imagine (Institute of molecular genetics and genetic engineering)
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Burkholderia cepacia YtnP and Y2-aiiA lactonases inhibit virulence of Pseudomonas aeruginosa via quorum quenching activity
No full textBurkholderia cepacia is well known as the causative agent of infections in humans where often shares niche with other pathogens, like Pseudomonas aeruginosa. Clinical isolate Burkholderia sp. BCC4135 was selected due to its strong quorum quenching (QQ) activity. Whole genome sequencing unveiled this isolate as B. cepacia with unique sequence type ST1485 and a myriad of genes belonging to resistome and virulome. Two QQ lactonases YtnP and Y2-aiiA originated from B. cepacia BCC4135 were cloned, expressed, and functionally characterized. They were active against a broad substrate spectrum of the N-acyl-homoserine lactones (AHLs). The YtnP lactonase was inactive, while Y2-aiiA was active against N-tetradecanoyl-DL-homoserine lactone (C14-HSL) which could imply the difference in their biological roles from the aspect of its quorum sensing (QS) autoregulation and interference with the QS systems of bacteria residing within the same niche. Both YtnP and Y2-aiiA were able to attenuate virulence potential of P. aeruginosa MMA83 clinical isolate declining its biofilm formation and virulence factors production. B. cepacia BCC4135 lactonases interfered with the las, rhl, and even pqs QS circuit of P. aeruginosa MMA83 transcription and the effect of combined enzymes was even more prominent. B. cepacia BCC4135 also employs the CepI/R QS system for governing its own virulence traits and possibly self-regulates the QQ/QS network through the different expression and activity of YtnP and/or Y2-aiiA. Our findings pointed out that BCC4135 lactonases could be exploited as an effective antivirulence drugs against P. aeruginosa and gave us a new insight into B. cepacia QQ/QS machinery
Silver(I) complexes with 1,10-phenanthroline-based ligands: The influence of epoxide function on the complex structure and biological activity
No full textIn a continuing search for a novel metal-containing antimicrobial agents, the present study reports the synthesis, characterization and biological evaluation of two silver(I) complexes with 1,10-phenanthroline-based ligands, [Ag(1,10-phen)(2)]CF3COO center dot H2O (Ag1) and [Ag(CF3COO)(5,6-epoxy-1,10-phen)](2)(Ag2), 1,10-phen is 1, 10-phenanthroline and 5,6-epoxy-1,10-phen is 5,6-epoxy-5,6-dihydro-1,10-phenanthroline. The complexes were characterized by different spectroscopic techniques (IR, H-1 and C-13 NMR and UV-Vis), while the crystal structure of Ag2 complex was determined by a single-crystal X-ray diffraction analysis. The spectroscopic data confirmed that the structure of Ag1 complex, with silver(I) ion tetrahedrally coordinated by two bidentate 1, 10-phen ligands, is in accordance to the previous report (S.E. Paramonov et al., 2003). The crystallographic results showed that in dinuclear Ag2 complex, both Ag(I) ions are coordinated bidentately by 5,6-epoxy-1, 10-phen and monodentately by trifluoroacetate, with presence of the short Ag center dot center dot center dot Ag contact of 2.963 angstrom. Both silver (I) complexes were evaluated in vitro for antimicrobial activity against four bacterial and four Candida species, showing selectivity towards the investigated species of Candida with minimal inhibitory concentrations (MICs) between 0.9 and 12.5 mu M. Moreover, Ag2 complex manifested significant antiproliferative properties in the case of a range of human cell lines, including human breast cancer (MDA-MB 231), which resulted from the presence of epoxy functional group in the ligand. The gel electrophoresis results obtained from the studies of Ag1 and Ag2 interactions with bacteriophage lambda DNA (XDNA) suggested that these complexes did not cause DNA degradation
Banat donkey, a neglected donkey breed from the central Balkans (Serbia)
