imagine (Institute of molecular genetics and genetic engineering)
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COMPARED EFFECTS OF DOXORUBICIN AND QUERCETIN COMBINED TREATMENT ON 2D AND 3D OSTEOSARCOMA MODEL SYSTEMS
No full textOsteosarcoma (OS) is an aggressive bone tumour affecting paediatric patients with a 5-year
survival rate of 66.5%. Standard treatment consists of surgical resection, chemotherapy, and
radiation for unresectable tumours. To improve and streamline current therapies for OS there is
a need for new, effective treatments. The aim of this study was to establish OS threedimensional
(3D) model able to reproduce key cancer features based on alginate microbeads
with immobilized human osteosarcoma cells (SAOS-2), as well as to examine of effects of
combined doxorubicin and quercetin treatment in two-dimensional (2D) and 3D models.
We successfully immobilized SAOS-2 cell in alginate microbeads (~ 1mm in diameter) and
cultivated them up to 21 days. The results of treatment showed that quercetin enhanced
doxorubicin’s effect on viability in 2D conditions, while this effect missing in 3D model.
However, analysis of the expression of genes associated with poor prognosis showed that
combined treatment in 3D model decreased the expression of pluripotency genes (SOX2,
Nanog), OS marker (SOX9) and resistance-related gene (ABCB1) compared to doxorubicin
treatment in 3D model. These results implying that combined treatment could potentially
improve the effectiveness of doxorubicin in osteosarcoma treatment.
Hence, further research is needed to understand the response in the 3D models upon combined
treatment that could be beneficial for OS treatment.VII Congress of the Serbian Genetic Society Zlatibor; October 2 to 5, 2024
GENERATING KNOCKOUT MUTANT IN THE GALU GENE OF CLINICAL ISOLATE ACINETOBACTER BAUMANNII 10593
No full textOne of the most important virulence factors of Acinetobacter baumannii, an opportunistic
pathogen that is one of the most significant causes of nosocomial infections worldwide, is the
production of a capsule on the cell surface. This structure presumably protects bacteria against
antimicrobial agents, disinfectants, desiccation, and the host immune system. Genes encoding
proteins involved in the biosynthesis and export of capsular polysaccharides are localised in the
capsule locus (KL). The construction of gene deletion mutants is an important technique to
evaluate the functional identification of genes in bacteria. This study describes the generation
of knockout mutant in the galU gene, which is involved in the biosynthesis of capsular simple
sugars. PCR amplicons of the upstream and downstream regions of the target gene were cloned
into a PCR cloning vector pJET and in the next step subcloned into a pEMGT plasmid, which
is suicidal in A. baumannii. The generated constructs were introduced into the selected isolate
of A. baumannii by triple conjugation and candidates were checked on a selective medium and
by PCR method. To induce the second step of recombination, a pSW-Apr plasmid was
introduced by conjugation into the candidates. The construction of the mutants was confirmed
by monitoring growth on a selective medium and finally by PCR method.
The capsular polysaccharides of A. baumannii require further investigation as potential targets
for novel antimicrobial therapeutics. In this work, gene deletion mutants were constructed that
can be used to study the contribution of the capsule to the virulence of A. baumannii.VII Congress of the Serbian Genetic Society Zlatibor; October 2 to 5, 2024
Control of Listeria monocytogenes and Staphylococcus aureus in meat and meat products by cell-free supernatant of Brevibacillus laterosporus BGSP7 and BGSP9
No full textThe presence of pathogens in food has increased awareness of food safety, but it also causes large economic losses. Fresh meat and meat products contain a sufficient quantity of proteins, lipids, water, and a favorable pH that stimulates the growth of various microorganisms, including pathogens.The aim of this study was to investigate the efficacy of Brevibacillus laterosporus BGSP7 (CFS-BGSP7) and BGSP9 (CFS-BGSP9) cell-free supernatants in the control of Listeria monocytogenes and Staphylococcus aureus in raw meat and meat products.Raw meat and meat products were sliced and then aseptically treated by immersion for 2 minutes into solutions containing: i) CFS-BGSP7; ii) CFS-BGSP9; iii) no treatment. The samples were then artificially contaminated with: Group I – L. monocytogenes (~4 log cfu g-1); Group II – S. aureus LMM322 (~4 log cfu g-1). Each sample was individually aseptically vacuum-packed and stored at 4°C for 8 weeks. The number of surviving bacteria in the samples were analyzed immediately after contamination with L. monocytogenes and S. aureus and at regular time-intervals: after 1, 3, 5 and 8 weeks of storage at 4°C.Meat samples treated with CFS-BGSP7 and CFS-SP9 showed a significant decrease in the cell counts of L. monocytogenes and S. aureus. When meat samples treated with CFS-BGSP7 and CFS-BGSP9 are compared, the results show a more intense reduction rate of both L. monocytogenes and S. aureus in all samples treated with CFS-BGSP7
AdeABC, AdeFGH, and AdeIJK efflux pumps as key factors in tigecycline resistance of Acinetobacter baumannii: a study from Western Balkan hospitals
No full textPurpose The present study investigated the role of resistance-nodulation-cell division (RND) efux pumps in tigecycline
resistance of Acinetobacter baumannii clinical isolates recovered from three Western Balkan countries (Serbia, Bosnia and
Herzegovina and Montenegro).
