Marina Milenković

Graphene quantum dots as autophagy-inducing photodynamic agents

The excellent photoluminescent properties of graphene quantum dots (GQD) makes them suitable candidates for biomedical applications, but their cytotoxicity has not been extensively studied. Here we show that electrochemically produced GQD irradiated with blue light (470 nm, 1W) generate reactive oxygen species, including singlet oxygen, and kill U251 human glioma cells by causing oxidative stress. The cell death induced by photoexcited GQD displayed morphological and/or biochemical characteristics of both apoptosis (phosphatidylserine externalization, caspase activation, DNA fragmentation) and autophagy (formation of autophagic vesicles, LC3-I/LC3-II conversion, degradation of autophagic target p62). Moreover, a genetic inactivation of autophagy-essential LC3B protein partly abrogated the photodynamic cytotoxicity of GQD. These data indicate potential usefulness of GQD in photodynamic therapy, but also raise concerns about their possible toxicity.

Multifunctional PLGA particles containing poly(l-glutamic acid)-capped silver nanoparticles and ascorbic acid with simultaneous antioxidative and prolonged antimicrobial activity.

A water-soluble antioxidant (ascorbic acid, vitamin C) was encapsulated together with poly(l-glutamic acid)-capped silver nanoparticles (AgNpPGA) within a poly(lactide-co-glycolide) (PLGA) polymeric matrix and their synergistic effects were studied. The PLGA/AgNpPGA/ascorbic acid particles synthesized by a physicochemical method with solvent/non-solvent systems are spherical, have a mean diameter of 775 nm and a narrow size distribution with a polydispersity index of 0.158. The encapsulation efficiency of AgNpPGA/ascorbic acid within PLGA was determined to be >90%. The entire amount of encapsulated ascorbic acid was released in 68 days, and the entire amount of AgNpPGAs was released in 87 days of degradation. The influence of PLGA/AgNpPGA/ascorbic acid on cell viability, generation of reactive oxygen species (ROS) in HepG2 cells, as well as antimicrobial activity against seven different pathogens was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated good biocompatibility of these PLGA/AgNpPGA/ascorbic acid particles. We measured the kinetics of ROS formation in HepG2 cells by a DCFH-DA assay, and found that PLGA/AgNpPGA/ascorbic acid caused a significant decrease in DCF fluorescence intensity, which was 2-fold lower than that in control cells after a 5h exposure. This indicates that the PLGA/AgNpPGA/ascorbic acid microspheres either act as scavengers of intracellular ROS and/or reduce their formation. Also, the results of antimicrobial activity of PLGA/AgNpPGA/ascorbic acid obtained by the broth microdilution method showed superior and extended activity of these particles. The samples were characterized using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, transmission electron microscopy, zeta potential and particle size analysis. This paper presents a new approach to the treatment of infection that at the same time offers a very pronounced antioxidant effect.

Evaluation of Hypericum perforatum oil extracts for an antiinflammatory and gastroprotective activity in rats

Oil extracts of Hypericum perforatum L. (Oleum Hyperici) were prepared in three different ways according to the prescriptions from traditional medicine. Variability of constituents and biological activity were evaluated in the obtained oil extracts. The carrageenan‐induced rat paw edema test has been used for screening the antiinflammatory activity, while the indomethacin‐induced rat gastric mucosa damage test was used for evaluation of gastroprotective activity. All examined oil extracts possessed antiinflammatory and gastroprotective activity. Among them, the oil extract prepared by maceration with 96% ethanol, followed by extraction with sunflower oil by heating on a water bath (extract 2), in a dose of 1.25 mL/kg p.o., exhibited the highest antiinflammatory effect (95.24 ± 11.66%) and gastroprotective activity (gastric damage score of 0.21 ± 0.12). The same oil extract had the highest content of quercetin and I3,II8‐biapigenin (129 ± 9 µg/mL and 52 ± 4 µg/mL, respectively). Quercetin and I3,II8‐biapigenin exhibited antiinflammatory activity similar to those of indomethacin as well as significant gastroprotective activity. The results provide evidence for the usage of Oleum Hyperici as an antiinflammatory and gastroprotective agent, which has been based previously only on ethnopharmacological claims. Copyright

