imagine (Institute of molecular genetics and genetic engineering)
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Anti-quorum sensing activity, toxicity in zebrafish (Danio rerio) embryos and phytochemical characterization of Trapa natans leaf extracts
No full textEthnopharmacological 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.Peer-reviewed manuscript: [https://imagine.imgge.bg.ac.rs/handle/123456789/1762]Supplementary data: [https://imagine.imgge.bg.ac.rs/handle/123456789/1768
Functional analyses of the FIIc.*64_*66del gene variant and its potential role in fetal loss etiology
No full textUgt1a1 (ta)(n) promoter genotype: diagnostic and population pharmacogenetic marker in Serbia
Get PDFThe UGT1A1 enzyme is involved in the metabolism of bilirubin and numerous medications. Unconjugated hyper-bilirubinemia, commonly presented as Gilbert syndrome (GS), is a result of decreased activity of the UGT1A1 enzyme, variable number of TA repeats in the promoter of the UGT1A1 gene affects enzyme activity. Seven and eight TA repeats cause a decrease of UGT1A1 activity and risk GS alleles, while six TA repeats contribute to normal UGT I Al activity and non-risk GS allele. Also, the UGT1A1 (TA)(n) promoter genotype is recognized as a clinically relevant phannacogenetic marker. The aim of this study was to access diagnostic value of UGTIAI (TA) n promoter genotyping in pediatric GS patients. Correlation of the UGT1A1(TA)(n) genotypes and level of unconjugated bilirubin at diagnosis and after hypocaloric and phenobarbitone tests in these patients was analyzed. Another aim of the study was to assess phannacogenetic potential ofUGT1A1 (TA)(n) variants in Serbia. Fifty-one pediatric GS patients and 100 healthy individuals were genotyped using different methodologies, polymerise chain reaction (PCR) followed by acrylamide electrophoresis, fragment length analysis and/or DNA sequencing. Concordance of the UGT1A1 (TA)(n) promoter risk GS genotypes with GS was found in 80.0% of patients. Therefore, UGT1A1 (TA)(n) promoter genotyping is not a reliable genetic test for GS, but it is useful for differential diagnosis of diseases associated with hyperbilirubinemia. Level of bilimbin in pediatric GS patients at diagnosis wasUGT1A1 (TA)(n) promoter genotype-dependent. We found that the frequency of phannacogenetic relevant UGT1A1 (TA)(n) promoter genotypes was 63.0%, pointing out that UGT1A1 (TA)(n) promoter genoty ping could be recommended for preemptive pharmacogendic testing in Serbia
Application of gum Arabic in the production of spray-dried chokeberry polyphenols, microparticles characterisation and in vitro digestion method
Get PDFDue to its high content of phenolics, black chokeberry has been proposed as a health-promoting material, especially use of chokeberry extract as a food or pharmaceuticals. Chokeberry extract was spray-dried using gum Arabic as a carrier. The optimal conditions for the efficient microencapsulation of chokeberry extract using spray-drying technique have been investigated. The aim of our study was to obtain microparticles with the best potential to improve functionality and stability of extracted chokeberry polyphenols, and to investigate the possibility of microbeads to protect active compounds during simulated digestion process. Morphological characteristics of microbeads were analyzed using FTIR and SEM techniques. Zeta potential, particle size and moisture content were determined. Released total phenolics and total anthocyanins content as well as concentration of individual anthocyanins were quantified before and after digestion process. Microparticles exhibited high encapsulation efficiency up to 87%, and high content of released polyphenols was achieved. After in vitro simulated digestion phenolic compounds decreased by 11-24%, exhibited higher protective effect of gum Arabic. Our results showed that chokeberry microparticles obtained by spray drying method could be useful supplements or functional food.
