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Trends in molecular characteristics and antimicrobial resistance of group B streptococci: a multicenter study in Serbia, 2015-2020
Get PDFGroup B Streptococcus (GBS) is a major cause of neonatal morbidity and mortality. Serbia has not fully implemented preventive measures against GBS neonatal diseases. Therefore, we aimed to assess the maternal GBS colonisation and invasive neonatal disease rate, to reveal the trends of antimicrobial resistance and serotype distribution of GBS from various patient groups. Randomly selected non-invasive (n=991) and all invasive GBS (n=80) collected throughout Serbia from 2015 to 2020 were tested for antimicrobial susceptibility, capsular typing, and hvgA detection. Overall, 877/5621 (15.6%) pregnant women were colonised with GBS. Invasive GBS infections incidence in infants (0.18/1000 live births) showed a decreasing trend (0.3 to 0.1/1000 live births). Type III was overrepresented in infants with invasive infections (n=35, 58.3%), whereas type V predominated among colonised adults (n=224, 25.5%) and those with noninvasive (n=37, 32.5%) and invasive infections (n=8, 40%). The hypervirulent clone III/ST17 was highly associated with invasive infections (n=28, 35%), particularly late-onset disease (n=9, 47.4%), showing an increase from 12.3 to 14.8%. The GBS resistance to erythromycin and clindamycin was 26.7% and 22.1%, respectively, with an upward trend. The emergence of the hypervirulent clone III/ST17 and the escalation in GBS resistance highlight an urgent need for continuous monitoring of GBS infections
Synthesis and biological profiling of novel isocoumarin derivatives and related compounds
Get PDFIn the continuation of our study of substituted isocoumarins a series of novel 3-azolyl isocoumarin and their thio derivatives, including some related lactone compounds was prepared and biologically profiled against C. albicans showing moderate activity with MIC values in range of 4-60 mu g mL(-1), in general. The additional characterisation of selected compounds was carried out by exploring their activity on CYP3A4 and CYP2D6 enzymes, while experiments on mutagenicity were performed by AMES test. The representative isocoumarins 3b, 4a and 4b showed lower inhibitory activity on CYP enzymes, when compared to the reference inhibitors, ketoconazole and quinidine. Compound 4a showed a higher mutagenic potential than the other two compounds. Further characterization included cytotoxicity profiling against normal MRC5 cells
Retinoic acid affects basic cellular processes and SOX2 and SOX18 expression in breast carcinoma cells
Get PDFGenetic and molecular heterogeneity, together with intrinsic and acquired resistance to therapy, represent the major obstacles to the successful treatment of different types of breast carcinoma. Increasing evidence demonstrates that SOX transcription factors in breast carcinomas could act both as oncogenes and tumor suppressors and have been associated with tumor stage and grade, poor prognosis, and therapy resistance. Both SOX2 and SOX18 overexpression has been correlated with poor prognosis in breast carcinomas, and these genes are recognized as potential antitumor targets. Our aim was to evaluate the effect of retinoic acid (RA), a well-known cyto-differentiating agent, on breast carcinoma cells in vitro and to investigate the potential of RA treatment to modify the expression of SOX2 and SOX18 genes. By applying various experimental approaches, we evaluated the effect of RA on basic cellular processes in SK-BR-3 and MCF7 breast carcinoma cell lines. We have shown that RA inhibits cell growth, reduces the number of Ki-67 positive cells, and causes cell-cycle arrest. RA effect was more prominent in SK-BR-3 cell line that lacks SOX2 expression, including a higher decrease in cell viability, reduction in colony formation, and significant remodeling of cellular structure. We have shown that RA treatment led to the downregulation of SOX2 expression in MCF7 cells and to the reduction of SOX18 expression in both cell lines. By functional analysis, we showed that the anti-proliferative effect of RA in both cell lines was not based on the activity of stemness marker SOX2, pointing to a SOX2-independent mechanism of action. The ability of RA to reduce SOX2/SOX18 expression raises the possibility that these genes can be used as biomarkers to distinguish RA-responders from non-responders. Together, our study shows that the response of breast carcinoma cell lines to RA treatment may vary, highlighting that the development of RA-based therapy should consider differences in breast carcinoma subtypes
De Novo Transcriptome Sequencing of Ramonda serbica: Identification of the Candidate Genes Involved in the Desiccation Tolerance
Get PDFRamonda serbica Panc. is a resurrection plant that can survive a long period of severe dehydration-desiccation.