Get PDFThe dominant donkey breed in the Balkans is the mid-sized Balkan donkey with a grey to chocolate coat color. Local breeders from Serbia, however, still maintain a few larger individuals of a lighter coat color, named Banat donkey, and speculate that they are descendants of a Spanish donkey heard that had been transferred to the Banat region by the Hapsburg Queen Maria Theresa in the XVIII century for a specific purpose, to work in local vineyards. We have previously found a unique nuclear gene-pool and a prevalence of mitochondrial Clade 2 haplotypes in several such animals. In this study, we: (i) perform a comparative analysis of 18 morphological traits of the Banat donkey (seven individuals), Balkan donkey (53 individuals from two sub-populations of this breed) and the potential hybrids (eight individuals), and demonstrate the morphological distinctiveness of the Banat donkey, highlighting the diagnostic traits for distinguishing the breed: hip height, croup width, body length and chest depth; (ii) reanalyse published nuclear microsatellite data for these groups, and reveal that, although severely depopulated, the genetically distinct Banat donkey is not severely affected by the loss of genetic diversity and inbreeding; (iii) demonstrate that previously published Banat donkey mitochondrial haplotypes, analyzed genealogically together with those reported in ancient and modern individuals from Spain, Italy, Turkey, Cyprus and Africa, are shared with three Spanish breeds and individuals belonging to Amiata and some other Italian breeds. A unique morphological feature present in Banat and Somali wild donkeys, but also in Amiata donkeys, black stripes on legs, suggests that the origin of Clade 2 donkeys may be much more complex than previously thought. Actions to preserve the Banat donkey, a valuable but critically endangered genetic resource ( lt 100 individuals), are urgent
Lactobacillus salivarius BGHO1 and Lactobacillus reuteri BGGO6-55 modify nutritive profile of Artemia franciscana nauplii in a strain ratio, dose and application timing-dependent manner
No full textThis study aimed to analyze an impact of Lactobacillus salivarius BGHO1 and Lactobacillus reuteri BGGO6-55 supplemented to Anemia franciscana cultivation medium on biochemical profile of hatched nauplii. Impacts of different BGHO1:BGGO6-55 ratios (75:25, 50:50 and 25:75) and their total concentrations (250 and 500 g/kg of Anemia cysts) at different timings of bacteria application (pre- and post-hatching) were examined. The effects were evaluated by quantification of naupliar peptide, soluble protein, phospho-, neutral and unsaturated lipid content. Uni and multifactorial analysis of variance (ANOVA) were used to estimate the effects of treatments relative to control and to model factor interactions, respectively Statistical analysis indicated that posthatching application of 75:25 strain ratio at high dose was associated with an increase of neutral lipid amount. Furthermore, factor interaction profiling identified positive correlation of lacto-bacilli concentration with the level of free amino acids/short peptides, phospho-, neutral and unsaturated lipids, but only at 50:50 strain ratio combination. Application of lower lactobacilli dose at 75:25 strain ratio caused an increase of soluble protein and phospholipid amount. Hypothetically, graded response of Artemia nauplii to lactobacilli supplementation was induced with different BGHO1 doses. It assumedly ranged from stress-response protein synthesis at lower doses to membrane permeability alterations and triglyceride-mediated defense mechanism activation at higher BGHO1 doses. In contrast to lactobacilli supplementation after Anemia hatching, pre-hatching application of lactobacilli was not associated with an increase of Anemia nutritive profile, though again there was a positive association of lactobacilli concentration with nutrient amount. This implicates interference of lactobacilli with the hatching process. Results presented here facilitate the design of future studies aiming to modify Artemia nutritive profile in accordance with nutritional demands of cultivated fish species
New minor groove covering DNA binding mode of dinuclear Pt(II) complexes with various pyridine-linked bridging ligands and dual anticancer-antiangiogenic activities