Methods A total of 37 A. baumannii isolates recovered from seven tertiary care hospitals in 2016 and 2022 were tested
against tigecycline using broth microdilution method. Then, efux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone
(CCCP) was used to determine the involvement of efux pumps in tigecycline resistance. Molecular typing was performed
by pulsed-feld gel electrophoresis (PFGE) and multiplex PCR-based determination of clonal lineage. Regulators of efux
pumps were analyzed for amino acid substitutions, while reverse transcription-quantitative PCR (RT-qPCR) enabled quantifcation of RND efux pumps expression.
Results All tested isolates were interpreted as resistant to tigecycline and showed reduced tigecycline minimum inhibitory
concentration (MIC) values in the presence of CCCP. PFGE analysis showed signifcant diversity among isolates grouped
in cluster I including IC2 (n=32) and IC3 (n=1) isolates, while cluster II was comprised of four IC1 isolates. The most
prevalent substitutions in AdeR were V120I and A136V and in AdeS G186V and N268H (n=33). The Q262R substitution
was detected in AdeL proteins of IC1 isolates, whereas no alterations were observed within AdeN. The expression of the
adeB, adeG, and adeJ genes in selected isolates was upregulated in fve (1.16- to 3-fold), sixteen (1.35- to 2.82-fold), and
twelve isolates (1.62- to 4-fold) compared to ATCC19606, respectively.
Conclusion This study revealed that overexpression of RND efux pumps underlies tigecycline resistance in A. baumannii
clinical isolates from the Western Balkan
Citrus flavonoids diosmin, myricetin and neohesperidin as inhibitors of Pseudomonas aeruginosa: Evidence from antibiofilm, gene expression and in vivo analysis
No full textCitrus flavonoids are group of bioactive polyphenols. Here, we investigated the potential of diosmin, myricetin and neohesperidin as possible inhibitors of Pseudomonas aeruginosa. This bacterium is a major clinical challenge due to its propensity to form resistant biofilm. The aims of this study were to examine flavonoids antibacterial activity using the microdilution method, assays intended to determine several antibiofilm mechanisms (crystal violet, congo red binding, extracellular DNA (eDNA) test and confocal laser scanning microscopy (CLSM) live/dead cell imaging), followed by virulence genes RT-qPCR analysis. Furthermore, we aimed to examine in vivo toxicity of the compounds as well as their efficacy in P. aeruginosa zebrafish embryo infection model. Minimal inhibitory concentrations of tested flavonoids towards P. aeruginosa were in range 0.05 – 0.4 mg/mL. A high potential of the compounds to disturb both the formation of the bacterial biofilm and its eradication was recorded, including significant reduction in biofilm biomass, exopolysaccharide and eDNA production. Biofilm treatment with diosmin resulted in the lowest percentage of live microbial cells as observed in the CLSM live/dead cell imaging. The lasI, pvdS, and rhlC genes were found to be downregulated in the presence of diosmin and myricetin. Only diosmin stood out as non-embryotoxic. Consequently, in vivo analysis using a zebrafish model of P. aeruginosa infection showed an antivirulence effect of diosmin. Our findings suggest that diosmin could be potential candidate for the development of new agent that target P. aeruginosa infections by reducing its virulence mechanisms
MikroRNK u lečenju glioblastoma
No full textGlioblastoma (GBM) is the most common and aggressive primary malignant brain tumor and is
characterized by high mortality and morbidity rates and unpredictable clinical behavior. The
disappointing prognosis for patients with GBM even after traditional treatments such as surgery
and postoperative radiation and chemotherapy has prompted the researchers to look for specific
targets to provide new insights into the development of modern therapies.
miRNAs are small non-coding RNA molecules with regulatory functions in all biological and
pathological processes. They play important roles in regulating gene expression and modulate up
to 60% of protein-coding genes in the human genome. Numerous factors, such as the amplification
or deletion of miRNA genes, aberrant transcriptional control of miRNAs, dysregulated epigenetic
modifications, and flaws in the machinery of miRNA biogenesis, contribute to the dysregulation
of miRNA expression in human cancer. Dysregulated miRNAs impact the hallmark of cancer,
such as maintaining proliferative signaling, eluding growth suppressors, preventing cell death,
initiating invasion and metastasis, and triggering angiogenesis.
