A New Approach in Identifying dss1 Suppressors by Whole Genome Sequencing of Ustilago maydis

Dss1, a small and intrinsically disordered protein, is one of the crucial regulators of homologous recombination (HR), a key DNA repair pathway that ensures error-free repair of double-strand breaks, thereby preserving genome stability. Dss1 is highly conserved in eukaryotes and is present in a model organism Ustilago maydis, known for its extreme resistance to ionizing and UV irradiation. This microorganism is a valuable model for investigating HR, which combines high genetic tractability and a DNA-repair system remarkably similar to humans’, including conserved BRCA2 ortholog Brh2 and its partner Dss1. Loss of dss1 function leads to the extreme sensitivity of the U. maydis mutant to genotoxic agents due to impaired DNA-repair and HR. The main aim of the research is to identify novel cellular factors involved in HR by searching for suppressors of dss1, secondary mutations that rescue genotoxic resistance of dss1 mutant. To uncover such suppressors, several independent Δdss1 suppressor strains were generated by random mutagenesis and phenotypically characterized. While complementation cloning, classical genetic approach, could not reveal the suppressor identity, we applied whole-genome sequencing in combination with a modified pooled-linkage analysis. This strategy is based on sequencing DNA from pools of meiotic segregants, grouped according to their expected phenotype. By separating putative suppressor and wt segregants, it becomes possible to track how often specific variant appear in each pool. Variants that consistently co-segregate with the expected suppressor phenotype show a shift in allele frequency, which points to genomic regions that are likely to carry the suppressor mutation. Although the analysis is ongoing, the study demonstrates the applicability of pooled-linkage sequencing as a complementary method to classical genetic tools. The approach has the potential to facilitate the identification of novel factors involved in HR in U. maydis, thus, representing an applicable framework for uncovering genetic networks that maintain genome integrity.Book of abstract: 8th edition of Young Biologists Matter Congress (Molecular Biology & Physiology) Thessaloniki, Greece, 29th September – 4th October 202

GENE EXPRESSION PROFILING OF ASTROCYTES DERIVED FROM IPSCS OF PATIENTS WITH 22Q11.2 DELETION SYNDROME

22q11.2 Deletion Syndrome (22q11.2DS), caused by microdeletion 22q11.2, is the most common microdeletion syndrome in humans. t is closely linked to an increased risk of neurodevelopmental disorders and provides a valuable model for investigating the molecular mechanisms underlying these disorders, which are still not fully understood. Here we analyzed transcriptomic profiling of 22q11.2DS astrocytes derived from iPSCs of mother and child with 22q11.2DS and one healthy control. RNA-seq was carried out on Illumina NovaSeq 6000 sequencer. Bioinformatic processing of raw data was conducted via NVIDIA platform, while differential gene expression analysis was performed in RStudio using DESeq2 R package. The obtained list of DEGs was used for pathway enrichment analysis by employing EnrichR and WikiPathways. 125 DEGs with lower expression and 287 DEGs with higher expression in 22q11.2DS astrocytes compared to the control were obtained. For genes with lower expression in 22q11.2DS astrocytes, 22q11.2 Copy Number Variation Syndrome and Axon Guidance were the top enriched pathways, while for genes with higher expression in 22q11.2DS astrocytes we did not identify biological pathways that are enriched in DEG lists more than would be expected by chance. We found 178 DEGs with lower expression and 205 DEGs with higher expression in astrocytes of symptomatic child with 22q11.2DS compared to non-symptomatic mother with 22q11.2DS. Employing EnrichR and WikiPathways we did not identify biological pathways that are enriched in DEG lists more than would be expected by chance. Our findings offer preliminary evidence of an altered transcriptomic landscape of 22q11.2DS astrocytes.Abstract book: FENS Regional Meeting 2025 , Oslo, Norway, 16-19 June 202

Analysis of Variants in Cytochrome P450 Superfamily Genes as Predictive Markers for Neoadjuvant Chemoradiotherapy in Rectal Cancer

