imagine (Institute of molecular genetics and genetic engineering)
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Optimization of ultrasound-assisted extraction of total polyphenolic compounds from Juglans nigra L. leaves
Get PDFThe ultrasound-assisted extraction of Juglans nigra L. leaves was optimized with respect to total phenolic content (TP) of the extracts by varying the concentration of aqueous ethanol solution (E) and different solvent-to-solid ratio (S). The influence and optimum of the operating parameters (E and S) was examined using response surface methodology (RSM). The statistical criteria indicated the adequacy, reliability and precision of the developed RSM model. RSM showed that maximum extraction yield of TP 28.59 mg g(-1) of draw plant was achieved at the optimal values of 50% E and 20 kg kg(-1) S. Using the modelled optimized conditions, the detected relative difference between the predicted and the experimental yield was +/- 2.3 %. The determined TP content in the extracts varied from 12.54 to 29.26 mg GAE g(-1) of dry plant indicated that J. nigra is a valuable source of phenolic substances. The extracts of J. nigra leaves obtained under optimal conditions showed good antioxidant activity (IC50 = = 18.91 +/- 0.03 mu g cm(-3)) which was determined by the scavenging effect on 2,2-diphenyl-1-picrylhydrazyl radical. The optimization of the TP extraction process is the important step in improving techno-economics of the potential commercial preparation of J. nigra extracts, as natural source of antioxidants
Honey bee viruses in Serbian colonies of different strength
Get PDFProtection of honey bees is of great economic importance because of their role in pollination. Crucial steps towards this goal are epidemiological surveys of pathogens connected with honey bee losses. In this study deformed wing virus (DWV), chronic bee paralysis virus (CBPV), acute bee paralysis virus (ABPV) and sacbrood virus (SBV) were investigated in colonies of different strength located in five regions of Serbia. The relationship between colony strength and virus occurrence/infection intensity were assessed as well as the genetic relationship between virus sequences from Serbia and worldwide. Real-time RT-PCR analyses detected at least one virus in 87.33% of colonies. Single infection was found in 28.67% colonies (21.33%, 4.00%, 2.67% and 0.67% in cases of DWV, ABPV, SBV and CBPV, respectively). In the majority of colonies (58.66%) more than one virus was found. The most prevalent was DWV (74%), followed by ABPV, SBV and CBPV (49.30%, 24.00% and 6.70%, respectively). Except for DWV, the prevalence of the remaining three viruses significantly varied between the regions. No significant differences were found between colony strength and either (i) the prevalence of DWV, ABPV, SBV, CBPV and their combinations, or (ii) DWV infection levels. The sequences of honey bee viruses obtained from bees in Serbia were 93-99% identical with those deposited in GenBank
Comparative toxicity evaluation of targeted anticancer therapeutics in embryonic zebrafish and sea urchin models
No full textCancer drug resistance and poor selectivity towards cancer cells demand the constant search for new therapeutics. PI3K-Akt-mTOR and RAS-MAPK-ERK signaling pathways are key mechanisms involved in cell survival, proliferation, differentiation, and metabolism and their deregulation in cancer can promote development of therapy resistance. We investigated the effects of targeted inhibitors (wortmannin, GSK690693, AZD2014 and tipifarnib) towards these two pathways on early zebrafish and sea urchin development to assess their toxicity in normal, fast proliferating cells. PI3K inhibitor wortmannin and RAS inhibitor tipifarnib displayed highest toxicity while GSK690693, a pan-Akt kinase inhibitor, exhibited a less significant impact on embryo survival and development. Moreover, inhibition of the upstream part of the PI3K-Akt-mTOR pathway (wortmannin/GSK690693 co-treatment) produced a synergistic effect and impacted zebrafish embryo survival and development at much lower concentrations. Dual mTORC1/mTORC2 inhibitor AZD2014 showed no considerable effects on embryonic cells of zebrafish in concentrations substantially toxic in cancer cells. AZD2014 also caused the least prominent effects on sea urchin embryo development compared to other inhibitors. Significant toxicity of AZD2014 in human cancer cells, its capacity to sensitize resistant cancers, lower antiproliferative activity against human normal cell lines and fast proliferating embryonic cells could make this agent a promising candidate for anticancer therapy