Quercetin Ameliorates Experimental Autoimmune Myocarditis in Rats

PURPOSE Experimental autoimmune myocarditis (EAM) in rats is an animal model of human giant cell myocarditis and post-myocarditis dilated cardiomyopathy. The pathogenesis of EAM has not been elucidated, but there is accumulating evidence that cytokines secreted from monocytes/macrophages and T cells play a crucial role in the induction and progression of disease. Flavonoids are a large group of polyphenolic compounds abundantly present in the human diet, which scavenge oxygen radicals and have anti-inflammatory activities. Having in mind in vivo beneficial effects of flavonoid quercetin in different animal models of immunoinflammatory diseases such as experimental autoimmune encephalomyelitis and adjuvant arthritis, on the one side, and its in vitro suppressive effect on production of tumor necrosis factor-alpha (TNF-α) on the other side, we investigated the effects of quercetin on EAM in rats. METHODS Myocarditis was induced in Dark Agouti (DA) rats by injection of porcine cardiac myosin and quercetin at doses of 10 or 20 mg/kg was orally administered from days 0 to 21 after induction of disease. The severity of myocarditis was evaluated by determination of heart weight/body weight ratio (Hw/Bw) and histopathological examination of hearts. The levels of cytokines (TNF-α, IL-12, IL-17 and IL-10) in serum and lymph node cells (LNC) culture supernatants were measured by ELISA. RESULTS The rats treated with 20 mg/kg of quercetin had significantly decreased incidence of EAM, Hw/Bw, macroscopic and microscopic scores of hearts. Further, in EAM rats treated with quercetin levels of TNF-α and IL-17 were significantly lower, while the level of IL-10 was significantly higher both in serum and culture supernatants of LNC stimulated with concanavalin A compared with vehicle-treated animals. CONCLUSIONS The present study suggests that quercetin ameliorates EAM, at least in part, by interfering production of proinflammatory (TNF-α and IL-17) and/or anti-inflammatory (IL-10) cytokines.

Chemical composition and antimicrobial activity of Thymus pannonicus All. (Lamiaceae) essential oil

This paper presents the results of a study on chemical composition and antimicrobial activity of Thymus pannonicus All. (Lamiaceae) essential oil from Vojvodina province (north of Serbia). The investigated oil was hydrodistilled from a flowering plant and analysed by GC and GC-MS. Fifty-three constituents were identified (>97% of total oil), with geranial (41.42%, w/w) and neral (29.61%, w/w) as the most prominent. The antimicrobial activity of the oil was evaluated using agar disc diffusion and broth microdilution method against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, two strains of Klebsiella pneumoniae and two strains of Candida albicans. The essential oil exhibited antimicrobial activity to varying degrees against all tested strains. The maximum activity of T. Pannonicus oil was observed against E. coli, S. aureus and both tested strains of C. Albicans (MIC = 50 µ/ml, each). Moderate activity was observed against P. aeruginosa and one of the tested strains of K. Pneumoniae (MIC = 200 µ/ml), while E. faecalis and the other strain of K. Pneumoniae expressed a higher degree of resistance (MIC > 200 µ/ml). This study confirms that essential oil of T. pannonicus possesses remarkable in vitro antimicrobial activity against several medicinally important pathogens. This is attributable to lemon-scented citral, a mixture of geranial and neral, which has well-documented antimicrobial activity against a range of bacteria and fungi.

mTOR-independent autophagy counteracts apoptosis in herpes simplex virus type 1-infected U251 glioma cells.

We investigated the role of autophagy, a stress-inducible lysosomal self-digestion of cellular components, in modulation of herpes simplex virus type 1 (HSV-1)-triggered death of U251 human glioma cells. HSV-1 caused apoptotic death in U251 cells, characterized by phosphatidylserine externalization, caspase activation and DNA fragmentation. HSV-1-induced apoptosis was associated with the induction of autophagic response, as confirmed by the conversion of cytosolic LC3-I to autophagosome-associated LC3-II, increase in intracellular acidification, presence of autophagic vesicles, and increase in proteolysis of the selective autophagic target p62. HSV-1-triggered autophagy was not associated with the significant increase in the expression of proautophagic protein beclin-1 or downregulation of the major autophagy suppressor mammalian target of rapamycin (mTOR). Moreover, the phosphorylation of mTOR and its direct substrate p70 S6 kinase was augmented by HSV-1 infection, while the mTOR stimulator Akt and inhibitor AMPK-activated protein kinase (AMPK) were accordingly activated and suppressed, respectively. An shRNA-mediated knockdown of the autophagy-essential LC3β, as well as pharmacological inhibition of autophagy with bafilomycin A1 or 3-methyladenine, markedly accelerated apoptotic changes and ensuing cell death in HSV-1-infected glioma cells. These data indicate that AMPK/Akt/mTOR-independent autophagy could prolong survival of HSV-1-infected U251 glioma cells by counteracting the coinciding apoptotic response.

Interaction of Lactobacillus fermentum BGHI14 with Rat Colonic Mucosa: Implications for Colitis Induction

ABSTRACT The present study was carried out to test the colonic mucosal response of rats to oral supplementation with Lactobacillus fermentum BGHI14 and to correlate the tissue reaction to trinitrobenzenesulfonate (TNBS)-induced colitis with mucosal barrier alterations caused by bacterial ingestion. An immune cell-mediated reaction of healthy colonic tissue was noticed after bacterial feeding. After prolonged bacterial treatment, the observed reaction had retreated to normality, but the mRNA levels of proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) remained elevated. These data point to the chronic low-grade inflammation that could be caused by long-term probiotic consumption. Although no detrimental effects of bacterial pretreatment were noticed in colitic rats, at least in the acute state of disease, the results obtained in our study point to the necessity of reassessment of existing data on the safety of probiotic preparations. Additionally, probiotic effects in experimental colitis models might depend on time coordination of disease induction with treatment duration.

