imagine (Institute of molecular genetics and genetic engineering)
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Cardiovascular variability and beta-ARs gene expression at two stages of doxorubicin - Induced cardiomyopathy
No full textUsing comprehensive analysis of heart rate (HRV) and blood pressure (BPV) short-term variability we estimated the time course of changes of autonomic nervous system remodeling in two stages of doxorubicin-induced cardiomyopathy (DCM). We also investigated the level of gene expression of cardiac beta-1 (beta-1AR) and beta-2 (beta-2AR) adrenoceptors. Experiments were performed in adult male Wistar rats equipped with indwelling catheters for BP recording and blood withdrawal. A 15 mg/kg total cumulative dose of doxorubicin was injected i.p. to rats to induce DCM or saline for control (n = 18). Rats were assessed for general toxicity, cardiovascular hemodynamic and echocardiography before treatment (n = 6), 35 days (DOX35; n = 6) and 70 days (DOX70; n = 6) post-treatment. HRV was evaluated by spectral analysis, Poincare plots, sample and approximate entropy. Expression of beta-1AR and beta-2AR mRNA was evaluated by RT-qPCR. Doxorubicin-treated rats exhibited poor general condition and lower survival than saline-treated rats. In DOX35 rats, there were no echocardiography signs of decompensation, no increase in serum cardiac troponins, but there was an increase of HRV and decrease of HR complexity. In these rats typical microscopic signs of cardiotoxicity were seen along with over-expression of beta-1AR mRNA. 70 days post-treatment echocardiography revealed signs of decompensation and serum cardiac troponin T was increased. At this stage BPV decreased. In conclusion, HRV increase matches transient over expression of cardiac beta-1AR mRNA in compensate stage of DCM while decompensate stage of DCM is characterized by a decrease of BPV and no changes in beta-1AR and beta-2AR gene expression
Poly (ε-caprolactone) microspheres for prolonged release of selenium nanoparticles
No full textPoly (ε-caprolactone) (PCL) microspheres as a carrier for sustained release of antibacterial agent, selenium nanoparticles (SeNPs), were developed. The obtained PCL/SeNPs microspheres were in the range 1–4 μm with the encapsulation efficiency of about 90%. The degradation process and release behavior of SeNPs from PCL microspheres were investigated in five different degradation media: phosphate buffer solution (PBS), a solution of lipase isolated from the porcine pancreas in PBS, 0.1 M hydrochloric acid (HCl), Pseudomonas aeruginosa PAO1 cell-free extract in PBS and implant fluid (exudate) from the subcutaneously implanted sterile polyvinyl sponges which induce a foreign-body inflammatory reaction. The samples were thoroughly characterized by SEM, TEM, FTIR, XRD, PSA, DSC, confocal microscopy, and ICP-OES techniques. Under physiological conditions at neutral pH, a very slow release of SeNPs occurred (3 and 8% in the case of PBS or PBS + lipase, respectively and after 660 days), while in the acidic environment their presence was not detected. On the other hand, the release in the medium with bacterial extract was much more pronounced, even after 24 h (13%). After 7 days, the concentration of SeNPs reached a maximum of around 30%. Also, 37% of SeNPs have been released after 11 days of incubation of PCL/SeNPs in the implant exudate. These results suggest that the release of SeNPs from PCL was triggered by Pseudomonas aeruginosa PAO1 bacterium as well as by foreign body inflammatory reaction to implant. Furthermore, PCL/SeNPs microspheres were investigated in terms of their biocompatibility. For this purpose, cytotoxicity, the formation of reactive oxygen species (ROS), and genotoxicity were evaluated on HepG2 cell line. The interaction of PCL/SeNPs with phagocytic cell line (Raw 264.7 macrophages) was monitored as well. It was found that the microspheres in investigated concentration range had no acute cytotoxic effects. Finally, SeNPs, as well as PCL/SeNPs, showed a considerable antibacterial activity against Gram-positive bacteria: Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 1228). These results suggest that PCL/SeNPs-based system could be an attractive platform for a prolonged prevention of infections accompanying implants. © 2018 Elsevier B.V.Peer-reviewed manuscript: [https://imagine.imgge.bg.ac.rs/handle/123456789/1614]Supporting information: [https://imagine.imgge.bg.ac.rs/handle/123456789/1615
Derivatives of rhamnolipids and 4-aminoquinoline as Pseudomonas aeruginosa and Serratia marcescens virulence inhibitors
