Effectiveness of magnetic fluid hyperthermia against Candida albicans cells.

Candida albicans is one of the most frequently isolated fungal pathogens causing opportunistic infections in humans. Targeted magnetic fluid hyperthermia (MFH) is a promising method in thermal therapy facilitating selective heating of pathogen cells like C. albicans. In the paper, we used meso-2,3-dimercaptosuccinic acid (DMSA)-coated magnetic nanoparticles (MNPs) and functionalised anti-C. albicans immunomagnetic nanoparticles (IMNPs) to investigate the potential of MFH in combating C. albicans cells in vitro. Using Mössbauer spectroscopy it was found that synthesised MNPs exhibited superparamagnetic phenomena. On the basis of calorimetric experiments, the maximum SAR (specific absorption rate) was found and a proper concentration of MNPs was established to control the temperature. MFH based on both DMSA-coated MNPs and functionalised anti-C. albicans IMNPs was more effective in combating C. albicans cells in vitro than thermostat hyperthermia. Especially promising results were obtained using functionalised IMNPs, which eradicated most of the pathogen colonies at the temperature of 43 °C.

A new look at the antibiotic amphotericin B effect on Candida albicans plasma membrane permeability and cell viability functions.

Amphotericin B (AmB) is an antifungal polyene for which the most accepted mode of action is formation of protein-like ion channels in the cell membrane. Patch-clamp research on Candida albicans protoplasts carried out in the outside-out configuration showed that application of 0.05 and 0.1 µM AmB caused a decrease in seal resistance. Such a phenomenon can be correlated with a decrease in membrane tightness. AmB applied at a 0.05 µM concentration also caused a decrease in the number of active TOK1 (two-pore outward rectifiers) potassium channels, but did not significantly change their open probability. The results indicate that in C. albicans protoplast AmB causes a decrease in cell membrane integrity by interaction with its lipid phase but not with ion channels. Fluorescence microscopy techniques showed that AmB treatment, in clinical concentrations, had no effect on the percentage of PI-positive protoplasts. AmB treatment in the concentrations tested did not cause a rapid reduction of the number of C. albicans protoplasts. However, there was a significant loss of replication competency and numerous morphological and physiological disorders, including cytoplasm shrinking, abnormal morphology of the nucleus and mitochondria, a sudden decrease in the MTT reduction level and oxidative stress. Our results show that the induction of yeast cell death by AmB, at therapeutic doses, is a multistage and long-term process involving multiple intracellular pathways.

Effect of Low Temperature on Changes in AGP Distribution during Development of Bellis perennis Ovules and Anthers.

Arabinogalactan proteins (AGPs) are a class of heavily glycosylated proteins occurring as a structural element of the cell wall-plasma membrane continuum. The features of AGPs described earlier suggest that the proteins may be implicated in plant adaptation to stress conditions in important developmental phases during the plant reproduction process. In this paper, the microscopic and immunocytochemical studies conducted using specific antibodies (JIM13, JIM15, MAC207) recognizing the carbohydrate chains of AGPs showed significant changes in the AGP distribution in female and male reproductive structures during the first stages of Bellis perennis development. In typical conditions, AGPs are characterized by a specific persistent spatio-temporal pattern of distribution. AGP epitopes are visible in the cell walls of somatic cells and in the megasporocyte walls, megaspores, and embryo sac at every stage of formation. During development in stress conditions, the AGP localization is altered, and AGPs entirely disappear in the embryo sac wall. In the case of male development, AGPs are present in the tapetum, microsporocytes, and microspores in normal conditions. In response to development at lower temperature, AGPs are localized in the common wall of microspores and in mature pollen grains. Additionally, they are accumulated in remnants of tapetum cells.

Influence of ultrasound on selected microorganisms, chemical and structural changes in fresh tomato juice.

The paper presents the possibility of applying ultrasonic technology for inactivation of mesophilic aerobic microorganisms, lactic acid bacteria, coliform bacteria, and yeast with the maintenance of the chemical and structural properties of tomato juice. The research was conducted on fresh tomato juice obtained from the Apis F1 variety. Pressed juice was exposed to high power ultrasound and frequency 20 kHz with three operational parameters: ultrasound intensity (28 and 40 W cm-2), treatment time (2, 5, and 10 min), and product storage time (1, 4, 7 and 10 days). The temperature of the juice during the sonication ranged from 37 to 52 °C depending on the intensity of ultrasound and time of treatment. Effectiveness of the tested microorganisms eradication in the juice depended on the amplitude and duration of the ultrasound treatment. It was shown that the juice exposed to an ultrasonic field with an intensity of 40 W cm-2 for 10 min was microbiologically pure and free from spoilage microorganism even after 10 storage days. No statistically significant differences in pH were found between the untreated juice and the sonicated samples. The ultrasonic treatment was found to change the content of lycopene in small degree (both an increase and a decrease, depending on the processing time) and to induce a small decrease in the vitamin C content. The study suggests that the ultrasonic treatment can be successfully implemented on an industrial scale for the production of not-from-concentrate (NFC) tomato juice.

Possibility to extend the shelf life of NFC tomato juice using cold atmospheric pressure plasma.