Desiccation induces cellular membrane integrity loss, protein aggregation, and denaturation, as well as
accelerated generation of reactive oxygen species. However, R. serbica can fully recover its metabolic functions
already one day upon watering [1]. The aim of our study was to obtain more insight into the desiccation
tolerance mechanisms by differential de novo transcriptomics of hydrated (HL) and desiccated leaves (DL).
For R. serbica transcriptome construction, the total high-quality RNA from mixed samples of five biological
replicates of HL and of DL separately, was extracted according to our previously optimised protocol [2].
Highly purified cDNA libraries were sequenced on an Illumina Hi-Seq platform. The ambiguous nucleotides,
adapter sequences, and low-quality sequences were trimmed, and the quality of the reads was checked before
and after the trimming. In total, 39608813 (with Q30=94%) and 37482969 (with Q30=94.1%) clean reads
were obtained in HL and DL, respectively, and used to perform transcriptome assembly by Trinity software.
After removing the redundancy, 189456 transcripts with 189003 unigenes were obtained (32.6% with the
length between 500-1kbp).
Comparative analysis revealed that a large portion of R. serbica sequences (49.1%) exhibited high homology
(according to obtained blast hits, e-value = 1e-5) with sequences found in the genome of another resurrection
plant Boea hygrometrica. Furthermore, among the obtained unigenes (merged data for HL and DL),
64.6% and 42.3% were annotated by NCBI non-redundant protein and nucleotide sequences database (db),
23% by PFAM db, 22.5% by Clusters of Orthologous Groups of proteins db, 48.02% by Swiss-Prot db, 23 %
KEGG db and 13.73 by Gene Ontology db. According to Blast2go analysis, the majority of annotated genes
of R. serbica were associated with translation, ribosomal structure, posttranslational modifications, protein
turnover, signalling pathways and cytoskeleton and encoded chaperonins and late embryogenesis abundant
(LEA) proteins.
Aiming to provide a list of candidates involved in the desiccation tolerance in R. serbica we analysed differentially
expressed genes in HL and DL. Genes associated with transmembrane transport, reproduction,
cell proliferation, and protein folding were up-regulated in HL compared with DL. On the other hand, genes
encoding proteins involved in cell wall architecture, LEA proteins and antioxidative defence were up-regulated
in DL. Taken together, our results imply a key role of genes responsible for leaf morphological changes
(wrapping and curling), and those encoding antioxidative enzymes (polyphenol oxidases and superoxide
dismutases), as well as LEA proteins, known to be a hallmark of desiccation tolerance in resurrection plants.Book of Abstracts: Belgrade BioInformatics Conference 202
Myeloid derived suppressor cells-therapy attenuates experimental autoimmune encephalomyelitis and modulates gut microbiota composition
Get PDFThe role of gut microbiota composition in efficacy of various immune-based therapies is increasingly recognized.
Thus, the aim of our study was to investigate if the efficacy of myeloid-derived suppressor cells
(MDSC)-Prostaglandin E2 (PGE2) therapy for multiple sclerosis (MS) correlates with gut microbiota composition
and function. MDSC generated from bone marrow cells in the presence of PGE2 were applied to spinal
cord homogenate/CFA-induced experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA)
rats, an animal model of MS. MDSC-PGE2 therapy resulted in a significant attenuation of EAE symptoms
over 30 days of disease monitoring. These results correlated with lower percentage of proinflammatory interferon-
gamma and interleukin-17 producing cells and higher percentage of anti-inflammatory IL-4 producing
cells in spinal cord and spleen. Gut microbial composition were studied using amplicon(16S rRNA)-based
metagenomic analyses of fecal samples collected prior to the induction of EAE and MDSC-PGE2 therapy application,
and at the peak of the disease. The induction of EAE resulted in a decrease of microbiota diversity,
whereas the MDSC-PGE2 therapy preserved the diversity in EAE-induced animals. The induction of EAE
