imagine (Institute of molecular genetics and genetic engineering)
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Poly (ε-caprolactone) microspheres for prolonged release of selenium nanoparticles
Get PDFPoly (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.This is the peer-reviewed version of the article: Filipović, N., Veselinović, L., Ražić, S., Jeremić, S., Filipič, M., Žegura, B., Tomić, S., Čolić, M., Stevanović, M., 2019. Poly (ε-caprolactone) microspheres for prolonged release of selenium nanoparticles. Materials Science and Engineering C 96, 776–789. [https://doi.org/10.1016/j.msec.2018.11.073]Supporting information: [https://imagine.imgge.bg.ac.rs/handle/123456789/1615]Published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1613
Is miR-133a marker of progressive chronic kidney disease?
Get PDFChronic kidney disease (CKD) is associated with a high incidence of cardiovascular (CV) disease and numerous risk factor have been implicated to play a role. Recent studies have shown that the levels of various microRNAs in the serum of patients with CKD have been altered and there are reports that miR-133a serum levels correlated with left ventricular hypertrophy in hemodialysis patients.The aim of this study was to investigate association between circulating miR-133a levels and development of de novo CV events in patients with CKD stage 3-5HD.METHODS: This study included 51 patients with diagnosed CKD and 7 healthy individuals as controls. According to the estimated glomerular filtration ratio (eGFR) patients were divided into three equal subgroups (n=17): patients with CKD stage 3b, 4, and 5HD. The level of miR-133a was measured in serum by quantitative real-time PCR. Echo parameters were measured by cardiac ultrasound at the beginning and after 12 months of follow-up. The following CV events were reported during 18 months of follow-up: cardiac death, myocardial infarction, cerebrovascular insult, exacerbation of existing and newly discovered angina pectoris and peripheral arterial disease
Frequency of genetic risk factors for thrombophilia and Gilbert syndrome in the population of Republic of Srpska, Bosnia and Hercegovina
No full textVariants in several genes (factor V-FV, prothrombin-PT and methylenetetrahydrofolate
reductase-MTHFR), such as FVL c.1691 G>A (Leiden), PT c.20210 G>A and MTHFR c.677 C>T,
have significant role in thrombophilia. UGT1A1 enzyme has a vital role in the metabolism of
bilirubin and drugs. UGT1A1*28 promotor variant, with 7 TA repeats in TATA promoter box,
is responsible for decreased gene expression and enzyme activity. UGT1A1*28 is a diagnostic
marker for Gilbert syndrome and an important pharmacogenetic marker for numerous
drugs, especially those used in the treatment of oncological patients. The aim of this study
was to determine frequency of four genetic markers of high clinical significance in healthy
population of Republic of Srpska, Bosnia and Hercegovina. In this study 100 healthy, unrelated
individuals (86 women and 14 men) from the population from all regions of Republic of
Srpska were included. We analyzed DNA samples using PCR-based methodology. Frequency
of homozygous genotype of FVL wild type allele was 95% and 5% of heterozygotes for
Leiden mutation were found. Frequency of homozygous genotype of prothrombin wild type
allele was 98% and 2% heterozygotes for prothrombin PT c.20210 G>A variant allele were
found. Frequency of homozygous genotype of MTHFR wild type allele was 50%, while 43%
heterozygotes and 7% homozygotes for MTHFR c.677 T allele were found. Allele frequencies
in healthy population were 2,5% for factor V Leiden mutation, 1% for prothrombin PT c.20210
G>A variant and 28,5% for MTHFR c.677 C>T variant. Genotypes UGT1A1 6/6 TA, 6/7 TA and
7/7 TA occur with a frequency of 37,19%, 37,19% and 25,6% respectively, and the frequency
of UGT1A1*28 allele in the general population is 44%. Our data of frequency of genetic risk
factors for thrombophilia and Gilbert syndrome are the first-of-its-kind for the population of
Republic of Srpska and they can be used as reference values in future research association
and pharmacogenetic studiesAbstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
“Mining” for the rare SERPINA1 variants in population of Serbia – analysis of the published data
No full textBackground: SERPINA1 is encoding for alpha-1-antitrypsin. Although some of rare SERPINA1
variants can be associated with alpha-1-antitrypsin deficiency (AATD), little is known about
their epidemiology and clinical phenotype, due to their low occurrence. Our aim was to
assess the availability of published data about rare SERPINA1 variants in population of Serbia.
Methodology: We used “alpha-1-antitrypsin” and “Serbia” as key-words to search PubMed.