Functional Characterization of Novel Phenylalanine Hydroxylase p.Gln226Lys Mutation Revealed Its Non-responsiveness to Tetrahydrobiopterin Treatment in Hepatoma Cellular Model
No full textTreatment with tetrahydrobiopterin (BH4) is the latest therapeutic option approved for patients with phenylketonuria (PKU)-one of the most frequent inborn metabolic diseases. PKU or phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene. Given that some PAH mutations are responsive to BH4 treatment while others are non-responsive, for every novel mutation that is discovered it is essential to confirm its pathogenic effect and to assess its responsiveness to a BH4 treatment in vitro, before the drug is administered to patients. We found a c.676C gt A (p.Gln226Lys) mutation in the PAH gene in two unrelated patients with PKU. The corresponding aberrant protein has never been functionally characterized in vitro and its response to BH4 treatment is unknown. Computational analyses proposed that glutamine at position 226 is an important, evolutionary conserved amino acid while the substitution with lysine probably disturbs tertiary protein structure and impacts posttranslational PAH modifications. Using hepatoma cellular model, we demonstrated that the amount of mutant p.Gln226Lys PAH detected by Western blot was only 1.2% in comparison to wild-type PAH. The addition of sepiapterin, intracellular precursor of BH4, did not increase PAH protein yield thus marking p.Gln226Lys as BH4-non-responsive mutation. Therefore, computational, experimental, and clinical data were all in accordance showing that p.Gln226Lys is a severe pathogenic PAH mutation. Its non-responsiveness to BH4 treatment in hepatoma cellular model should be considered when deciding treatment options for PKU patients carrying this mutation. Consequently, our study will facilitate clinical genetic practice, particularly genotype-based stratification of PKU treatment
The use of canine mesenchymal stem cells for the autologous treatment of osteoarthritis
No full textMesenchymal stem cells (MSCs) hold enormous potential for cell-based therapy in the treatment of various diseases, particularly those which currently cannot be cured and result in poor outcomes or invasive surgery. Here we present results of the application of autologous, culture-expanded, adipose tissue (AT)derived MSCs for the osteoarthritis (OA) treatment of 10 canine patients. The stemness of isolated cells has been confirmed by their ability to differentiate into osteo-and chondrocytic lineages. The clinical effect of a single injection of AT-MSCs into the symptomatic joints was evaluated by a veterinarian for five disabilities characteristic of OA at 30, 60 and 90 days after treatment, which has been designated as the initial evaluation period. Functional outcomes for all analysed characteristics improved significantly at the end of this evaluation compared with the baseline. In addition, for 5 of these 10 patients, an extended follow-up study was performed from 1 to 4 years after the initial evaluation period. We detected long-lasting positive effects on two out of five analysed characteristics. The results demonstrate that the use of autologous AT-MSCs is a successful approach to canine OA therapy
Oral treatment with Lactobacillus rhamnosus 64 during the early postnatal period improves the health of adult rats with TNBS-induced colitis
No full textThe current study investigated a potential modulating effect of orally applied Lactobacillus rhamnosus 64 (LB64) during the early postnatal period (day of life: similar to 3-30), during young adult period (day of life: 31-70) or throughout experiment, on parameters of trinitrobenzenesulfonic acid (TNBS)-induced colitis in adult rats. Treatment with LB64 during early postnatal, but not during young adult period reduced clinical damage score, neutrophil and macrophage infiltration into colon, the level of cytokine and myeloperoxidase (MPO) activity, but had no influence on other parameters of oxidative damage. Early postnatal treatment with LB64 also increased the diversity of fecal Bifidobacteria and Eubacteria, and improved maturation of ileal villi in 30-days old rats. When LB64 is applied during a critical period early in life, it affects immune system functioning of adults, probably by interactions with the mucosal immune system of the gastrointestinal tract that provides immune system maturation and shapes the overall immune response