No full textNew anticancer platinum(II) compounds simultaneously targeting tumor cells and tumor-derived neoangiogenesis, with new DNA interacting mode and large therapeutic window are appealing alternative to improve efficacy of clinical platinum chemotherapeutics. Herein, we describe three novel dinuclear [{Pt(en)Cl}(2)(mu-L)](2+) complexes with different pyridine-like bridging ligands (L), 4,4 '-bipyridine (Pt1), 1,2-bis(4-pyridyl)ethane (Pt2) and 1,2-bis(4-pyridyl)ethene (Pt3), which highly, positively charged aqua derivatives, [{Pt(en)(H2O)}(2)(mu-L)](4+), interact with the phosphate backbone forming DNA-Pt adducts with an unique and previously undescribed binding mode, called a minor groove covering. The results of this study suggested that the new binding mode of the aqua-Pt(II) complexes with DNA could be attributed to the higher anticancer activities of their chloride analogues. All three compounds, particularly complex [{Pt(en)Cl}(2)(mu-4,4 '-bipy)]Cl-2 center dot 2H(2)O (4,4 '-bipy is 4,4 '-bipyridine) (Pt1), overcame cisplatin resistance in vivo in the zebrafish-mouse melanoma xenograft model, showed much higher therapeutic potential than antiangiogenic drug sunitinib malate, while effectively blocking tumor neovascularization and melanoma cell metastasis. Overall therapeutic profile showed new dinuclear Pt(II) complexes could be novel, effective and safe anticancer agents. Finally, the correlation with the structural characteristics of these complexes can serve as a useful tool for developing new and more effective anticancer drugs
Nature-inspired synthetic analogues of quorum sensing signaling molecules as novel therapeutics against Pseudomonas aeruginosa infections
No full textThe rapid emergence of antibiotic-resistant pathogens presents a global healthcare challenge. Bacteria control their virulence, motility, and biofilm formation, all of them being required for establishing pathogenicity, through a cell density-dependent communication system known as quorum sensing (QS). QS comprises production of extracellular signaling molecules, their detection, and population-wide response involving regulation of the virulence genes expression. Inhibition of QS affects virulence and reduces harmful effects to the host and as such presents a promising strategy to fight antibiotic-resistant infections. Multiresistant Pseudomonas aeruginosa belogns to the group of most critical pathogens for which the introduction of new therapeutics is imperative. In the search for novel therapeutics nature continues to be inexhaustible source of bioactive scaffolds, which provide the bases for structure-based rational drug design enabling further exploitation of diverse natural structures. This review describes bacterial QS systems, highlights strategies for their manipulation, overviews nature-inspired antivirulence molecules primarily against P. aeruginosa, and discusses their perspectives
The role of silicic acid in modulation of oxidative stress responses in cucumber (Cucumis sativus L.) exposed to toxic copper
Get PDFBakar (Cu) je esencijalan mikrolement za biljke, dok je prisustvo Cu u višku fitotoksično i izaziva oksidativni stres. Silicijum (Si) ne pripada grupi esencijalnih već korisnih elemenata, a jedini je element koji ispoljava protektivan efekat na biljke izložene stresnim uslovima. Uloga Si u stresu izazvanom toksičnim koncentracijama Cu nije dovoljno istražena. Cilj ovog rada bio je da se ispitaju mehanizmi delovanja Si u takvim uslovima kod krastavca, akumulatora Si. Rezultati su pokazali da primena Si povećava toleranciju biljaka na toksičnost Cu. Mehanizmi delovanja Si su usmereni ka snižavanju ekspresije gena odgovornih za usvajanje Cu i, posledično, smanjenju koncentracije Cu u biljkama, kao i ka ublažavanju disbalansa drugih esencijalnih mikroelemenata, pre svega gvožđa. Na taj način, Si je smanjio hlorozu i povećao biomasu listova biljaka. Si je doprineo preraspodeli Cu na nivou ćelija korena, povećavajući imobilizaciju Cu u ćelijskim zidovima. Povećanom akumulacijom liganada koji vezuju Cu, kao što su organske kiseline (citrat i malat u korenu, a akonitat u listu) i aminokiseline (nikocijanamin i histidin u listu), Si je ublažio toksične efekte Cu unutar ćelije. Pored toga, stimulacijom aktivnosti antioksidativnih enzima, smanjen je nivo oksidativnog stresa. Primena Si je uticala na glavne regulatore homeostaze Cu, mikro RNK (miR398 i miR408), čija je snižena ekspresija u korenu omogućila povišenu ekspresiju njihovih targeta: Cu/Zn superoksid-dismutaze 2 (CSD2) i lakaze 3. U skladu sa tim, akumulacija Cu-proteina, CSD2 u korenu i plastocijanina u listu, kao primarnih depoa Cu jona je izraženija kod biljaka gajenih sa Si. Razumevanje mehanizama delovanja Si u biljkama će doprineti njegovoj široj primeni u cilju povećanja tolerancije biljaka na stres.Copper (Cu) is an essential microelement