Background: Patients with locally advanced rectal cancer (LARC) often receive neoadjuvant chemoradiotherapy (nCRT) based on 5-fluorouracil (5-FU). Cytochrome P450 (CYP) enzymes, involved in drug metabolism and carcinogenesis, may influence response to therapy. Variability in CYP gene expression makes them promising biomarkers for predicting treatment outcomes in rectal cancer. Material and methods: Genomic DNA from 38 LARC patients treated with standard nCRT (fluorouracil + leucovorin + radiotherapy) was analyzed. Clinical response was assessed by tumor regression grade (TRG). Seven patients with extreme responses – good (3 TRG1 and 2 TRG2) and poor (2 TRG5) were selected for whole-exome sequencing (WES). Candidate predictive variants were identified based on their differential presence between responder and nonresponder groups and biological relevance (localization in coding region or regulatory elements and enzyme-altering effects). Selected variants were validated in 29 moderate-response patients (15 TRG3 and 14 TRG4) by targeted sequencing. Results: Two genetic variants were selected according to the criteria outlined above: rs149012039, which is located within the CYP2D7 pseudogene and represents a frameshift variant, and rs3093200, which is located in the first exon of the CYP4F2 gene and has a damaging effect on the protein. Validation in the remaining samples showed that neither variant was present in patients with a moderate response to therapy. Conclusions: The presence of variants rs149012039 and rs3093200 in individuals with a good clinical response and their absence in individuals with a moderate or poor response supports their potential as predictive biological markers for response to nCRT in rectal cancer

FROM HIVE TO HEALTH: PROBIOTIC POTENTIAL OF APILACTOBACILLUS KUNKEEI AND ITS EFFECT ON HUMAN VITAMIN D RECEPTOR EXPRESSION

Objective: The Vitamin D receptor (VDR) plays a key role in maintaining calcium and phosphate homeostasis, modulating the immune response, and mediating interactions between the gut microbiota and the host. Dysfunction of the VDR has been linked to alterations in gut microbiota composition and the development of autoimmune diseases. While traditional probiotics are typically isolated from fermented dairy products, there is growing interest in alternative sources, including bee products and the gut microbiota of honeybees. Among bee-associated bacteria, the fructophilic lactic acid bacterium Apilactobacillus kunkeei has emerged as a promising candidate for probiotic applications. Methods: Ten strains of A. kunkeei isolated from the guts of healthy honey bees (Apis mellifera) from the Vojvodina region (Serbia) were tested for probiotic properties and safety attributes for potential human use. Their effect on the expression of VDR and VDR-related genes was examined using an in vitro model of the human intestinal epithelial barrier (Caco-2 cell line). Additionally, the genomes of two selected strains were analyzed. Results: All examined strains exhibited strong antimicrobial activity against human gut pathogens. Among them, only strains A1, A4, A28, and A56 demonstrated mucin-binding ability. Notably, A. kunkeei strains A1 and A55 were capable of modulating the expression of VDR and tight junction-related genes in the Caco-2 cell line. Both strains also showed sensitivity to all tested antibiotics. Conclusions: These findings highlight the potential of A. kunkeei A1 and A55 strains to modulate VDR function and support their further development as safe and effective probiotics for human use

