imagine (Institute of molecular genetics and genetic engineering)
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Silver(I) and gold(III) complexes with miconazole: The influence of the metal ion on the antimicrobial activity of the coordinated azole
No full textTo develop a new antimicrobial agent, we used the clinically approved antifungal azole, miconazole (mcz), as a ligand for the synthesis of silver(I) and gold(III) complexes. The new complexes [Ag(NO3-O)(mcz-N)2] (1) and [AuCl3(mcz-N)] (2) were synthesized and characterized by 1H NMR, IR and UV–Vis spectroscopy and mass spectrometry, while the crystal structure of 1 was determined by single-crystal X-ray diffraction analysis. From the results obtained, it can be concluded that in both complexes, mcz is monodentately coordinated to the silver(I) and gold(III) ions through the imidazole nitrogen atom N3. In the solid state, complex 1 contains two mcz ligands and monodentately coordinated nitrate in the third position, while in the case of 2 gold(III) ion is coordinated by one mcz and three chlorido ligands, resulting in the expected square-planar arrangement around the metal center. DFT and TDDFT calculations were employed to elucidate the electronic structures and thermodynamic stability of the synthesized complexes in solution to complement the experimental findings. The coordination of mcz to silver(I) and gold(III) ions leads to an enhancement of its activity against Gram-negative Escherichia coli and Pseudomonas aeruginosa strains, while against the panel of Staphylococcus aureus and Candida species, only 2 shows improved activity compared to mcz. Both complexes 1 and 2 were tested in vitro for their antimycobacterial activity against the strain Mycobacterium tuberculosis H37Rv and showed good growth inhibition with minimum inhibitory concentration (MIC) values of 3.12 and 8.69 μM, respectively, with complex 1 being twice effective as mcz (MIC = 7.50 μM). Complex 2 significantly reduced the production of pyocyanin, a virulence factor in P. aeruginosa controlled by quorum sensing, while this effect was not observed for 1
Origin and Genealogy of Rare mtDNA Haplotypes Detected in the Serbian Population
No full textBackground: The Balkan Peninsula has served as an important migration corridor between Asia Minor and Europe throughout humankind’s history and a refugium during the Last Glacial Maximum. Past migrations such as the Neolithic expansion, Bronze Age migrations, and the settlement of Slavic tribes in the Early Middle Ages, are well known for their impact on shaping the genetic pool of contemporary Balkan populations. They have contributed to the high genetic diversity of the region, especially in mitochondrial DNA (mtDNA) lineages. Serbia, located in the heart of the Balkans, reflects this complex history in a broad spectrum of mtDNA subhaplogroups. Methods: To explore genetic diversity in Serbia and the wider Balkan region, we analyzed rare mtDNA subclades—R0a, N1a, N1b, I5, W, and X2—using publicly available data. Our dataset included already published sequences from 3499 HVS-I/HVS-II and 1426 complete mitogenomes belonging to West Eurasian and African populations, containing both contemporary and archaeological samples. We assessed the parameters of genetic diversity found in different subclades across the studied regions and constructed detailed phylogeographic trees and haplotype networks to determine phylogenetic relationships. Results: Our analyses revealed the observable geographic structure and identified novel mtDNA subclades, some of which may have originated in the Balkan Peninsula (e.g., R0a1a5, I5a1, W1c2, W3b2, and X2n). Conclusions: The geographic distribution of rare subclades often reveals patterns of past population movements, routes, and gene flows. By tracing the origin and diversity of these subclades, our study provided new insights into the impact of historical migrations on the maternal gene pool of Serbia and the wider Balkan region, contributing to our understanding of the complex genetic history of this important European crossroads
Hydrazone-bridged 5-nitrofuran and piperidine/piperazine derivatives: Synthesis, DFT studies, and evaluation of anticancer and antimicrobial activity