Various miRNAs play roles in the pathogenesis and development of GBM, and many miRNAs
have been confirmed to be associated with the inhibition of GBM. Many miRNAs are dysregulated
in GBM and act as tumor suppressors or oncogenes by altering proliferation, cell cycle
progression, aggressiveness, migration, tumor cell differentiation and apoptosis of GBM. miRNAs
are also recognized as a GBM microenvironment modulators.
As novel molecules, miRNAs represent an attractive candidate for targeted therapies against GBM
because miRNAs direct the posttranscriptional regulation of entire signaling network of genes
within the cells involved in various pathogenesis events. miRNAs may be used as important
genetic markers of the biological activities of GBM cells, such as proliferation, migration,
invasion, conventional radiotherapy and chemotherapy resistance, as well as biomarkers of GBM
prognosis. The use of miRNAs as therapeutic interventions is based on the idea that the expression
of miRNAs is dysregulated in GBM and that the malignant phenotype can be restored by targeting
miRNAs. The most common strategy is to ablate the function of miRNAs or to restore the
expression of silenced miRNAs. Both of these approaches may facilitate new discoveries for GBM
therapeutics. In addition, miRNAs in combination with conventional chemo- and radiotherapy also
show a promising outcome for GBM treatment.
It is clear that the focus of future research need to be on understanding of the effects that miRNAs
have on the transcriptional network and how they are related to the remarkable capacity of GBM
to grow rapidly while avoiding the immune system. The advancements in understanding of
miRNA biology, together with the expanding area of the field of nanotechnology poised to address
the critical issue of delivery, can moved us closer to the possibility for successful miRNA-based
treatment for GBM. miRNAs can be new goals in diagnosis and therapy, ultimately leading to a
new age of personalized medicine for cancer patients
Isolation of glyphosate-degrading bacteria from soil samples from Serbia
No full textMicrobial biodegradation is a promising approach towards the removal
of contaminants from the environment [1]. Our research focuses on the discovery of
new microorganisms and enzymes degrading glyphosate, a widely used pesticide.
Microorganisms were isolated from forest and agricultural soil samples from Serbia,
using different media including ones with glyphosate as sole source of carbon. Seven
best candidates were identified by 16S rRNA sequencing, revealing the genera
Cupriavidus, Lelliottia and Pseudescherichia, which have not previously been
reported to degrade glyphosate. Biotransformations of glyphosate using whole cells
were monitored with TLC and HPLC analysis to confirm the degradation and
investigate the presence of other degradation products, possibly hinting at a novel
mechanism of degradation.EU-CONEXUS EENVIRO Research Conference, 29-31 October 2024 - Bucharest, Romani
Chemical engineering aspects of a perfusion-based 3D in vitro osteosarcoma model
No full textOsteosarcoma is an aggressive primary bone cancer affecting 4-5 per million people.
Dominantly utilised models in osteosarcoma research, including cell monolayers and animals,
have limited accuracy in evaluating drug efficiency as less than 10% of drug candidates reach
the market. In contrast, 3D in vitro models being physiologically relevant hold great promise
to advance anticancer drug discovery. Our aim was to characterize in more depth previously
established 3D in vitro osteosarcoma model based on scaffolds and a perfusion bioreactor by
integrating a chemical engineering approach with standard biological methods of
characterization. Murine osteosarcoma K7M2-wt cells were seeded onto macroporous
composite scaffolds (2 wt.% alginate, 2 wt.% hydroxyapatite) at the density 15x106 cells/cm3
of scaffold volume. Cells were then cultivated for 7 days in a “3D Perfuse“ bioreactor
(Innovation Center of Faculty of Technology and Metallurgy, Belgrade, Serbia) under a
continuous medium flow of 0.27 ml/min (corresponding to the medium velocity of 40 μm/s),
while static cultures served as a control. Biological assays indicated that perfusion conditions
positively affected cell metabolic activity and expression of other cancer cell hallmarks such
as cellular self-aggregation into spheroid-like forms and extracellular matrix secretion. Cell
aggregates (n ~ 300) were quantitatively characterised in terms of their size, shape and number
demonstrating that aggregates were more numerous, more uniformly distributed throughout
the scaffold volume and were slightly larger under perfusion conditions compared to those in
static cultures (140 μm vs. 116 μm in diameter). Additionally, under perfusion, aggregates had
a more irregular shape which could be attributed to the presence of shear stresses (estimated