Get PDFRezistencija bakterija na antibiotike je rastući problem globalnih razmera tako da su pronalaženje i uvođenje novih terapijskih opcija u borbu protiv bakterijskih infekcija postali prioriteti i fundamentalnih i primenjenih istraživanja. U potrazi za jedinjenjima prirodnog porekla sa anti-virulentnom aktivnošću izdvojen je nepatogeni soj iz rizosfere biljke dobračice (Glechoma hederacea), Lysinibacillus sp. BV152.1, čiji su metaboliti inhibirali formiranje biofilmova P. aeruginosa PAO1. Hemijske analize su pokazale da aktivnu komponentu etil acetatnog ekstrakta čini smeša ramnolipida. Uporednom analizom anti-biofilm aktivnosti utvrđeno je da su di-ramnolipidi soja Lysinibacillus sp. BV152.1 bolji inhibitori procesa formiranja biofilmova (adhezije i sazrevanja) kod P. aeruginosa PAO1 od komercijalno dostupnih ramnolipida, poreklom iz P. aeruginosa. Amidnom derivatizacijom di-ramnolipida poboljšana je njihova anti-biofilm aktivnost, gde je najaktivniji derivat di-ramnolipida iz Lysinibacillus sp. BV152.1 imao morfolinsku grupu i inhibirao je formiranje biofilmova P. aeruginosa PAO1 za 80% u koncentraciji od 100 μg/ml, a isti derivat u koncentraciji od 50 μg/ml inhibirao je formiranje biofilmova S. marcescens za 88%. Hinolini, jedinjenja sa širokim spektrom bioloških aktivnosti, a takođe i autoinduceri PQS signalnog puta međubakterijske komunikacije P. aeruginosa predstavljaju dobru osnovu za razvoj anti-virulentnih jedinjenja. Derivati aminohinolina sintetisani u ovoj studiji nisu pokazali značajnu antibakterijsku aktivnost prema Gram negativnim patogenima. Najveću inhibitornu aktivnost na formiranje biofilmova kod P. aeruginosa i S. marcescens imala su jedinjenja koja su sadržala atom hlora ili CF3 grupu na poziciji C(7) i alifatični lanac sa 12 C atoma na poziciji C(4) (jedinjenja 5 i 10), sa BFIC50 koncentracijama od 69 μM odnosno 63 μM. Ova jedinjenja su prvi derivati hinolina za koje je utvrđena sposobnost da inhibiraju biofilmove S. marcescens. Detaljnom analizom odnosa strukture i aktivnosti jedinjenja 5 i 10 je pokazano da dužina alifatičnog lanca i lipofilnost jedinjenja imaju najveći uticaj na formiranja biofilomova kod S. marcescens. Jedinjenje 10 je izdvojeno kao najpotentniji inhibitor proizvodnje piocijanina kod P. aeruginosa hinolinske prirode opisan do sada...Bacterial resistance to antibiotics is a growing problem on a global scale, so finding and introducing new therapeutic options to combat bacterial infections has become a priority in both fundamental and applied research. In a search for structurally new compounds with anti-virulent activity, novel non-pathogenic strain was isolated from the rhizosphere of a Glechoma hederacea plant, named BV152.1, whose metabolites inhibited the formation of P. aeruginosa PAO1 biofilms. Chemical analyzes have shown that the active compound of ethyl acetate extract is a mixture of rhamnolipids. Comparative analysis of their anti-biofilm activity revealed that the di-rhamnolipids from the strain Lysinibacillus sp. BV152.1 are better inhibitors of P. aeruginosa PAO1 biofilm adhesion and maturation, than commercially available rhamnolipids, originated from P. aeruginosa. The amide derivatization of di-rhamnolipids enhanced the anti-biofilm activity of these compounds, where the most active di-rhamnolipid derivative from Lysinibacillus sp. BV152.1 had a morpholine group and inhibited the formation of P. aeruginosa PAO1 biofilms by 80% at a concentration at 100 μg/ml. The same derivative at 50 μg/ml inhibited formation of S. marcescens biofilm by 88%. Quinolines, compounds with a wide range of biological activities, and autoinducers of the quorum sensing PQS signaling pathway of P. aeruginosa represent a good basis for the development of compounds with anti-virulent activity. The aminoquinoline derivatives synthesized in this study did not show significant antibacterial activity against Gram-negative pathogens, making them suitable chemical structures for the development of anti-virulent agents. The highest inhibitory activity of P. aeruginosa and S. marcescens biofilm formation showed compounds containing a chlorine atom (compound 5) or CF3 group (compound 10) at position C(7) and an aliphatic chain of 12 C atoms at position C(4) with BFIC50 concentrations of 69 μM and 63 μM, respectively. These compounds are the first quinoline derivatives identified to have the ability to inhibit S. marcescens biofilms formation. Detailed analysis of the structure and activity relationships of compounds 5 and 10 showed that the aliphatic chain length and lipophilicity of the compounds had the greatest influence on inhibition of biofilm formation in S. marcescens..