for plants, although it is phytotoxic in excess, causing oxidative stress. Silicon (Si) does not belong to the essential but beneficial elements, and it is the only known element with protective effect to plants exposed to stress. The role of Si in plants exposed to Cu-stress has not been thoroughly studied so far. The aim of this study was to investigate the Si-mediated mechanisms under such conditions in cucumber plants, a Si-accumulating species. The results showed that Si application enhanced plant tolerance to Cu in excess. Si-mediated mechanisms were directed towards downregulation of the genes involved in Cu uptake, thus decreasing Cu concentration in the treated plants, as well as alleviating imbalance of other microelements, particularly Fe. Consequently, Si alleviated chlorosis supporting higher leaf biomass production in these plants. Si contributed to the redistribution of Cu at the root cell level, by increasing the immobilization of Cu in the cell walls. Higher accumulation of Cu-binding ligands such as organic acids (citrate and malate in the roots and aconitate in the leaves) and amino acids (nicotianamine and histidine in the leaves) diminished Cu-toxicity effects inside the cells. Additionally, a decreased level of oxidative stress was achieved by Si-stimulated activities of antioxidative enzymes. Si-application downregulated the expression of micro RNAs (miR398 and miR408), the main regulators of Cu homeostasis, thus enabling higher expression of their target genes: Cu/Zn superoxide dismutase 2 (CSD2) and laccase 3. Accordingly, accumulation of Cu-proteins, CSD2 in the roots and plastocyanine in the leaves, as “buffering sinks” for Cu, was obtained in the plants grown with Si. Understanding the Si-mediated mechanisms in plants will contribute to its broader application with the aim to increase plant tolerance to stress
Application of targeted next generation sequencing for the mutational profiling of patients with acute lymphoblastic leukemia
Get PDFlt b gt Uvod: lt /b gt Akutna limfoblastna leukemija (ALL) je najčešće maligno oboljenje kod dece, dok je kod odraslih njena učestalost mnogo niža. U današnjoj kliničkoj praksi kao najvažnije metode stratifikacije pacijenata u određene grupe rizika koriste se metode identifikacije citogenetičkih aberacija i malog broja molekulanih markera. Tehnologija sekvenciranja nove generacije (SNG) obezbeđuje veliku količinu podataka koji doprinose razjašnjavanju mutacionog profila dečje (dALL) i adultne ALL (aALL). lt b gt Metode: lt /b gt Uzorci DNK iz 34 dALL i aALL pacijenata analizirani su primenom SNG ciljanog sekvenciranja ("TruSeq Amplicon Cancer Panel - TSACP") kojim se sekvenciraju "hotspot" mutacije u 48 gena povezanih sa kancerom. lt b gt Rezultati: lt /b gt Identifikovano je ukupno 330 varijanti u kodirajućim regionima, od kojih je samo 95 njih za posledicu imalo potencijalnu promenu u proteinu. Posmatrano kod pojedinačnih pacijenata, detektovane mutacije su pretežno remetile Ras/RTK signalni put (STK11, KIT, MET, NRAS, KRAS, PTEN). Pored toga, identifikovano je 5 pacijenata sa istom mutacijom u HNF1A genu, koja je uzrokovala poremećaje u Wnt i Notch signalnom putu. Kod dva pa cijenta otkrivene su varijante u NOTCH1 genu. Nije detektovano istovremeno prisustvo varijanti u HNF1A i NOTCH1 genu, dok su geni uključeni u Ras/RTK signalni put pokazali tendenciju ka akumuliranju mutacija. lt b gt Zaključak: lt /b gt Naši rezultati pokazuju da ALL sadrži Mali broj mutacija, bez značajnih razlika između dALL i aALL (medijana po pacijentu 2 odnosno 3). Detektovane mutacije izazivaju poremećaje u nekoliko ključnih signalnih puteva, prvenstveno Ras/RTK kaskade. Ova studija doprinosi ukupnom znanju o mutacionom profilu ALL, što vodi ka boljem razumijevanju molekularne osnove ovog oboljenja.lt b gt Background: lt /b gt Acute lymphoblastic leukemia (ALL) is the most common cancer in children, whereas it is less common in adults. Identification of cytogenetic aberrations and a small number of molecular abnormalities are still the most important risk and therapy stratification methods in clinical practice today. Next generation sequencing (NGS) technology provides a large amount of data contributing to elucidation of mutational landscape of childhood (cALL) and adult ALL (aALL). lt b gt Methods: lt /b gt We analyzed DNA samples from 34 cALL and aALL patients, using NGS