as ~2 mPa on average). Following the chemical engineering approach, the mass transport of
oxygen and nutrients was modelled under static and perfusion conditions and then correlated
with spheroid distribution across the scaffold. Overall, the chemical engineering perspective
provided a comprehensive insight into the 3D model features, highlighting the advantages of
perfusion cultures.Twenty-Second Young Researchers Conference – Materials Science and Engineering December 4 – 6, 2024, Belgrade, Serbi
ABVD protokol nije adekvatan za sve pacijente sa uznapredovalim klasičnim Hočkinovim limfomom - petogodišnje iskustvo jednog centra iz svakodnevne kliničke prakse
No full textAdvanced-stage classical Hodgkin lymphoma (AScHL) is a therapeutic challenge due to chemoresistance. This study aims to present real-world data on the application of the ABVD regimen (doxorubicin, bleomycin, vinblastine, dacarbazine) in patients with AScHL. Methods: This retrospective study examines the clinical and laboratory parameters, as well as the treatment and outcome of patients diagnosed with AScHL, in the period between 2016 and 2020. Results: The cohort consisted of 49 patients with AScHL. Median follow-up was 47 months (range: 1 - 79). The most important clinical and laboratory characteristics are summarized in Table 1. All patients were initially treated with ABVD. The overall response rate was 72.3% (complete response = 61.7%; partial response = 10.6%), while 27.7% of patients exhibited refractoriness. Additionally, 10.6% relapsed at a later stage. Of the investigated parameters (Table 1), only an elevated erythrocyte sedimentation rate (ESR) ≥ 50 mm in the first hour) was associated with shorter progression-free survival (PFS), (median PFS = 19 months vs. not reached (NR), in patients with ESR < 50 mm in the first hour; p = 0.039), while the presence of bulky disease was associated with shorter overall survival (OS), (p = 0.044). Also, refractory patients had significantly shorter OS (median OS = 54 months vs. NR in patients who achieved remission; p = 0.004). The median PFS and OS were not achieved; fouryear PFS and OS were 61% and 89%, respectively. Patients treated with autologous transplantation (AT) in relapsed/refractory disease had a longer PFS (p = 0.02), but not a longer OS. Brentuximab vedotin (BV) was successfully used in 4/14 patients, of whom three patients received it as consolidation treatment after AT. Conclusion: A significant number of patients with AScHL cannot be cured with ABVD, thus more intensive treatment or innovative therapies are warranted.Uznapredovali klasični Hočkinov limfom (UkHL) predstavlja terapijski izazov zbog značajnog procenta neuspeha prve terapijske linije. Cilj rada je da se opiše lečenje bolesnika sa UkHL-om u svakodnevnoj kliničkoj praksi ABVD protokolom. Metode: U ovoj retrospektivnoj studiji su ispitivani kliničko-laboratorijski parametri, lečenje i ishod bolesnika kod kojih je UkHL dijagnostikovan u periodu od 2016. do kraja 2020. godine. Rezultati: Kohortu je činilo 49 bolesnika sa UkHL-om čija je medijana praćenja bila 47 meseci (opseg: 1 - 79). Najvažnije kliničko-laboratorijske karakteristike su sumirane u Tabeli 1. Svi bolesnici su inicijalno lečeni ABVD protokolom. Ukupni odgovor je iznosio 72,3% (kompletni odgovor = 61,7%; parcijalni odgovor = 10,6%), dok je ostalih 27,7% pacijenata ispoljilo refraktornost. Još 10,6% bolesnika je relapsiralo naknadno. Od ispitivanih parametara (Tabela 1) samo je povišena sedimentacija eritrocita (SE ≥ 50 mm u prvom satu) uticala na kraće preživljavanje bez progresije (PBP), (medijana PBP = 19 meseci naspram nedostignuta kod bolesnika sa SE < 50mm u prvom satu; p = 0,039), dok je prisustvo velike tumorske mase (engl. bulky tumor mass/bulky disease) bilo povezano sa kraćim ukupnim preživljavanjem (UP), (p = 0,044). Takođe, primarno refraktorni bolesnici su imali značajno kraće UP (medijana UP = 54 meseca naspram nedostignuta kod bolesnika sa postignutom remisijom; p = 0,004). Medijana PBP-a i UP-a nije dostignuta; četvorogodišnje PBP i UP iznosili su 61%, odnosno 89%. Pacijenti koji su lečeni autolognom transplantacijom (AT) u relapsiranoj/refraktornoj (R/R) bolesti imali su duže PBP (p = 0,02), ali ne i UP. Brentuksimab vedotin (BV) je uspešno primenjen kod četiri od 14 bolesnika, kod troje u konsolidaciji nakon autologne transplantacije. Zaključak: Značajan broj bolesnika sa UkHL-om ne može da bude izlečen ABVD protokolom, već je neophodno za njih obezbediti intenzivnije lečenje ili inovativne terapije
ProteoCure: A European network to fine-tune the proteome
No full textProteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion. Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential. The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tuning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s)