Antifungal potential of bacterial rhizosphere isolates associated with three ethno-medicinal plants (poppy, chamomile, and nettle)
No full textThe objective of the present study was to isolate Actinobacteria, preferably Streptomyces spp. from the rhizosphere soils of three ethno-medicinal plants collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and Urtica dioica) and to screen their antifungal activity against Candida spp. Overall, 103 sporulating isolates were collected from rhizosphere soil samples and determined as Streptomyces spp. Two different media and two extraction procedures were used to facilitate identification of antifungals. Overall, 412 crude cell extracts were tested against Candida albicans using disk diffusion assays, with 42% (43/103) of the strains showing the ability to produce antifungal agents. Also, extracts inhibited growth of important human pathogens: Candida krusei, Candida parapsilosis, and Candida glabrata. Based on the established degree and range of antifungal activity, nine isolates, confirmed as streptomycetes by 16S rRNA sequencing, were selected for further testing. Their ability to inhibit Candida growth in liquid culture, to inhibit biofilm formation, and to disperse pre-formed biofilms was assessed with active concentrations from 8 to 250 mu g/mL. High-performance liquid chromatographic profiles of extracts derived from selected strains were recorded, revealing moderate metabolic diversity. Our results proved that rhizosphere soil of ethno-medicinal plants is a prolific source of streptomycetes, producers of potentially new antifungal compounds
Construction and identification of recombinant lentivirus overexpression and RNA interference vector containing cardiac adriamycin reactive protein gene
No full textObjective: To construct the recombinant lentiviral vector containing cardiac adriamycin responsive protein (CARP) gene and small-hairpin RNA (shRNA) targeting CARP gene and to pack the lentivirus, and to lay the foundation for further study on the function and mechanism of CARP in adriamycin (ADR) induced cardiomyopathy. Methods: After the rat CARP gene was amplified by PCR and shRNAs targeting CARP were designed and synthesized, they were inserted into the shuttle plasmids GV-358 or GV-248. respectively. After confirmed by sequencing, the recombinant shuttle vectors containing CARP gene or shRNA and auxiliary packaging plasmid Helper 1 0 and Helper 2 0 were co transfected into the IIEK293T cells for virus packaging and amplification; the viral titer was detected by end point dilution. The IIEK293T cells were infected with the recombinant lentiviruses. and the green fluoresence intensity was observed by fluorescence mircroscope. The H9C2 cells were infected with the recombinant lentivirues and divided into control group. CARP overexpression group and shRNA group; Western blotting method was used to detect the CARP expression levels in the cells in various groups. Results: The DNA sequencing results showed that the sequences of CARP overexpression and shRNA vectors were in accordance with the designed sequences. The expression of green fluorescence protein was seen under fluorescence microscope after transfection of the vectors of the IIEK293T cells. After infection of the H9C2 cells, the expression level of CARP protein in CARP overexpression group was 5. 3 times higher than that in control group ( P=0. 01); while it was down-regulated by 53% in CARP shRNA group compared with control group ( P= 0 02). Conclusion: The lentivirus expression vectors carrying CARP or shRNA targeting CARP are successfully constructed and the lentiviruses obtained could significantly interfere the expression of CARP in the II9C2 cells
Influence of variants in folate metabolism genes on 6-mercaptopurine induced toxicity during treatment for childhood acute lymphocytic leukemia