No full textIn the present study, we designed eleven novel compounds (PNH1-PNH11) by combining three pharmacophores, namely piperidine/piperazine, 5-nitrofuran, and hydrazone, that were frequently reported in the chemical structures of antimicrobial and anticancer agents. The target compounds were obtained by reacting 5-nitrofuran-2-carbaldehyde and in-house synthesized hydrazide derivatives carrying piperidine/piperazine moiety. After confirming the proposed structures by various spectral techniques, PNH1-PNH11 were tested for their anticancer and antimicrobial activities. Based on the biological data obtained, PNH4 ((E)-4-(4-(4-methoxyphenyl)piperazin-1-yl)-N'-((5-nitrofuran-2-yl)methylene)benzohydrazide) appeared to be the most attractive derivative in this series as an effective cytotoxic agent with concurrent antibacterial activity. Molecular docking studies within nitroreductase were applied to support the antibacterial activity mechanism of PNH4 and to explain its superior activity compared to the other synthesized compounds. Furthermore, natural bond orbital (NBO) analysis, potential energy surface (PES) scanning investigations, HOMO-LUMO energies, and molecular electrostatic potential (MEP) and contour maps calculations were carried out to gain insights into the structural properties, chemical reactivity, and stability of the most active compound using density functional theory (DFT) at the B3LYP functional using basis set 6–31G(d,p)
Diagnostic Potential of CD44, CD133, and VDR in Epithelial Ovarian Tumors: Association with Histopathology Parameters
No full textCancer stem cells (CSCs) significantly contribute to heterogeneity, malignancy, and therapy resistance in ovarian cancer. Recent studies emphasize the role of the vitamin D receptor (VDR) in regulating cell differentiation and stemness in various types of cancer. This study aims to determine the expression levels of CD44, CD133, and VDR in epithelial ovarian tumors (EOTs) and to compare these levels across different tumor types, including benign, atypical proliferative tumors, and five types of malignant phenotypes, in order to evaluate their potential as diagnostic tools for malignancy. Tissue samples from 218 patients diagnosed with EOT were analyzed. Clinical and histopathologic parameters were recorded. Quantitative immunohistochemical tissue microarray analysis was used to assess the expression levels of CD44, CD133, and VDR using two different scoring systems. Comparisons were made between benign tumors (n = 45), atypical proliferative tumors (n = 42), and ovarian carcinomas (n = 131), including high-grade serous (HGSC) and non-HGSC subtypes. Ovarian cancer, especially HGSC, showed a significantly higher expression of CD44 and VDR (p < 0.05) compared to atypical proliferative tumors and benign tumors. The expression of CD133 was highest in atypical proliferative tumors (p < 0.05). A moderate positive correlation was found between CD44, CD133, and VDR in all groups, with significant correlations with tumor grade and FIGO stage in ovarian cancer (p < 0.05). The increased expression of CD44 and VDR in aggressive ovarian cancer, along with elevated CD133 levels in atypical proliferative tumors, highlights the complexity of tumor biology. These markers may serve as valuable targets for the diagnosis of ovarian cancer
Functional Ambidexterity of an Ancient Nucleic Acid-Binding Domain
No full textThe helix-hairpin-helix (HhH) motif is an ancient and ubiquitous nucleic acid-binding element that has emerged as a model system for studying the evolution of dsDNA-binding domains from simple peptides that phase separate with RNA. We analyzed the entire putative evolutionary trajectory of the HhH motif – from a flexible peptide to a folded domain – for functional robustness to total chiral inversion. Against expectations, functional “ambidexterity” was observed for both the phase separation of HhH peptides with RNA and binding of the duplicated (HhH)2-Fold to dsDNA. Moreover, dissociation kinetics, mutational analysis, and molecular dynamics simulations revealed an overlap between the binding modes adopted by the natural and mirror-image proteins to natural dsDNA. The similarity of several dissociation phases upon chiral inversion may reflect the history of (HhH)2-Fold binding, with the ultimate emergence of a high-affinity binding mode, supported by a bridging metal ion, depopulating but not displacing more primitive (potentially ambidextrous) modes. These data underscore the surprising functional robustness of the HhH protein family and suggest that the veil between worlds with alternative chiral preferences may not be as impenetrable as is often assumed
Exploring the chemical space of new monobactams
No full textAntimicrobial resistance is a major global health threat, with drug-resistant bacteria causing 4.7
million deaths annually. β-Lactams are the most widely used antibacterials, but resistance arises
from PBP mutations and β-lactamases. Monobactams, a subclass with a monocyclic core, resist
hydrolysis by metallo-β-lactamases, with aztreonam being the only approved drug in this class.