miR-183 and miR-21 expression as biomarkers of progression and survival in tongue carcinoma patients
No full textMicro RNAs (miRNAs) have a major role in human cancerogenesis.The current study investigated the prognostic significance of miR-183 and miR-21 expression in tongue carcinoma patients. For qPCR of miR-183 and miR-21 expression, total RNA isolated from 60 fresh-frozen tissue of tongue carcinomas was converted into cDNA by TaqMan MicroRNA Reverse Transcription Kit and quantified by TaqMan MicroRNAs Expression Assays. Fold changes in the miRNAs expression, normalized to RNU6B, were determined using 2(-Delta Delta Ct) method, and dichotomized into high and low according to cut-off values derived from ROC curve analysis. miR-183 emerged as promising discriminatory biomarker of poor outcome. Tissue over-expression of miR-183, observed in 68.3% of tongue carcinomas, was associated with clinical stage (p = 0.037), tumor size (p = 0.036), and high alcohol intake (p = 0.034).The patients with miR-183 over-expression had significantly shorter overall survival (p = 0.006) and a 5.666 times higher risk of poor outcome (p = 0.005), while miR-21 over-expression carried a tendency towards poorer survival (p = 0.073). However, multivariate analysis revealed that the recurrences were independent adverse prognostic factors, while miR-183 over-expression lost its significance. Our results suggests that over-expression of miR-183 in tumor tissue could be a potential marker of clinical stage and a poor survival of tongue carcinoma patients and may be associated with high alcohol consumption. Oncogenic miRNAs, such as the investigated miR-183 and miR-21, could be novel prognostic biomarkers of tumor progression and adverse clinical outcome in oral cancer, as well as novel therapeutic targets in cancer
Novel Kombucha Beverage from Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum, with Antibacterial and Antioxidant Effects
No full textKombucha is a nonalcoholic beverage traditionally made by fermenting black tea using a combination of yeast and acetic acid bacteria (AAB) cultures. Ganoderma lucidum hot water extract (HWE) was used-to our knowledge for the first time-to prepare a novel, health-promoting kombucha product. During the 11-day fermentation, pH, total acidity, and the numbers of yeasts and AAB were monitored. It was found that sweetened G. lucidum HWE was a good medium for yeast and AAB growth. The desired acidity for the beverage was reached on the second day (3 g/L) of the fermentation process; the maximum established acidity was 22.8 +/- 0.42 g/L. Fourier transform infrared analysis revealed that the vacuum-dried beverage is a mixture of various compounds such as polysaccharides, phenols, proteins, and lipids. Total phenolic content of the liquid sample was 4.91 +/- 0.2338 mg gallic acid equivalents/g, whereas the vacuum-dried sample had a smaller amount of phenolics (2.107 +/- 0.228 mg gallic acid equivalents/g). Established half-maximal effective concentrations for DPPH scavenging activity and reducing power were 22.8 +/- 0.17 and 10.61 +/- 0.34 mg/mL, respectively. The antibacterial testing revealed that activity does not originate solely from synthesized acetic acid. The liquid G. lucidum beverage was the most effective against the tested bacteria, with the lowest minimum inhibitory concentration (0.04 mg/ml,) against Staphylococcus epidermidis and Rhodococcus equi, and a minimum bactericidal concentration (0.16 mg/mL) against Bacillus spizizenii, B. cereus, and R. equi. The vacuum-dried sample was less effective, with the lowest minimum bactericidal concentration against the Gram-positive bacteria R. equi (1.875 mg/mL) and against the Gram-negative bacteria Proteus hauseri (30 mg/mL)
Synthesis, structural characterization and antimicrobial activity of silver(I) complexes with 1-benzyl-1H-tetrazoles