NOVEL SERBIAN WHITE SOFT CHEESE IMPROVE GUT EPITHELIAL BARRIER AND AUTOPHAGY IN VITRO

Objective: The application of natural starter cultures in fermented dairy products is gaining momentum due to their dual role in improving product quality and promoting host health. This study examines the potential of novel natural starter cultures used in the production of white soft cheese to modulate gut epithelial barrier function and autophagy in vitro. Methods: White soft cheese was produced using two artisanal starter strains: Lactococcus lactis subsp. lactis BGTRK4-21 and Lactobacillus plantarum BGGO7-29, both isolated from traditional Serbian dairy products. In vitro digestion of cheese samples aged 1, 10, 20, and 30 days was incubated with Caco-2 cells, and the expression of tight junction proteins, autophagy-related genes, as well as genes involved in antioxidant defense and antimicrobial peptide production, was quantified using RT-qPCR. Results: Exposure to digested cheeses significantly upregulated the expression of CLDN4, OCLN, BECN1, MAP1LC3B, p62, HBD1, SOD2, and GSTH genes in a time-dependent manner, with the most pronounced effects observed in 10- and 20-day-old cheese samples. These results suggest enhanced intestinal barrier integrity, activation of autophagy pathways, and improved cellular antioxidant and antimicrobial defense mechanisms. Conclusions: White soft cheese produced with selected natural starter cultures exerts beneficial effects on intestinal epithelial cells, indicating its potential as a functional food. These findings support the development of traditional dairy products with added probiotic-like benefits, contributing to gut health through barrier enhancement and autophagy modulation

Personalized Medicine Begins at Birth: Newborn Screening for Spinal Muscular Atrophy in Serbia as a Model of Individualized Care

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality, with disease-modifying therapies achieving maximal benefit when administered presymptomatically, underscoring the medical, ethical, and public health imperative for newborn screening (NBS). In 2021, Serbia launched its first genetic NBS initiative for SMA, centralized at the Faculty of Biology, University of Belgrade, a center of SMA diagnostics and research since 1997. Over a 17-month pilot study, 12,000 newborns across two maternity hospitals were screened using dried blood spots analyzed by qPCR for SMN1 absence, with confirmatory MLPA testing and SMN2 copy number determination. Following pilot study success, the national SMA screening program began on September 15, 2023, now encompassing 52 public and 6 private maternity hospitals. By September 09, 2025, 119,735 newborns had been screened, identifying 19 infants with SMA; 17 received immediate therapy—6 with 2 SMN2 copies, 7 with 3 copies, and 4 with 4 copies—while 2 infants with 5 SMN2 copies remain under observation. Treated infants remain largely asymptomatic. A multidisciplinary Expert SMA Commission ensures individualized treatment decisions, integrating genetic, clinical, and laboratory data. The program establishes Serbia’s first reliable SMA incidence estimate (1:6,302 births) and demonstrates that presymptomatic diagnosis, structured screening workflows, and personalized therapy can transform SMA from a severe, life-limiting disease into a manageable condition. Serbia’s experience provides a compelling model for integrating precision medicine into national health systems through coordinated collaboration among academia, patient advocacy, industry, and government, illustrating how early genetic diagnosis and tailored interventions can fundamentally change disease trajectories.Spinalna mišićna atrofija (SMA) predstavlja vodeći genetički uzrok smrtnosti odojčadi, pri čemu terapije koje menjaju tok bolesti imaju najveći efekat kada se primene presimptomatski. To naglašava medicinski, etički i javnozdravstveni značaj neonatalnog skrininga. Srbija je 2021. pokrenula prvi genetički neonatalni skrining za SMA, centralizovan na Biološkom fakultetu Univerziteta u Beogradu, koji od 1997. ima ekspertizu u istraživanjima i dijagnostici SMA. U 17-mesečnoj studiji izvodljivosti testirano je 12,000 novorođenčadi iz dva porodilišta. Uzorci suvih krvavih mrlja analizirani su metodom qPCR radi otkrivanja odsustva gena SMN1, uz potvrdno testiranje i određivanje broja kopija SMN2 metodom MLPA. Nakon završetka studije izvodljivosti, nacionalni program skrininga za SMA počeo je 15. septembra 2023. i danas obuhvata 52 državna i 6 privatnih porodilišta. Do 9. septembra 2025. testirano je 119,735 novorođenčadi i identifikovano je 19 SMA pozitivnih beba. Od toga je 17 odmah započelo terapiju—6 sa dve kopije SMN2, 7 sa tri kopije i 4 sa četiri kopije—dok su dva novorođenčeta sa pet kopija pod kliničkim nadzorom. Lečene bebe su uglavnom ostali asimptomatski. Multidisciplinarna Stručna komisija za SMA donosi individualizovane odluke o terapiji, kombinujući genetičke, kliničke i laboratorijske podatke. Program je dao prvu pouzdanu procenu učestalosti SMA u Srbiji (1:6,302) i pokazao da presimptomatska dijagnoza, jasno definisani protokoli i personalizovana terapija mogu SMA pretvoriti iz teškog, životno ograničavajućeg oboljenja u (iz)lečivu bolest. Srpsko iskustvo pruža model za integraciju precizne medicine u nacionalne zdravstvene sisteme kroz saradnju akademske zajednice, pacijenata, industrije i države