No full textPurpose: To analyze influence of variants in TYMS, MTHFR, SLC19A1 and DHFR genes on 6-mercaptopurine (MP) induced toxicity during maintenance phase of treatment for childhood acute lymphocytic leukemia (ALL). Methods: One-hundred twenty-seven children with ALL that received maintenance therapy were involved in this study. All patients were treated according to Berlin-Frankfurt-Muenster (BFM) based protocols. Myelotoxicity and hepatotoxicity were evaluated using surrogate markers (median 6-MP dose, number of leukopenic episodes and levels of bilirubin and transaminases on each visit). Results: Higher number of leukopenic episodes, as a surrogate marker of 6-MP myelotoxicity, was found in carriers of TYMS 3R3R and 3R4R genotypes (p=0.067) as well as in TYMS 3R6bp+ (28bp VNTR, 6bp indel) haplotype carriers (p=0.015). Carriers of DHFR CATAG (-680, -675, -556, -464, -317) haplotype were also found to have higher number of leukopenic episodes (p=0.070). SLC19A1 c.80A allele (p=0.079) and TYMS 2R6bp+ (5'UTR VNTR, 6bp indel) haplotype carriers (p=0.078) had fewer leukopenic episodes. No difference in genotype frequencies between the control group of volunteered blood donors and childhood ALL patients was found. Conclusions: Variants in TYMS, SLC19A1 and DHFR genes are potential biomarkers of myelotoxicity and could be used for 6-MP therapy individualization in maintenance phase of childhood ALL treatment, alongside with well-established TPMT variants
Silver(I) complexes with 4,7-phenanthroline efficient in rescuing the zebrafish embryos of lethal Candida albicans infection
No full textFive novel silver(I) complexes with 4,7-phenanthroline (4,7-phen), [Ag(NO3-O)(4,7-phen-mu-N4,N7)](n) (1), [Ag(ClO4-O)(4,7-phen-mu-N4,N7)](n) (2), [Ag(CF3COO-O)(4,7-phen-mu-N4,N7)](n) (3), [Ag-2(H2O)(0.58)(4,7-phen)(3)](SbF6)(2) (4) and {[Ag-2(H2O)(4,7-phen-mu-N4,N7)(2)](n)(BF4)(2)}(n) (5) were synthesized, structurally elucidated and biologically evaluated. These complexes showed selectivity towards Candida spp. in comparison to the tested bacteria and effectively inhibited the growth of four different Candida species, particularly of C. albicans strains, with minimal inhibitory concentrations (MICs) in the range of 2.0-10.0 mu M. In order to evaluate the therapeutic potential of 1-5, in vivo toxicity studies were conducted in the zebrafish model. Based on the favorable therapeutic profiles, complexes 1, 3 and 5 were selected for the evaluation of their antifungal efficacy in vivo using the zebrafish model of lethal disseminated candidiasis. Complexes 1 and 3 efficiently controlled and prevented fungal filamentation even at sub-MIC doses, while drastically increased the survival of the infected embryos. Moreover, at the MIC doses, both complexes totally prevented C. albicans filamentation and rescued almost all infected fish of the fatal infection outcome. On the other side, complex 5, which demonstrated the highest antifungal activity in vitro, affected the neutrophils occurrence of the infected host, failed to inhibit the C. albicans cells filamentation and showed a poor potential to cure candidal infection, highlighting the importance of the in vivo activity evaluation early in the therapeutic design and development process. The mechanism of action of the investigated silver(I) complexes was related to the induction of reactive oxygen species (ROS) response in C. albicans, with DNA being one of the possible target biomolecules
GENETIC PREDICTION OF BIOGEOGRAPHIC ANCESTRY WITHIN EUROPE
No full textOver the past decade, there has been growing interest of forensic DNA community in genetic
prediction of human biogeographic ancestry. Irrespective of forensic applications, predicting
of human ancestry based on DNA analysis might also contribute to better understanding of
population history and individual’s genealogy. Nevertheless, efficient inferences of ancestry
rely upon proper ancestry informative markers (AIMs), capable of differentiating between
populations inhabiting the region of interest. This presentation will focus on searching for
possible AIMs specific for Europe, with a special emphasis put on the markers relevant to
central and eastern parts of the continent. Advantages and disadvantages of prediction
based on haploid and autosomal markers will be discussed. A panel of 224 autosomal AIMs,
recently developed by the NEXT Consortium and its collaborators will be presented, in terms
of their performance in prediction of ancestry within Central and Eastern Europe.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY
