathione decreased. The results in this study indicated that the CAPP treatment in A. flavus targeted both cell surface structures, cell wall, and plasma membrane, inflicting injury on hyphal cells which led to subsequent oxidative stress and finally cell death at higher CAPP doses.

New silver complexes with bioactive glycine and nicotinamide molecules - Characterization, DNA binding, antimicrobial and anticancer evaluation.

This study introduces a pair of newly synthesized silver complexes, [Ag2(HGly)2]n(NO3)2n (1) and [Ag(Nam)2]NO3·H2O (2) (Gly - glycine, Nam - nicotinamide), that were prepared and characterized by relevant methods in solid state (elemental, spectral, thermal and structural analysis) and their stability in solution was verified by 1H NMR measurements. Moreover, suitable reaction conditions were observed by potentiometry depending on pH in case of binary system Ag-Gly. X-ray analysis confirmed argentophilic interactions in complex 1 with an Ag1-Ag2 distance of 2.8018(6) Å. Antimicrobial testing indicates higher growth inhibition effect of complex 1 than complex 2. Moreover the effectivity of both complexes against bacteria (Staphylococcus aureus and Escherichia coli) is superior (or similar) to that of the commercially available Ag(I) sulfadiazine, AgSD (used, for example, in Dermazine cream). The binding of the Ag(I) complexes to calf thymus DNA was investigated using electronic absorption, fluorescence and circular dichroism spectrophotometry. The Stern-Volmer quenching constants obtained from the linear quenching plot were estimated in the range from 2.01×103 to 20.34×103M-1. The results of topoisomerase I and topoisomerase II (Topo I and Topo II) inhibition assay suggested that complex 2 inhibits the enzyme activity of both enzymes at a concentration of 2µM. The cytotoxicity of both complexes on L1210 leukemia cells was revealed to be approximately three times higher than that of cisplatin. Moreover, the new Ag(I) complexes also induced apoptosis of the leukemia cells. The high DNA binding activity of these complexes is considered to be responsible for their cytotoxic effects.

Changes in metabolome and in enzyme activities during germination of Trichoderma atroviride conidia.

The aim of this work was to study the metabolic changes during germination of Trichoderma atroviride conidia along with selected marker enzyme activities. The increase in proteinogenic amino acid concentrations together with the increase in glutamate dehydrogenase activity suggests a requirement for nitrogen metabolism. Even though the activities of tricarboxylic acid cycle enzymes also increased, detected organic acid pools did not change, which predisposes this pathway to energy production and supply of intermediates for further metabolism. The concentrations of many metabolites, including the main osmolytes mannitol and betaine, also increased during the formation of germ tubes. The activities of H(+)-ATPase and GDPase, the only marker enzymes that did not have detectable activity in non-germinated conidia, were shown with germ tubes.

Transient excretion of succinate from Trichoderma atroviride submerged mycelia reveals the complex movements and metabolism of carboxylates.

Submerged growth of Trichoderma atroviride CCM F 534 on glucose-containing medium was accompanied by the excretion of organic acids (succinate, citrate, alpha-ketoglutarate, fumarate, aconitate). The excretion of succinate was transient. After 48-72 h cultivation, millimolar amounts of succinate disappeared from the medium. We studied the mechanism of the removal of succinate from the medium and demonstrated the activation of the inward transport of succinate by submerged mycelia. This transport was carrier-mediated, had a low solute specificity, and was driven by proton-motive force. The last aspect was provided by the activation of the H(+)-ATPase, as documented by measurements of ATPase activity and expression of the pma gene. The disruption of the pma gene abolished the capacity of the mycelia to re-uptake succinate but not its production. Results show that excreted carboxylates could serve as alternative nutrients in the late phase(s) of submerged growth, explain why inward transport system(s) for carboxylates are induced, and indicate that the inward-directed transport could interfere with the production of carboxylic acids by fungi.

Study of Trichoderma viride metabolism under conditions of the restriction of oxidative processes.