in control group associated with a higher relative abundance of Peptococcaceae, but the lower levels of Veillonellaceae
and different groups of Prevotellaceae, known to produce immunosuppressive short chain fatty
acid (SCFA), and Lactobacillus reuteri, known for its anti-inflammatory function. In contrast, there were no
changes in levels of these immunoregulatory taxa in EAE-animals treated with MDSC-PGE2 therapy. Also,
SCFA producing Ruminococcaceae, and Coriobacteriaceae, known to metabolize phytoestrogens to immunosuppressive
metabolites were more abundant in EAE-animals treated with MDSC-PGE2 therapy. Predicted
metabolic profiling obtained by PICRUSt2 revealed that pathways involved in biosynthesis of polyamines,
metabolites known to contribute to homeostasis of gastrointestinal mucosa, were enriched in MDSC-PGE2
treated animals. Considering these results, the modification of gut microbiota composition and function
could further increase efficacy of MDSC-PGE-2 based therapy of autoimmune diseases.Book of Abstracts: Belgrade BioInformatics Conference 202
Toxicity investigation of CeO2 nanoparticles coated with glucose and exopolysaccharides levan and pullulan on the bacterium Vibrio fischeri and aquatic organisms Daphnia magna and Danio rerio
No full textCerium oxide nanoparticles (nCeO2) have widespread applications, but they can be hazardous to the environment. Some reports indicate the toxic effect of nCeO2 on tested animals, but literature data are mainly contradictory. Coating of nCeO2 can improve their suspension stability and change their interaction with the environment, which can consequently decrease their toxic effects. Herein, the exopolysaccharides levan and pullulan, due to their high water solubility, biocompatibility, and ability to form film, were used to coat nCeO2. Additionally, the monosaccharide glucose was used, since it is a common material for nanoparticle coating. This is the first study investigating the impact of carbohydrate-coated nCeO2 in comparison to uncoated nCeO2 using different model organisms. The aim of this study was to test the acute toxicity of carbohydrate-coated nCeO2 on the bacterium Vibrio fischeri NRRL B-11177, the crustacean Daphnia magna, and zebrafish Danio rerio. The second aim was to investigate the effects of nCeO2 on respiration in Daphnia magna which was performed for the first time. Finally, it was important to see the relation between Ce bioaccumulation in Daphnia magna and Danio rerio and other investigated parameters. Our results revealed that the coating decreased the toxicity of nCeO2 on Vibrio fischeri. The coating of nCeO2 did not affect the nanoparticles’ accumulation/adsorption or mortality in Daphnia magna or Danio rerio. Monitoring of respiration in Daphnia magna revealed changes in CO2 production after exposure to coated nCeO2, while the crustacean's O2 consumption was not affected by any of the coated nCeO2. In summary, this study revealed that, at 200 mg L−1, uncoated and carbohydrate-coated nCeO2 are not toxic for the tested organisms, however, the CO2 production in Daphnia magna is different when they are treated with coated and uncoated nCeO2. The highest production was in glucose and levan-coated nCeO2 according to their highest suspension stability.This is the peer-reviewed version of the article: Milenković I, Radotić K, Despotović J, Lončarević B, Lješević M, Spasić SZ, Nikolić A, Beškoski VP. Toxicity investigation of CeO2 nanoparticles coated with glucose and exopolysaccharides levan and pullulan on the bacterium Vibrio fischeri and aquatic organisms Daphnia magna and Danio rerio. Aquatic Toxicology. 2021;236:105867; [https://doi.org/10.1016/j.aquatox.2021.105867]The published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1449
crISPr-cas9 technology: from basic research to clinical application
Get PDFTehnologije za manipulaciju molekula DNK su omogućile brojna otkrića i prodore u biomedicinskim naukama.
Ipak, metode za uvođenje ciljanih promena u genomu su do skora bile relativno komplikovane i nepristupačne
najvećem broju naučnika. Primenom CRISPR-Cas9 tehnologije počela je revolucija u
biomedicinskim naukama zato što su metode za genomsko inženjerstvo postale dostupne gotovo svakoj laboratoriji.