Vrnjačka Banja; October 13 to 17, 2019
WHOLE MITOCHONDRIAL GENOME DIVERSITY IN SERBIAN POPULATION: PHYLOGENETIC AND FORENSIC ASPECTS
No full textMitochondrial DNA (mtDNA) is used in forensics for over three decades and is particularly
suitable when STR profiling cannot be performed due to the degraded and/or scarce nuclear
DNA. Traditionally, mtDNA typing is based on ~600 bp of the hypervariable segments I and
II (HVS-I and HVS-II) of the control region (CR, ~1100 bp). Nowadays, it is possible to use
variability of complete mtDNAs which enables maximum resolution of distinct maternal
lineages. However, the number of complete mitogenomes in reference databases such as
EMPOP is still insufficient, and that hampers their wider usage in forensic casework. In order
to fill in the gap in the reference database, which, considering Slavic-speaking populations,
currently comprises only mitogenomes of East and West Slavs, we present population data
for 226 Serbian mitogenomes, representatives of South Slavs from the Balkan Peninsula. We
support previous findings on both high levels of genetic diversity in the Serbian population
and patterns of genetic differentiation among Serbian and ten studied European populations.
However, increased genetic differentiation was observed among Serbian and two European
populations (Russians and Poles) with our high resolution data. We demonstrate that
the inclusion of indel polymorphisms into analysis contributed towards nearly complete
resolution of mtDNA haplotypes (97.1% vs. 86.3% without indels), and that the random
match probability was as low as 0.53%. Bayesian skyline analysis of Serbian mitogenomes
revealed population expansion after the Last Glacial Maximum and during the Migration
period (IV-IX century A.D.). Phylogenetic analysis of the Serbian and relevant West Eurasian
haplotypes contributed towards the improvement of the worldwide mtDNA phylogeny to
the certain extent, which is essential for the interpretation of the mtDNA casework. Lineages
of a putative Balkan origin as well as those shared among Serbian and other European
populations were observed.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
PROTECTIVE ROLE OF EXOPOLYSACCHARIDES PRODUCED BY Lactobacillus plantarum BGAN8 AGAINST CADMIUM INDUCED DAMAGE ON Caco-2 CELLS
No full textOne of the most toxic heavy metals is cadmium which was classified as a human
carcinogen. Cadmium is capable of induction of numerous acute and chronic disorders.
Several studies have shown that inflammation and oxidative stress play a key role in
the toxicity and carcinogenicity. Some reports have shown that species belonging to
Lactobacillus sp. present in the human gastrointestinal tract and in fermented foods
have the ability to bind and detoxify heavy metal ions. There are evidences that
exopolysaccharides (EPS) can be used as tools for removing heavy metal ions by
detoxication, which prevents their absorption from the gastrointestinal tract by host
cells.
Lactobacillus plantarum BGAN8 showed the best cadmium binding ability, among
others EPS producing strains from Laboratory for Molecular Microbiology collection
(LMM collection). So, the aim of this study was to determine the role of isolated and
purified EPS from Lactobacillus plantarum BGAN8 (EPS-AN8) in protection of
cadmium induced damage like inflammation and oxidative stress in Caco-2 cells.
The subtoxic dose of CdCl2 (the concentration that has killed less than 10% of Caco-2
cells) was determined by LDH and MTT assay. Caco-2 cells were treated with subtoxic
dose of CdCl2 and EPS-AN8. The changes in expression of target genes were
determined by quantitative PCR, while the changes in permeability of Caco-2 cells
were tracked by lucifer yellow assay.
Treatment of differentiated CdCl2 treated Caco-2 cells monolayer with EPS-AN8
alleviated inflammation and oxidative stress (decreased level of expression of mRNA
IL-8, NQO1 genes) and also decreased level of lucifer yellow flux. In conclusion, EPSAN8
mighAbstract book: Immunology at the confluence of multidisciplinary approaches, Hotel Mona Plaza Belgrade December 6th-8th, 201