targeted sequencing TruSeq Amplicon - Cancer Panel (TSACP) which targets mutational hotspots in 48 cancer related genes. lt b gt Results: lt /b gt We identified a total of 330 variants in the coding regions, out of which only 95 were potentially protein-changing. Observed in individual patients, detected mutations predominantly disrupted Ras/RTK pathway (STK11, KIT, MET, NRAS, KRAS, PTEN). Additionally, we identified 5 patients with the same mutation in HNF1A gene, disrupting both Wnt and Notch signaling pathway. In two patients we detected variants in NOTCH1 gene. HNF1A and NOTCH1 variants were mutually exclusive, while genes involved in Ras/RTK pathway exhibit a tendency of mutation accumulation. lt b gt Conclusions: lt /b gt Our results showed that ALL contains low number of mutations, without significant differences between cALL and aALL (median per patient 2 and 3, respectively). Detected mutations affect few key signaling pathways, primarily Ras/RTK cascade. This study contributes to knowledge of ALL mutational landscape, leading to better understanding of molecular basis of this disease
Ononis spinosaL., an edible and medicinal plant: UHPLC-LTQ-Orbitrap/MS chemical profiling and biological activities of the herbal extract
No full textThis study explored the chemical profile of the aerial parts ofOnonis spinosaand further investigated its biological activities. Chemical profiling of the extract revealed the presence of 63 different compounds: phenolic acids, flavonoid glycosides and aglycones, isoflavonoid glycosides and aglycones, and other related compounds. Our results revealed that the extract was active against 8 strains of free floating bacteria. It showed anti-biofilm potential againstStaphylococcus aureusand was able to supress the production of staphyloxanthin inS. aureusat sub-minimal inhibitory concentrations. Its antioxidant activity was evaluated by using several assays (phosphomolybdenum, DPPH, ABTS, CUPRAC, FRAP, and metal chelating assay), which showed that the extract exhibited a dose dependent activity. Inhibition of AChE, BChE, amylase, glucosidase and tyrosinase was achieved by the extract, demonstrating its anti-enzymatic activity. The antiproliferative potential of the extract towards human cancer cell lines (HepG2, MCF-7, SiHa and A172) was determined by using the crystal violet assay. Ki67, a marker of proliferation was downregulated in the A172 glioblastoma cell line
The Genetic Landscape and Epidemiology of Phenylketonuria
Get PDFPhenylketonuria (PKU), caused by variants in the phenylalanine hydroxylase (PAH) gene, is the most common autosomal-recessive Mendelian phenotype of amino acid metabolism. We estimated that globally 0.45 million individuals have PKU, with global prevalence 1:23,930 live births (range 1:4,500 [Italy]-1:125,000 [Japan]). Comparing genotypes and metabolic phenotypes from 16,092 affected subjects revealed differences in disease severity in 51 countries from 17 world regions, with the global phenotype distribution of 62% classic PKU, 22% mild PKU, and 16% mild hyperphenylalaninemia. A gradient in genotype and phenotype distribution exists across Europe, from classic PKU in the east to mild PKU in the southwest and mild hyperphenylalaninemia in the south. The c.1241A gt G (p.Tyr414Cys)-associated genotype can be traced from Northern to Western Europe, from Sweden via Norway, to Denmark, to the Netherlands. The frequency of classic PKU increases from Europe (56%) via Middle East (71%) to Australia (80%). Of 758 PAH variants, c.1222C gt T (p.Arg408Trp) (22.2%), c.1066-11G gt A (IVS10-11G gt A) (6.4%), and c.782G gt A (p.Arg261Gln) (5.5%) were most common and responsible for two prevalent genotypes: p.[Arg408Trp];[Arg408Trp] (11.4%) and c.[1066-11G gt A];[1066-11G gt A] (2.6%). Most genotypes (73%) were compound heterozygous, 27% were homozygous, and 55% of 3,659 different genotypes occurred in only a single individual. PAH variants were scored using an allelic phenotype value and correlated with pre-treatment blood phenylalanine concentrations (n = 6,115) and tetrahydrobiopterin loading test results (n = 4,381), enabling prediction of both a genotype-based phenotype (88%) and tetrahydrobiopterin responsiveness (83%). This study shows that large genotype databases enable accurate phenotype prediction, allowing appropriate targeting of therapies to optimize clinical outcome