Ova tehnologija je prešla veliki put od osnovnih istraživanja u vezi sa prokariotskim genomima pre
nekoliko decenija, preko otkrića mehanizma stečenog imuniteta bakterija, da bi danas postala dominantna
tehnologija za genomsko inženjerstvo. Fokus ovog rada je na praktičnom aspektu primene CRISPR-Cas9
tehnologije i njenim poređenjem u odnosu na alternative za uvođenje dvolančanih prekida u genomu
(TALEN i ZFN nukleazama). Detaljno će se obraditi upotreba CRISPR-Cas9 tehnologije u bazičnim (naučnim)
istraživanjima i u medicini. Razmatraće se i svi nedostaci trenutnih tehnologija za genomsko inženjerstvo,
uključijući uvođenje nespecifičnih promena u genomu i načini prevazilaženja ovih nedostataka. Kroz brojne
primere upotrebe CRISPR-Cas9 tehnologije će biti približeno zašto je ova metoda značajna ne samo za biomedicinske
nauke, već za celokupno društvo.Technologies for DNA manipulation have enabled numerous breakthroughs in the field of biomedical sciences.
Until recently, methods for genome editing have been too complicated and practically unavailable
for the vast majority of research laboratories. CRISPR-Cas9 technology has started a revolution in the biomedical
sciences since it enabled the use of genome engineering methods in almost every research laboratory.
This technology has gone a long way from its discovery in bacterial genomes, through being identified
as a part of the bacterial immune system, to the application it is most known today – genome engineering.
This paper will focus on the practical aspects of using CRISPR-Cas9 and its comparison to similar methods
for genome engineering (using TALEN and ZFN nucleases). This paper will cover the benefits and drawbacks
of CRISPR-Cas9 genome editing including off-target cleavage, and the possibilities to overcome
these drawbacks. The use of CRISPR-Cas9 technology in basic research and clinical studies will be covered
in detail. Current research related to CRISPR-Cas9 technology will be covered to emphasize the importance
of this method not only for life sciences, but for society as a whole.Zahvaljujem se Prof. Dr Dušanki Savić-Pavićević, Dr Meliti Vidaković i Dr Snežani Kojić na izuzetno korisnim sugestijama
tokom pisanja rada. Autor je član „Returning expert“ programa (Centrum für internationale Migration und Entwicklung,
SR Nemačka)
Biogenic silencers of Pseudomonas aeruginosa virulence
Get PDFPseudomonas aeruginosa jedan je od najznačajnijih uzročnika unutarbolničkih infekcija čiji je terapijski tretman
konvencionalnim antibioticima sve češće neefikasan usled rezistencije na antibiotike. Inovativni vidovi
kontrole infekcija, poput utišavanja međućelijske komunikacije bakterija, a time i onemogućavanja virulencije
i inhibicije patogenog fenotipa su stoga od izuzetnog značaja. U ovom radu biće predstavljena istraživanja
koja su bazirana na prirodnom svojstvu bakterija koje dele ekološke niše da sarađuju, ali i kompetiraju,
na osnovu čega su analizirane Delftia tsuruhatensis i Burkholderia cepacia koje tokom infekcija kolokalizuju
sa P. aeruginosa. Pokazano je da D. tsuruhatensis 11304 produkuje C18-HSL koji inhibira virulenciju P. aeruginosa
i rekonstituiše osetljivost na antibiotike, a takođe je po prvi put u literaturi opisano prisustvo dihidroksi-
C18-HSL u biološkim uzorcima. Opisane su i laktonaze vrste B. cepacia BCC4135 koje degraduju
autoinducere komunikacije P. aeruginosa i inhibiraju ekspresiju faktora virulencije. Utvrđena je njihova supstratna
specifičnost i ukazano na različitu biološku funkciju u zavisnosti od lokalizacije.Pseudomonas aeruginosa is a leading cause of nosocomial infections, whose therapeutic treatment with
conventional antibiotics is increasingly ineffective due to antibiotic resistance. Inovative approaches of infection
control, such as silencing the bacterial quorum sensing system and thus virulence and pathogenic
phenotype inhibition are of great importance. In this study, there will be presented research based on natural
feature of bacteria that share the same ecological niche to coordinate, but also to compete, based on
which Delftia tsuruhatensis and Burkholderia cepacia that colocalize with P. aeruginosa during infections were
analysed. D. tsuruhatensis 11304 has been shown to produce C18-HSL which inhibits P. aeruginosa virulence
and reconstitutes antibiotic susceptibility, and the presence of dihydroxy-C18-HSL in biological samples has
also been described for the first time in the literature. B. cepacia BCC4135 lactonases that degrade autoinducers
of P. aeruginosa quorum sensing system and inhibit virulence factor expression have also been reported.