Cold Atmospheric pressure Plasma (CAP) is a non-thermal method used in food processing. CAP generated with the use of nitrogen in a Glide-arc device for 300 to 600 s exhibited high potential for microbial decontamination and did not induce substantial changes in the physicochemical properties of NFC tomato juice. Samples exposed to cold atmospheric plasma had mostly an intact structure, as revealed by digital microscopy. The investigations indicate that CAP can be applied for biological and chemical waste-free decontamination of food and extension of its shelf life.

Evaluation of selected microbial and physicochemical parameters of fresh tomato juice after cold atmospheric pressure plasma treatment during refrigerated storage.

The Cold Atmospheric pressure Plasma (CAP) technology is an emerging technology used for conditioning and microbiological decontamination of biomaterials including food. A novel tool for inactivation of juice background spoilage microorganisms, as well as high count of inoculated yeast while maintaining physicochemical properties in tomato juice - CAP technology was utilized in this study. Dry matter content and pH were not significantly influenced by CAP generated in GlidArc reactor. Small increase of lycopene, and slight loss of vitamin C content were observed.

Synergistic antifungal interactions of amphotericin B with 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol.

Amphotericin B (AmB) is a very potent antifungal drug with very rare resistance among clinical isolates. Treatment with the AmB formulations available currently is associated with severe side effects. A promising strategy to minimize the toxicity of AmB is reducing its dose by combination therapy with other antifungals, showing synergistic interactions. Therefore, substances that display synergistic interactions with AmB are still being searched for. Screening tests carried out on several dozen of synthetic 1,3,4-thiadiazole derivatives allowed selection of a compound called 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol (abbreviated as C1), which shows strong synergistic interaction with AmB and low toxicity towards human cells. The aim of the present study was to investigate the type of in vitro antifungal interactions of the C1 compound with AmB against fungal clinical isolates differing in susceptibility. The results presented in the present paper indicate that the C1 derivative shows strong synergistic interaction with AmB, which allows the use of a dozen to several dozen times lower AmB concentration necessary for 100% inhibition of the growth of pathogenic fungi in vitro. Synergistic interactions were noted for all tested strains, including strains with reduced sensitivity to AmB and azole-resistant isolates. These observations give hope for the possibility of application of the AmB - C1 combinatory therapy in the treatment of fungal infections.

Antifungal effects of a 1,3,4-thiadiazole derivative determined by cytochemical and vibrational spectroscopic studies.

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 µg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and ß(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of ß(1→3) glucans and ß(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses.

Amphotericin B-copper(II) complex shows improved therapeutic index in vitro.

The AmB-Cu(II) complex has recently been reported as an antifungal agent with reduced aggregation of AmB in aqueous solutions, increased anti C. albicans activity and lower toxicity against human cells in vitro. In the present work, investigations of the activity of the AmB-Cu (II) complex against fungal pathogens with varying susceptibility, including C. albicans and C. parapsilosis strains and intrinsically resistant A. niger, and cytotoxicity in normal human dermal fibroblasts (NHDF) in vitro were performed. For better understanding of the mechanism of reduced cytotoxicity and increased fungicidal activity, the influence of the AmB-Cu (II) complex on membrane integrity and accumulation of cellular reactive oxygen species (ROS) and mitochondrial superoxide was compared with that of conventional AmB. In the sensitive C. albicans and C. parapsilosis strains, the AmB-Cu(II) complex showed higher fungicidal activity (the MIC value was 0.35-0.7µg/ml for the AmB-Cu (II) complex, and 0.45-0.9µg/ml for Fungizone) due to increased induction of oxidative damage with rapid inhibition of the ability to reduce tetrazolium dye (MTT). In the NHDF cell line, the CC50 value was 30.13±1.53µg/ml for the AmB-Cu(II) complex and 17.46±1.24µg/ml for (Fungizone), therefore, the therapeutic index (CC50/MIC90) determined in vitro was 86.09-43.04 for the AmB-Cu(II) complex and 38.80-19.40 for Fungizone. The lower cytotoxicity of the AmB-Cu(II) complex in human cells resulted from lower accumulation of cellular and mitochondrial reactive oxygen species. This phenomenon was probably caused by the induction of successful antioxidant defense of the cells. The mechanism of the reduced cytotoxicity of the AmB-Cu(II) complex needs further investigation, but the preliminary results are very promising.

Amphotericin B-copper(II) complex as a potential agent with higher antifungal activity against Candida albicans.

Amphotericin B (AmB) is a polyene antibiotic produced by Streptomyces nodosus used for more than 50 years in the treatment of acute systemic fungal infections. It exhibits a broad spectrum of activity against fungal and protozoan pathogens with relatively rare resistance. The aim of this study was to prepare and evaluate the utility of the AmB-Cu(2+) complex as a potential compound with a high fungicidal activity at lower concentrations, compared with conventional AmB. It was hypothesized that insertion of copper ions into fungal cell membranes, together with the AmB-Cu(2+) complex bypassing the natural homeostatic mechanisms of this element, may contribute to the increased fungicidal activity of AmB. The analysis of results indicates the increased antifungal activity of the AmB-Cu(2+) complex against Candida albicans in comparison with the pure AmB and Fungizone. Additionally, it was stated that the increased antifungal activity of the AmB-Cu(2+) complex is not the sum of the toxic effects of AmB and Cu(2+) ions, but is a result of the unique structure of this compound.