[1] Targeting biofilms is a promising strategy for combating resistance, as they contribute to
infection persistence and antibiotic tolerance. [2]
We aimed to develop potent inhibitors of PBP1b from Streptococcus pneumoniae by synthesizing
a focused library of monobactams with diverse R1 side chains (Figure 1). Structural analysis of
obtained co-crystal structures revealed potential for more potent enzyme inhibition through
additional hydrogen and halogen bonding with a key threonine residue. Despite nanomolar PBP
inhibition, these compounds did not exhibit strong antibacterial activity. To increase efficacy, we
synthesized two series of aztreonam chimeras. The first contained nitroxide conjugates that
mimic nitric oxide, while the second contained acylhomoserine lactone (AHL) and
acylhomocysteine lactone conjugates designed to inhibit quorum sensing. Conjugates were
synthesized using diverse linkers that formed either only amide bonds – R2 (nitroxides) or also
sulfonic acid esters – R3 (AHLs). Some compounds were effective against Gram-positive
bacteria—unusual for monobactams—while others showed MIC values comparable to
aztreonam against ESKAPE pathogens. Additionally, they inhibited biofilm formation in clinical
isolates of Escherichia coli, Pseudomonas aeruginosa PAO1, and Acinetobacter baumannii,
highlighting their dual mode of action.V International Conference on Anti-infectives 2025, (ICA 2025) May 20 - 22, 2025, SaarlandPoster 6
Establishing transgenic zebrafish model expressing the A30P α-synuclein mutation for Parkinson’s disease research
No full textParkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide. Major characteristic of PD is the pathological aggregation of α-synuclein protein in the brain. Among the inherited α-synuclein mutations, associated with PD, the A30P mutation is known to stimulate the formation of toxic oligomers and fibrils. To better understand the molecular mechanisms underlying PD and to promote the development of novel therapies, animal models, such as zebrafish, expressing human α-synuclein variants are used. Our study aimed to establish a new transgenic zebrafish model expressing mutated the A30P α-synuclein, facilitating in vivo investigation of the mechanisms of early aggregation processes. Here we present a generation of new transgenic zebrafish line stably overexpressing human αsynuclein with amino acid substitution A30P. The plasmid UAS:Dendra2-Hsa.SNCA, myl7:EGFP, coding for human α-synuclein tagged with Dendra2 as well as a transgenesis marker expressing EGFP under a heart-specific promoter, was used as a template for site-directed mutagenesis. Tol2 transgenesis system was composed of the A30P plasmid (UAS:Dendra2Hsa.SNCA_A30P, myl7:EGFP) and Tol2 RNA. They were microinjected into embryos at one-cell stage and embryos were screened three days post fertilization for EGFP-positive signal in the heart. EGFP-positive larvae are raising to adulthood, to identify carriers. Once the animals reach sexual maturity, outcrosses with the pan-neuronal driver line PanN:GAL4, will enable targeted expression of the mutated α-synuclein in the nervous system, facilitating future studies on oligomer-induced neurotoxicity relevant to PD pathogenesis and allowing for testing of potential new treatments.BeCELS 2025: Belgrade Conference for Early-Career Life Scientists, taking place on Friday, September 5, 2025, at the Institute of Molecular Genetics and Genetic Engineering (IMGGE) in Belgrad
The effects of FAAH inhibitor, URB597, on dopamine content, synthesis and degradation in the striatum of the rats exposed to chronic unpredictable stress
No full textAnhedonia is a key symptom of major depressive disorder, often linked to disrupted activation of reward-related pathways in the striatum and impaired reward learning, associated with decreased dopaminergic signaling. Since conventional antidepressants are not always effective, alternative therapeutic approaches are being explored. Enhancing endocannabinoid signaling through pharmacological inhibition of fatty acid amide hydrolase (FAAH) has emerged as a promising treatment option. The goal of our study was to determine the effects of FAAH inhibition on dopamine content in the striatum in the animal model of depression. Wistar rats of both sexes, two months old, were subjected to chronic unpredictable stress (CUS) for six weeks. During the last two weeks, animals were injected either with URB597, a selective FAAH inhibitor, or with vehicle. Experimental rats were sacrificed after six weeks, and their striatum was dissected. Dopamine concentration in striatal tissue was determined using a commercially available ELISA kit, while protein levels of tyrosine hydroxylase (TH) and monoamine oxidase (MAO) were examined using Western blot. Our results have shown a significant decrease in dopamine levels in the striatum in CUS animals, while URB597 treatment increased dopamine levels in stressed animals of both sexes. Protein levels of TH were reduced in CUS males and unaffected in CUS females. MAO protein levels were elevated in stressed animals, while URB597 treatment reduced them in stressed animals of both sexes. This research suggests that FAAH inhibition can mitigate disrupted dopamine turnover in depression, supporting its potential as a novel strategy for treating anhedonia in depression.BeCELS 2025: Belgrade Conference for Early-Career Life Scientists, taking place on Friday, September 5, 2025, at the Institute of Molecular Genetics and Genetic Engineering (IMGGE) in Belgra