45S5Bioglass®-based scaffolds coated with selenium nanoparticles or with poly(lactide-co-glycolide)/selenium particles: Processing, evaluation and antibacterial activity

In the bone tissue engineering field, there is a growing interest in the application of bioactive glass scaffolds (45S5Bioglass(®)) due to their bone bonding ability, osteoconductivity and osteoinductivity. However, such scaffolds still lack some of the required functionalities to enable the successful formation of new bone, e.g. effective antibacterial properties. A large number of studies suggest that selenium (Se) has significant role in antioxidant protection, enhanced immune surveillance and modulation of cell proliferation. Selenium nanoparticles (SeNp) have also been reported to possess antibacterial as well as antiviral activities. In this investigation, uniform, stable, amorphous SeNp have been synthesized and additionally immobilized within spherical PLGA particles (PLGA/SeNp). These particles were used to coat bioactive glass-based scaffolds synthesized by the foam replica method. Samples were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and transmission electron microscopy (TEM). SeNp, 45S5Bioglass(®)/SeNp and 45S5Bioglass(®)/PLGA/SeNp showed a considerable antibacterial activity against Gram positive bacteria, Staphylococcus aureus and Staphylococcus epidermidis, one of the main causative agents of orthopedic infections. The functionalized Se-coated bioactive glass scaffolds represent a new family of bioactive, antibacterial scaffolds for bone tissue engineering applications.

Antimicrobial and antioxidant properties of methanol extracts of two Athamanta turbith subspecies

The methanol extracts of fruits and leaves of Athamanta turbith subsp. hungarica (Borbás) Tutin (Umbelliferae) and A. turbith subsp. haynaldii (Borbás & Uechtr.) Tutin were analyzed for their antimicrobial and antioxidant properties. Phenolic compounds (flavonoids and phenolic acids) of the extracts were examined using HPLC. All the extracts were characterized by the presence of caffeic acid derivates, luteolin and its glycosides, with luteolin 7-O-glucoside as one of the main compounds. Luteolin 7-O-rutinoside was detected only in A. turbith subsp. haynaldii extracts. Investigation of antimicrobial activity was performed against six bacteria and two fungal strains, using the agar diffusion technique and broth microdilution assay. The extracts of investigated A. turbith subspecies exerted similar antimicrobial activity, whereas the best activity was detected against Candida albicans. In order to investigate antioxidant properties, ferric ion reducing antioxidant power (FRAP), radical scavenging capacity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical (HO·), and the effect on lipid peroxidation (LP) were examined. All the examined extracts showed moderate antioxidant capacity, whereas the fruit extracts were more active than the extracts of leaves. Also, the extracts of A. turbith subsp. hungarica exerted higher antioxidant capacity than corresponding A. turbith subsp. haynaldii extracts. The relationship between estimated activity and chemical composition of the extracts is discussed.

Beneficial Effects of Dimethyl Fumarate on Experimental Autoimmune Myocarditis

BACKGROUND Fumaric acid esters (FAE) have been proven to be effective for the systemic treatment of psoriasis and multiple sclerosis, Th1 cell-mediated chronic inflammatory diseases, but their effect on autoimmune myocarditis has not yet been addressed. We investigated the effect of dimethyl fumarate (DMF) on myosin-induced experimental autoimmune myocarditis (EAM). METHODS Dark Agouti (DA) rats immunized with porcine cardiac myosin were orally treated with 5 and 15 mg/kg body weight (bw) DMF either from days 0-10 (early treatment groups) or from days 10-21 (late treatment groups) after induction of EAM. All rats were sacrificed on day 21 after immunization and hearts were evaluated macroscopically and microscopically. Levels of TNF-alpha and IL-10 in serum and lymph node cells culture supernatants were detected by ELISA. RESULTS Both early and late treatment with 15 mg/kg body weight (bw) DMF markedly reduced the severity of myocarditis by comparing the incidence, heart weight/bw ratio, macroscopic and microscopic scores, and number of OX-6+ cells in the myocardium. Further, levels of tumor necrosis factor-alpha (TNF-alpha) in serum and culture supernatants of lymph node cells stimulated with ConA or myosin were significantly lower in DMF-treated EAM animals compared with vehicle-treated EAM rats. There was no significant difference in serum levels of interleukin-10 between DMF- and vehicle-treated EAM rats. CONCLUSIONS These results show for the first time that DMF ameliorates experimental autoimmune myocarditis and may be acted, at least in part, by interfering with the production of TNF-alpha.