Their substrate specificity was determined and different biological function depending on their
localization was indicated.Jedan deo ovog rada realizovan je na Dipartimento di Scienze Chimiche, Università di Napoli Federico
II, Napulj, Italija pod rukovodstvom prof dr Antonio Molinaro i dr Flaviana Di Lorenzo, kojima se ovom prilikom
zahvaljujem. Hvala dr sci med Zorici Vasiljević sa Instituta za zdravstvenu zaštitu majke i deteta Srbije ‘’Dr Vukan Čupić‘’
koja je obezbedila kliničke izolate korišćene u ovom radu, kao i dr Milanu Kojiću i drugim saradnicima Laboratorije za
molekularnu mikrobiologiju Instituta za molekularnu genetiku i genetičko inženjerstvo za izuzetnu pomoć tokom
izrade eksperimenata
Methoxy‐Substituted Hydroxychalcone Reduces Biofilm Production, Adhesion and Surface Motility of Acinetobacter baumannii by Inhibiting ompA Gene Expression
No full textAn increasing lack of available therapeutic options against Acinetobacter baumannii urged researchers to seek alternative ways to fight this extremely resistant nosocomial pathogen. Targeting its virulence appears to be a promising strategy, as it offers considerably reduced selection of resistant mutants. In this study, we tested antibiofilm potential of four synthetic chalcone derivatives against A. baumannii. Compound that showed the greatest activity was selected for further evaluation of its antivirulence properties. Real‐time PCR was used to evaluate mRNA expression of biofilm‐associated virulence factor genes (ompA, bap, abaI) in treated A. baumannii strains. Also, we examined virulence properties related to the expression of these genes, such as fibronectin‐ and collagen‐mediated adhesion, surface motility, and quorum‐sensing activity. The results revealed that the expression of all tested genes is downregulated together with the reduction of adhesion and motility. The conclusion is that 2′‐hydroxy‐2‐methoxychalcone exhibits antivirulence activity against A. baumannii by inhibiting the expression of ompA and bap genes, which is reflected in reduced biofilm formation, adhesion, and surface motility.This is the peer reviewed version of the article: Ušjak, D., Dinić, M., Novović, K., Ivković, B., Filipović, N., Stevanović, M., Milenković, M.T., n.d. Methoxy-Substituted Hydroxychalcone Reduces Biofilm Production, Adhesion and Surface Motility of Acinetobacter baumannii by Inhibiting ompA Gene Expression. Chemistry & Biodiversity n/a. [https://doi.org/10.1002/cbdv.202000786]Poster: [https://imagine.imgge.bg.ac.rs/handle/123456789/2820]Supporting information: [https://imagine.imgge.bg.ac.rs/handle/123456789/2822
In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship
Get PDFDesiccation or extreme water loss leads to protein denaturation, aggregation, and degradation and impairs membrane lipid fluidity, resulting in loss of membrane integrity at the cellular level. The induction of late embryogenesis abundant proteins (LEAPs) is considered an essential component of desiccation tolerance strategy in so-called resurrection plants. This heterogeneous group of hydrophilic, non-globular proteins is characterised by a high structural plasticity that allows them to adopt a random conformation in aqueous solutions that transforms into α-helices during dehydration [1]. Therefore, LEAPs can interact with various ligands and partners, including ion sequestration and stabilisation of membranes and enzymes during freezing or drying [2].Our new transcriptome database of an endemic resurrection species Ramonda serbica allowed us to identify 164 members of the LEA gene family. LEAPs of this sample data have an average primary sequence similarity and identity of 10% and 6%, respectively, but with a high variance (141 and 108), which means that the sample proteins can be classified based on domain homology. The averaging is based on multiple sequence alignment and the variance is estimated using pairwise sequence alignment scores. Accordingly, all identified LEAPs were clustered into six groups based on protein families (PFAM). Among these groups, LEAPs differ significantly in their secondary structure, disorder propensity and aggregation potential. Furthermore, we built homology models using PDB structures as templates. For each group, an ensemble of superimposed 3D homology models was analyzed. The information obtained from the representative structural models is key to understanding the function of LEAPs and the regulation of their intrinsic structural disorder-to-order transition during desiccation. This will pave the way for the identification of LEAPs endogenous partners and their targets in the cell and provide further insights into the protective mechanisms of desiccation tolerance.Related to conference paper: [https://imagine.imgge.bg.ac.rs/handle/123456789/1836