Trichoderma viride was capable of growth and conidiation in the presence of high concentrations of the uncoupler 3,3',4',5-tetrachlorosalicylanilide (up to 100 micromol x L(-1) and of the respiration inhibitor mucidin (up to 100 micromol x L(-1) ) in both submerged and surface cultivation. When vegetative mycelia were cultivated on the solid Czapek-Dox medium with yeast autolysate under an anaerobic and CO2-containing atmosphere, the growth was observed only rarely but the microorganism survived as long as 3 months under these conditions. Major products of metabolism of both aerobic and anaerobic submerged mycelia were identified by means of 1H-NMR measurements. Major products excreted to the medium under aerobic conditions were succinic and citric acids. Major metabolites present in the submerged mycelia were gamma-aminobutyric (and glutamic) acid and alanine. Under anaerobic conditions, citric acid was not excreted into the medium but ethanol appeared. Its production could not be increased upon increasing the sugar concentration. The appearance of secondary metabolites was found to be modified by oxygen availability during the mycelial growth. Results suggest that the vegetative form of T. viride is capable of fermentative metabolism characterized by the production of ethanol and succinate and that the excretion of carboxylic acids is developmentally regulated and modified by oxygen availability.

Characterization of microorganisms isolated from lignite excavated from the Záhorie coal mine (southwestern Slovakia).

Microorganisms were isolated from lignite freshly excavated in the Záhorie coal mine (southwestern Slovakia) under conditions excluding contamination with either soil or air-borne microorganisms. The isolates represented both Prokarya and Eukarya (fungi). All were able to grow on standard media, although some microorganisms were unstable and became extinct during storage of coal samples. Bacteria belonged to the genera Bacillus, Staphylococcus, and Rhodococcus, according to both morphological criteria and ITS sequences. Several bacterial isolates were resistant to antibiotics. The presence of anaerobic bacteria was also documented, although they have not yet been identified. Fungal isolates were typified by using their ITS sequences. They belonged to the genera Trichoderma (Hypocrea), Penicillium, Epicoccum, Metarhizium (Cordyceps), and Cladosporium. Several fungi produced compounds with antibiotic action against standard bacterial strains. The evidence for the presence of microorganisms in native lignite was obtained by means of fluorescence microscopy, scanning electron microscopy, and electron microprobe analysis. Results demonstrated that microorganisms were able to survive in the low-rank coal over a long time period.

Developmental changes in Trichoderma viride enzymes abundant in conidia and the light-induced conidiation signalling pathway.

The expression of glutamic acid decarboxylase gene and the laccase activity were measured during the development of surface-cultivated Trichoderma viride mycelia in order to examine their up-regulation by light. The results show that the changes in activity of GAD induced by light observed previously are caused by transcriptional regulation of gad gene expression in both submerged mycelia and aerial mycelia after photoinduction. The expression of tga gene encoding a T. viride G(alpha) protein was found not to be up-regulated by light and was also present in the non-conidiating mutant of T. viride suggesting that this protein is not involved in the regulation of conidiation in this fungus, or that it plays a role is in later stages of conidia development. The activity of laccase was also not light-inducible and may be related to the maturation of conidia.

Changes in properties of glutamate transport in Trichoderma viride vegetative mycelia upon adaptation to glutamate as carbon source.

The U-(14)C-labelled glutamate uptake was measured in both sucrose- and glutamate-grown mycelia of Trichoderma viride. The biomass yield was five-fold lower with glutamate as a sole carbon source. The rate of glutamate transport measured at a glutamate concentration of 1 mM remained unchanged in glutamate-grown mycelia whereas the properties of the glutamate transport were substantially changed compared to sucrose-grown mycelia. The glutamate uptake in both sucrose- and glutamate-grown mycelia was inhibited by an uncoupler (3,3',4',5-tetrachlorosalicylanilide) but the inhibitory efficiency was higher in the latter. The affinity of the permease to glutamate increased approximately five-fold in the glutamate-grown mycelia (about 76 microM compared to about 16 microM). The pH optimum for glutamate uptake was 4 in sucrose-grown mycelia but the glutamate-grown mycelia had two pH optima, one at pH 4 and the second between pH 6 and 7. The inhibition of glutamate uptake by other amino acids yielded different inhibitory patterns in the two mycelia under study. The glutamate uptake in mycelia of different ages also showed differences in both transport rate and temporal pattern. The results show that the growth of mycelia on glutamate led to the appearance of an additional permease with different properties and suggest that only this permease is operating in mycelia grown on glutamate.