Bimetallic alginate nanocomposites: New antimicrobial biomaterials for biomedical application
No full textTwo bimetallic (Zn/Cu) alginate based nanocomposites, impregnated with carbonate or phosphate mineral phase, were prepared by a facile procedure. Mineralized samples exhibited different morphologies and properties when compared to the non-mineralized sample. Antimicrobial testing against Escherichia coil, Staphylococcus aureus and Candida albicans showed that mineralized samples are more efficient than non-mineralized in elimination of microorganisms. The results of this study suggest that bimetallic mineralized alginates could be potentially used as affordable, easy to produce antimicrobial materials
Fingerprinting of the antioxidant status in Alyssum markgrafii shoots during nickel hyperaccumulation in vitro
No full textThis study investigated the role of antioxidant system of Alyssum markgrafii, during long-term exposure to 0.5 or 1 mM NiCl2 x 6H(2)O in vitro. Applied methodology included sample preparation protocol which reduces oxidation of key metabolites along with novel luminescent method and well-established photometric procedures. During 5-week treatments, plants accumulated 1121 and 2470 ppm of Ni2+ respectively, followed by severe growth retardation, chlorophyll degradation and peroxidation of lipids. These effects were more pronounced after 1 mM Ni2+ treatment and additionally accompanied by increased water loss. Activities of luminol-converting peroxidases and glutathione reductase upon 0.5 mM treatment were increased while catalase and superoxide dismutase were diminished. The fact that these two groups of enzymes run in antiparallel might suggest functional redistribution between antioxidant enzymes rather than orchestrated action to prevent oxidative damage. Total antioxidant capacity (TAC) was also increased after 0.5 mM treatment which coincided with increased GR activity and elevated glutathione content indicating this low molecular weight antioxidant as an important factor associated with nickel tolerance. This study also emphasizes the possible important role of luminol-converting peroxidases in nickel hyperaccumulation, although they are not considered as antioxidant enzymes sensu stricto since some of them can also produce reactive oxygen species as well
Analysis of the CYP2C19 genotype associated with bleeding in Serbian STEMI patients who have undergone primary PCI and treatment with clopidogrel
No full textBleeding is one of the possible adverse events during clopidogrel therapy. The CYP2C19 gene is the most significant genetic factor which influences response to clopidogrel treatment. We aimed to examine the contribution of the CYP2C19 gene to bleeding occurrence during clopidogrel therapy in Serbian patients with ST segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI). This case-control study included 53 patients who experienced bleeding and 55 patients without bleeding. Bleeding events were defined and classified using the Bleeding Academic Research Consortium (BARC) criteria. All patients were prescribed daily doses of clopidogrel during the 1-year follow-up after PCI. The CYP2C19*17 (c.-806C gt T, rs12248560), rs11568732 (c.-889T gt G, CYP2C19*20), CYP2C19*2 (c.681G gt A; rs4244285) and CYP2C19*3 (c.636G gt A; rs4986893) variants were analysed in all 108 patients. Additionally, sequencing of all nine exons, 5'UTR and 3'UTR in the rs11568732 carriers was performed. Association between bleeding (BARC type ae lt yen gt 2) and the CYP2C19*17 variant was not observed [odds ratio (OR), 0.53; 95% confidence interval (CI), 0.2-1.1; p = 0.107). The rs11568732 variant showed significant association with bleeding (OR, 3.7; 95% CI, 1.12-12.44; p = 0.025). Also, we found that the rs11568732 variant appears independently of haplotype CYP2C19*3B, which is contrary to the previous findings. Our results indicate the absence of CYP2C19*17 influence and turn the attention to the potential significance of the rs11568732 variant in terms of adverse effects of clopidogrel. However, it is necessary to conduct an independent conformation study in order to verify this finding. Also, an analysis of the functional implication of the rs11568732 variant is necessary in order to confirm the significance of this variant, both in relation to its influence on gene expression and in relation to its medical significance
QSAR modeling of dihydrofolate reductase inhibitors as a therapeutic target for multiresistant bacteria
No full textAntibacterial resistance is a growing public health threat of major concern around the world so development of new therapeutic approaches to prevent bacterial multidrug resistance has become a primary consideration for medicinal chemistry research. QSAR models for the dihydrofolate reductase inhibition with 2,4-diamino-5-(substituted-benzyle)-pyramidine derivatives were developed with further computer-aided design of new derivatives with desired activity. The Monte Carlo method has been used as a computational tool for QSAR modeling. For the representation of molecular structure and optimal descriptor calculation, the simplified molecular input line entry system (SMILES) together with the molecular graph (hydrogen-suppressed graph-HSG, hydrogen-filled graph-HFG, and the graph of atomic orbitals-GAO) was used. One-variable models have been calculated for one data split into training, test, and validation set. The impact of Morgan's extended connectivity index on built QSAR models and outliers was determined. Statistical parameters for the best QSAR model are satisfying. Structural indicators (molecular fragments) responsible for the increase and the decrease of the stated activity are defined, and with the application of defined structural alerts, the computer-aided design of new derivatives with desired activity is presented. Computational experiments presented and applied in this research can satisfactorily predict desired endpoint and can be used further for computer-aided antibacterial drug design
Collaboration in the actions of Brh2 with resolving functions during DNA repair and replication stress in Ustilago maydis
Get PDFCells maintain a small arsenal of resolving functions to process and eliminate complex DNA intermediates that result as a consequence of homologous recombination and distressed replication. Ordinarily the homologous recombination system serves as a high-fidelity mechanism to restore the integrity of a damaged genome, but in the absence of the appropriate resolving function it can turn DNA intermediates resulting from replication stress into pathological forms that are toxic to cells. Here we have investigated how the nucleases Mus81 and Gen1 and the helicase Blm contribute to survival after DNA damage or replication stress in Ustilago maydis cells with crippled yet homologous recombination-proficient forms of Brh2, the BRCA2 ortholog and primary Rad51 mediator. We found collaboration among the factors. Notable were three findings. First, the ability of Gen1 to rescue hydroxyurea sensitivity of dysfunctional Blm requires the absence of Mus81. Second, the response of mutants defective in Blm and Gen1 to hydroxyurea challenge is markedly similar suggesting cooperation of these factors in the same pathway. Third, the repair proficiency of Brh2 mutant variants deleted of its N-terminal DNA binding region requires not only Rad52 but also Gen1. and Mus81. We suggest these factors comprise a sub pathway for channeling repair when Brh2 is compromised in its interplay with DNA.Published version: [https://imagine.imgge.bg.ac.rs/ha]ndle/123456789/1190This is the peer reviewed version of the paper: Kojic, M., Milisavljevic, M., & Holloman, W. K. (2018). Collaboration in the actions of Brh2 with resolving functions during DNA repair and replication stress in Ustilago maydis. DNA Repair, 63, 47–55.[ https://doi.org/10.1016/j.dnarep.2018.01.010
Analysis of mitochondrial DNA variability of residents of the Republic of Serbia : population genetics, phylogenetic and phylogeographic perspectives.