Results: The search retrieved 24 studies and rare variants were reported in 10. Study about
AAT polymorphism in healthy adult population identified 17 heterozygous carriers (9 of P and
8 F allele). Among healthy newborns the frequencies were 0.031 for P and 0.0073 for other
variants. Three studies addressed the identification and functional characterization of two
variants, G320R and V321F, detected in patients with emphysema and lung cancer. In the
study enrolling 50 patients with premature development of chronic obstructive pulmonary
disease, 2 cases were detected (Mmalton and Q0amersfoort). Two studies on patients with
lung cancer reported frequencies for P (0.0076) i.e. other rare variants (0.00027). When
patients with monoclonal gammopathies were studied 5 carriers of rare variants were found.
Finally, in a group of children with liver disease, the frequency of P variant was 0.0112, while
for other rare variants it exceeded 0.0056.
Conclusion: Currently available literature provides certain data about the presence of rare
SERPINA1 variants in different age and health-status groups in the population of Serbia.
Nevertheless, further studies are warranted to assure a more comprehensive evaluation of
their epidemiology and clinical features.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Long noncoding RNA GAS5 in induction remission phase of pediatric acute lymphoblastic leukemia treatment
No full textIntroduction and Aim: Growth arrest specific 5 (G AS5) is a lo ng noncoding RNA that has
a role in cell growth arrest and apoptosis. GAS5 also interacts with the glucocorticoid
receptor, which makes it a possible pharmacotranscriptomic modulator of glucocorticoid
(GC) treatment response. We intended to find correlations between GAS5 expression and
GC therapy response in pediatric acute lymphoblastic leukemia (ALL) and elucidate the
molecular mechanism of GC effects on the GAS5 signaling pathway.
Material and Method: Peripheral blood mononuclear cells were collected from 29 patients
on the day of diagnosis (0), day 15 and day 33 of ALL therapy. GAS5 expression was measured
using RT-qPCR. HeLa cells were transfected with GAS5 expression vector and NF-κB DNAbinding
activity was analyzed using EMSA.
Results: For each ALL patient, GAS5 expression was higher on day 15 in comparison to its level
at diagnosis (p<0.0005), but it was still significantly higher than at diagnosis for the majority
of patients (p=0.001). On day 33, the level of GAS5 expression decreased in comparison
with day 15. In patients with a higher number of blasts on day 8, reflecting poor therapy
response, higher GAS5 expression on day 0 (p=0.016) and a lower ratio of day 15/diagnosis
expression levels (p=0.009) were detected. Thus, GAS5 expression profile influences GC
therapy response in pediatric ALL.
Discussion: The results presented in this study suggest that GAS5 could be aAbstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Mobile integrons: sensing the cell physiology
No full textMobile class 1 integrons have been recognized as genetic elements involved in
rapid dissemination of genes encoding for antibiotic resistance. Despite the fact
that the dynamics of gene cassette recombination depends on expression of the
class 1 integrase (intI1) gene; the regulation of intI1gene expression has not been
sufficiently explained so far. Previously, a binding motif of LexA protein was identified
within the promoter of the intI1 gene. When the SOS response is activated, LexA
undergoes autoproteolytic inactivation and the intI1 gene expression is activated.
This control allows a subtle coupling of integron activity with bacterial physiology.
However, initially simplistic understanding of the intI1 gene expression regulation was
revised when significant differences in regulation among different bacterial species
were revealed: (i) control through the cAMP receptor protein in Vibrio cholerae; (ii)
nucleoid proteins, Fis, IHF and H-NS affect expression in E. coli; (iii) stringent response
regulates the intI1 expression in E. coli biofilms independently of the SOS response.
Our study was performed on Pseudomonas spp. model. We have shown the role of
RpoS and PsrA proteins in positive regulation of integrase transcription during the
stationary phase of growth. Additionally, we determined that the activity of the lexA
gene promoter was decreased in rpoS and psrA mutants. Thus we proposed that
PsrA indirectly regulates the intI1 gene promoter activity through regulation of the
lexA gene expression in co-operation with additional regulators. Also, Tn5 transposon
mutagenesis and subsequent site-directed mutagenesis enabled identification of a
novel two-component system (TCS) regulating intI1 transcription in Pseudomonas
spp. The role of this TCS in complex network of the intI1 expression regulation, as
well as in regulation of other cell processes, will be determined by wild-type and TCS
mutant transcriptome sequencing.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Bacteriophages and phage-encoded depolymerases
No full textBacteriophages are the most numerous biological entities on the planet, occupying every
habitat on Earth. Although they induce high mortality on bacterial realm, phages are seen
as the driving forces of bacterial evolution and diversification. As natural killers of bacteria,
their potential in treating bacterial infections was acknowledged immediately after their
discovery, a century ago. However, with the lack of understanding of phage biology and the
emergence of antibiotics, the “phage therapy” was abandoned in the West. The perception
changed with modern day rise of antibiotic resistance. Advantages of phage therapy
include its potency against bacterial biofilms, structures underlining much of the reported
resistance to antibiotics. Biofilm matrix consists of extracellular polymeric substances (EPS)
which provide many advantages to bacterial cells especially in terms of resistance to various
agents. But as with every other bacterial adaptation, phages evolved ways to subvert it and
developed enzymes that degrade EPS polysaccharides, allowing them access to surface
receptors. These enzymes, depolymerases, are phage-encoded and usually located on tail
fibers of phage particles, but can also be released from bursting bacterial cells as soluble
enzymes.