An angiogenesis-independent mechanism of action for anti-VEGFA therapies
No full textVEGF-A is one of the most potent pro-angiogenic factors and it is often upregulated in a variety of tumors. Although VEGF-signaling is mostly related to angiogenesis, VEGF-A has also been shown to regulate tumor cell survival and migration. However, research is mainly focused on the impact of anti-angiogenic therapies on endothelial cells, not exploring their effect on other cell populations. Here, we investigated the impact of bevacizumab, an anti-VEGF-A therapy, on innate immune cell populations present in the tumor microenvironment of zebrafish xenografts. By using a bevacizumab sensitive breast cancer cell line (Hs578T), in which bevacizumab impairs angiogenesis and shrinks tumor size, we show that bevacizumab can modulate the number of tumor-associated-macrophages and switch them towards a pro-inflammatory M1-like phenotype. Strikingly, depletion of macrophages leads to the same phenotype as bevacizumab, i.e reduction of tumor size and impairment of angiogenesis, suggesting that VEGF-A seems to be involved in breast cancer survival through recruitment of tumor-associated-macrophages, independently of angiogenesis. To test this, we used and human in vitro heterologous system of co-culturing human Hs578T with monocyte-derived macrophages from healthy donors. Here we show that bevacizumab, in the absence of endothelial cells, can reduce the number of tumor cells and alter macrophage polarization profile. With this, we show that bevacizumab can induce its anti-tumor activity through macrophages. To explore the angiogenesis-independent molecular mechanismof-action of bevacizumab, we are currently analyzing single-cell-RNA sequencing data. To translate this research, we aim to test the clinical relevance of these findings in a cohort of bevacizumab-treated patients.BeCELS 2025: Belgrade Conference for Early-Career Life Scientists, taking place on Friday, September 5, 2025, at the Institute of Molecular Genetics and Genetic Engineering (IMGGE) in Belgrad
3D bioactive environment for osteosarcoma research
No full textCurrent standard osteosarcoma (OS) treatment involves tumor resection combined with neoadjuvant and adjuvant chemotherapy. Despite ongoing efforts to advance these therapies, progress has been slow due to limited translation of in vitro and in vivo findings into the patient care. Recently, three-dimensional (3D) culture models have emerged as relevant and reliable platforms for cancer research. This study focused on development and characterization of macroporous alginate-based scaffolds with incorporated bioactive glass (BAG) particles, as cell carriers, to mimic OS environment. Two types of BAGs (SBA2 and 47.5B) with different compositions, were evaluated regarding cytocompatibility with murine OS K7M2-wt cells using MTT assay, as well as bioactivity in terms of BAG transformation into hydroxyapatite (HAP) assessed by scanning electron microscopy (SEM) and energy-dispersive X-ray (EDS) analysis. Then, composite scaffolds were produced by controlled gelation of mixtures containing 2 wt.% Na-alginate, 2 wt.% BAG, and different CaCl2 concentrations, followed by freeze-drying, and rehydration. The obtained results showed that both BAG types were cytocompatible, while the phase transformation occurred on days 2 and 5 for 47.5B and SBA2 BAGs, respectively. The best candidate scaffolds were obtained with BAG/alginate mixtures containing 0.06 wt.% CaCl2. The selected scaffolds were seeded with OS cells (15×106 cells/cm³) achieving a seeding efficiency of up to 92%. During 7-day cultivation the cells in both scaffold types (i.e. with 47.5B and SBA2) retained viability, metabolic activity, and ability to proliferate, while spontaneously forming aggregates. Overall, alginate scaffolds with embedded BAG particles supported OS cell behavior similar to that observed in vivo.BeCELS 2025: Belgrade Conference for Early-Career Life Scientists, taking place on Friday, September 5, 2025, at the Institute of Molecular Genetics and Genetic Engineering (IMGGE) in Belgrad