No full textсавременим људским популацијама Европе резултат су веома комплексних еволуционих, демографских и културолошких процеса који су се одвијали на простору Европе током више хиљада година. Међу најважнијим процесима издвајају се пост-глацијална реколонизација Европе, ширење пољопривреде током неолита (неолитска транзиција) и бројне миграције људских популација, попут миграција индоевропских народа током бронзаног доба и Велике сеобе народа у раном средњем веку. Балканско полуострво је имало важну улогу током целокупне историје људске врсте на евроазијском континенту због тога што је представљало важан миграцијски коридор преко којег су се одвијале миграције од праисторије па све до савременог доба. Такође, током последњег леденог максимума Балканско полуострво је представљало један од глацијaлних рефугијума са којег је отпочела пост-глацијaлна реколонизација Европе. Имајући у виду да српска популација насељава централни део Балканског полуострва, у овој популацији се могу очекивати генетички трагови комплексних демографских процеса.Предмет истраживања ове докторске дисертације било је испитивање варијабилности митохондријске ДНК (мтДНК) у савременој популацији Србије са популационо генетичког, филогенетског и филогеографског аспекта. У ту сврху анализирана је варијабилност првог и другог хиперваријабилног сегмента (HVS-I и HVS-II) и полиморфизми кодирајућег региона мтДНК код 172 испитаника чији су преци по женској линији из последње две генерације декларисаног српског етницитета. Поред тога, одабрано је и комплетно секвенцирано 58 митогенома који су анализирани у циљу бољег разумевања еволуције појединих мтДНК хаплогрупа, као и утврђивања доприноса различитих миграција у обликовању садашњег обрасца варијабилности митохондријског генског пула српске популације.У српској популацији заступљене су већином мтДНК хаплогрупе/подхаплогрупе карактеристичне за европске популације, а њихове учесталости и параметри генетичке разноврсности налазе сe у распону вредности детектованих у осталим европским популацијама. Српска популација није генетички диференцирана у односу на друге јужнословенске популације и у свим анализама заузима централну позицију између јужнословенских популација источног и западног дела Балканског полуострва. Највећи проценат HVS-I и HVS-I/HVS-II хаплотипова становништво Србије дели са географски блиским популацијама Балканског полуострва из јужнословенске групе, где су детектовании потенцијално приватни хаплотипови...Genetic structure and clinal distribution of genetic variation in contemporary human populations in Europe are the results of interplay between exceptionally complex evolutionary, demographic and cultural processes which took place in Europe during several thousand years. Among the most important processes are post-glacial recolonization of Europe, spread of farming during the Neolithic period (Neolithic transition) and numerous migrations of human populations such as Bronze Age migrations of Indo-Europeans and migrations during the Migration Period that occurred in the Early Middle Ages. The Balkan Peninsula played an important role during the entire history of humankind in Europe and Asia because it served as an important migration corridor starting from prehistoric to present days. Furthermore, during the last glacial maximum, this region was one of the glacial refugia from which the post-glacial recolonization of Europe was initiated. Since Serbian population inhabits the central part of the Balkan Peninsula, this population may harbour genetic traces of past complex demographic processes that were occurring in the Balkans over time.The subject of this doctoral thesis was the study of the mitochondrial DNA (mtDNA) variability in a contemporary population of Serbia from population genetics, phylogenetic and phylogeographic aspects. For that purpose, the variability of HVS-I and HVS-II regions and polymorphisms within the coding region of mtDNA have been assessed in 172 individuals whose Serbian maternal ancestry was confirmed for at least two generations. Furthermore, 58 selected mitogenomes were completely sequenced in order to provide new insights into the evolution of particular mtDNA haplogroups and to assess the contribution of various human migrations in modelling contemporary maternal gene pool of the Serbian population.Serbian population comprises mainly mtDNA haplogroups/subhaplogroups typical for European populations and their frequencies and levels of genetic diversity are mostly in the range of those detected in other European populations. Serbian population is not genetically differentiated from other south-Slavic populations and in all analyses it occupies a central position among south-Slavic populations from the eastern and western part of the Balkan Peninsula. The highest percentage of HVS-I and HVS-I/HVS-II haplotypes Serbian population shares with south-Slavic populations from the Balkan Peninsula, where potential private haplotypes have been observed as well. In general, European populations were grouped in a manner reflecting their geographic distribution. Furthermore, Slavic populations were grouped according to their linguistic affiliation. Rather high values of all parameters of genetic diversity of rare mtDNA subhaplogroups in the Balkan Peninsula are concordant with the previously demonstrated refugial character of this region..