We isolated several phages active against multidrug-resistant Acinetobacter baumannii,
Klebsiella pneumoniae and Pseudomonas aeruginosa, three members of the WHO’s list
of bacteria for which new antibiotics are urgently needed. The phages were isolated from
Belgrade wastewaters, purified, screened for host range against laboratory collection and
characterized based on plaque and virion morphology. A. baumannii phages NOVI and ISTD
produced halos around plaques on majority of sensitive strains, indicating the presence
of potent depolymerase(s). Their adsorption rate, one step growth curve and SDS–PAGE
profiles were determined as well as their antibiofilmAbstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Bacillus Cereus NM1 isolated from soil produces two-component lantibiotic thusin, active against multiresistant pathogens
No full textIncreasing number of multidrug resistant pathogens in the world generates a need for
discovering new antimicrobial compounds. Bacteriocins, bacterial antimicrobial peptides,
are considered as potential alternatives to antibiotics. Understanding of how these peptides
work (mechanism of action) or better characterization of bacteriocins is one of the main
goals in the field.
The aim of this study was to (i) isolate cultivable bacteria from soil, (ii) screen for production
of antimicrobial peptides (iii) identify bacterial strains with antimicrobial activity against
various pathogens and (iv) characterize antimicrobial compound/s.
For isolation of bacteria, serial tenfold dilutions were spread onto LB plates. After incubation
at 37°C for 48h, fifty colonies per plate were randomly selected, inoculated into LB broth
and incubated overnight at 37°C. Among the randomly selected isolates from soil, only
one - NM1 - showed a strong inhibitory activity against Staphylococcus aureus, Listeria
monocytogenes and clinical Streptococcus agalactiae strains. It was identified by 16S rRNA
gene sequencing as Bacillus cereus. Bacteriocin was extracted from the supernatant and
purified to homogeneity by C18 solid phase extraction and reversed phase HPLC. Based on
the amino acid sequence and molecular mass of the active fraction, the bacteriocin from
NM1 was identified as thusin, a two-component lantibiotic isolated previously from Bacillus
thurigiensis BGSC4BT1. Spontaneously resistant mutants of S. agalactiae were isolated on
thusin. Whole genome sequencing of Wt and three resistant mutants was performed
in order to identify mutations causing resistant phenotype.
In sum, we characterized bacteriocin thusin from B. cereus NM1, and showed its
broad spectrum of antimicrobial activity. Also, we isolated spontaneous mutants of
S. agalactiae resistant to thusin, however the localization (mapping) of the mutations
is still in progress.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
Autochthonous and regional vine varieties – genetic potential and clonal selection
No full textAs a country which strives to join the European Union, Serbia is looking forward to the
possibility of using the EU CMO measures for development of the viticulture and wine
production sector, primarily the measure for restructuring and conversion of vineyards with
replacement of vineyards with autochthonous and regional vine varieties. On the other
hand, the increasing popularity of wines from international vine varieties and grubbing-up
of vineyards in the previous period caused permanent loss of numerous old vineyards with
autochthonous and regional vine varieties and disappearance of some rare vine varieties. At
this moment there are only about 31 autochthonous and regional wine varieties/group of
varieties from the total of 200 commercially cultivated vine varieties in Serbia. Because of
these reasons, it is necessary to examine and preserve genetic resources of autochthonous
and regional vine varieties and to select the best clones through a detailed research and
certification scheme.
The Center for Viticulture and Oenology, through the network of its seven centers in the
territory of Serbia, carries out evaluations of vineyards with valuable and endangered genetic
material and selection of vineyards according to priorities, and, starting two years ago,
genetic and phytosanitary clonal selection of some important autochthonous and regional
vine varieties.
This paper presents the results of research conducted in about 2000 vineyards within the
Serbian wine-growing areas with the purpose of determining the potential for selection of
vine biotypes, as well as research on selection of potential clones of some autochthonous
and regional vine varieties.Abstracts of the 6th CONGRESS OF THE SERBIAN GENETIC SOCIETY Vrnjačka Banja; October 13 to 17, 2019