Get PDFHerein, we report the synthesis and structural characteristics of three tetrazole-containing compounds, 1-benzyl-1H-tetrazole (bntz), 1-benzyl-1H-tetrazol-5-amine (bntza) and 1-(4-methoxybenzyl)-1H-tetrazol-5-amine (mbntza) and the corresponding silver(I) complexes of the general formula [Ag(NO3-O)(L-N4)(2)](n), L = bntz (1), bntza (2) and mbntza (3). Silver(I) complexes 1-3 and 1-benzyl-1H-tetrazoles have been studied in detail by NMR, IR and UV-Vis spectroscopic methods and the structures of 1 and 2 have been determined by single-crystal X-ray diffraction analysis. The results of these analyses revealed a monodentate coordination of the ligands to Ag(I) ion via the N4 tetrazole nitrogen. The antimicrobial potential of silver(I) complexes 1-3 was evaluated against the broad panel of Gram-positive and Gram-negative bacteria and fungi, displaying their remarkable inhibiting activity with MIC (minimal inhibitory concentration) values in the range 2-8 and 0.16-1.25 mu g/mL (3.8-16.3 and 0.31-2.15 mu M), respectively. On the other hand, 1-benzyl-1H-tetrazoles used for the synthesis of the silver(I) complexes were not active against the investigated strains, suggesting that the activity of the complexes originates from the Ag(I) ion exclusively. Moreover, silver(I) complexes 1-3 have good therapeutic potential, which can be deduced from their moderate cytotoxicity on the human fibroblast cell line MRC5, with IC50 values falling in the range 30-60 mu g/mL (57.7-103.4 mu M).Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1144]This is the peer-reviewed version of the article: Andrejević, T. P., Nikolić, A. M., Glišić, B., Wadepohl, H., Vojnović, S., Zlatović, M., Petković, M., Nikodinović-Runić, J., Opsenica, I. M., & Djuran, M. (2018). Synthesis, structural characterization and antimicrobial activity of silver(I) complexes with 1-benzyl-1H-tetrazoles. Polyhedron, 154, 325–333. [https://doi.org/10.1016/j.poly.2018.08.001
Anti-quorum sensing activity, toxicity in zebrafish (Danio rerio) embryos and phytochemical characterization of Trapa natans leaf extracts
Get PDFEthnopharmacological relevance: Trapa natans L. (water chestnut or water caltrop) is a widespread aquatic plant, which has been cultivated for food and traditional medicine since ancient times. Pharmacological studies showed that water chestnut exhibits the wide range of biological activities, such as antimicrobial, antioxidative, analgesic, anti-inflammatory, as well as antiulcer. Aim of the study: Evaluation of anti-virulence potential and toxicity of T. natans methanol (TnM), acetone (TnA) and ethyl acetate (TnEA) leaf extracts. Materials and methods: The anti-quorum sensing activity of Tn extracts was addressed by measuring their effects on biofilm formation, swarming motility and pyocyanin and elastase production in Pseudomonas aeruginosa. Specific P. aeruginosa biosensors were used to identify which of the signaling pathways were affected. The lethal and developmental toxicity of extracts were addressed in vivo using the zebrafish (Danio rerio) model system. The phenolic composition of T. natans leafs extracts was analyzed by a linear ion trap-OrbiTrap hybrid mass spectrometer (LTQ OrbiTrapMS) and UHPLC system configured with a diode array detector (DAD) hyphenated with the triple quadrupole mass spectrometer. Results: Subinhibitory concentrations of Tn leaf extracts (0.2 MIC) inhibited pyocyanin and elastase production up to 50% and 60%, respectively, and reduced swarming zones, comparing to non-treated P. aeruginosa. TnA inhibited biofilm formation by 15%, TnM showed a stimulatory effect on biofilm formation up to 20%, while TnEA showed no effect. The bioactive concentrations of TnM and TnA were not toxic in the zebrafish model system. Twenty-two phenolic compounds were tentatively identified in TnM, where thirteen of them were identified in T. natans for the first time. Tn extracts, as well as their major components, ellagic and ferulic acids, demonstrated the ability to interfere with P. aeruginosa Las and PQS signaling pathways. Conclusions: This study demonstrates anti-virulence potential of Tn leaf extracts against medically important pathogen P. aeruginosa and confirms the ethnopharmacological application of this plant against microbial infections.Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1111]This is the peer reviewed version of the paper: Aleksić, I., Ristivojević, P., Pavić, A., Radojević, I., Comić, L. R., Vasiljević, B., Opsenica, D., Milojkovic-Opsenica, D., & Šenerović, L. (2018). Anti-quorum sensing activity, toxicity in zebrafish (Danio rerio) embryos and phytochemical characterization of Trapa natans leaf extracts. Journal of Ethnopharmacology, 222, 148–158. [https://doi.org/10.1016/j.jep.2018.05.005]Supplementary data: [https://imagine.imgge.bg.ac.rs/handle/123456789/1768