Trichinella spiralis extracellular vesicles induce anti-inflammatory and regulatory immune responses in vitro

The helminth Trichinella spiralis, through its excretory-secretory (ES L1) products, induces immune regulatory mechanisms that modulate the host’s immune response not only to itself, but also to bystander antigens, foreign or self in origin, which can result in the alleviation of inflammatory diseases. Under the influence of ES L1, dendritic cells (DCs) acquire a tolerogenic phenotype and the capacity to induce Th2 and regulatory responses. Since ES L1 products represent a complex mixture of proteins and extracellular vesicles (TsEVs) the aim of this study was to investigate the impact of TsEVs, isolated from ES L1 products, on phenotypic and functional characteristics of DCs and to elucidate whether TsEVs could reproduce the immunomodulatory effects of the complete ES L1 product. Monocyte-derived DCs treated with TsEVs acquired semi-matured phenotypes, characterized by low expression of human leukocyte antigen – DR isotype (HLA-DR), cluster of differentiation (CD) 86 (CD86), and CD40, moderate expression of CD83 and C–C chemokine receptor type 7 (CCR7), and increased expression of tolerogenic markers indoleamine 2,3‐dioxygenase 1 (IDO-1) and immunoglobulin-like transcript 3 (ILT3), together with the unchanged production of IL-12 and IL-23, and elevated production of IL-10 and transforming growth factor (TGF)-β, compared with controls. Gene expression analysis of TsEV-treated DCs revealed elevated levels of mTOR, Ahr, NF-κB2, RelB, SOCS1 and SOCS3, which participate in signaling pathways involved in DC maturation and the subsequent regulation of release of both anti-inflammatory and pro-inflammatory cytokines. TsEVs promoted the capacity of DCs to drive polarization of Th2 and anti-inflammatory responses, and impaired their capacity to induce Th1/Th17 polarization. Moreover, TsEV-treated DCs possessed a high capacity to induce conventional FoxP3 + regulatory T cells, as well as unconventional T regulatory (Tr1) cells. Tolerogenic properties of TsEV-treated DCs were retained even after challenge with a pro-inflammatory stimulus. These findings highlight the potential of TsEVs to induce immune tolerance, suggesting their potential use as therapeutics for the treatment of inflammatory disorders

Silver(I) complexes with antifungal drug econazole: Structural characterization and antimicrobial activity study

Two new silver(I) complexes with the antifungal agent econazole (ecz) of the general formula [Ag(еcz)2]X, X = CF3SO3− (Ag1) and PF6− (Ag2), were synthesized and structurally characterized by spectroscopic (1H NMR, IR and UV–Vis) and electrochemical methods. The crystal structure of Ag2 was determined by single crystal X-ray diffraction analysis, confirming an ideal linear geometry of the silver(I) ion in this complex. In addition, by utilising Hirshfeld surface analysis (HSA), it was verified that the crystal structure of Ag2 is stabilized by H⋯H, H⋯Cl, C⋯H, and H⋯F interactions. Density functional theory (DFT) calculations provided additional evidence for formation of the complexes in the solid state and their stability in solution. The coordination of ecz to the silver(I) ion endowed this agent with antibacterial activity against Gram-positive (Pseudomonas aeruginosa and Escherichia coli) and Gram-negative (Staphylococcus aureus and Listeria monocytogenes) bacteria. The most significant antibacterial activity of Ag1 and Ag2 was observed against S. aureus with 56- and 73-fold improvement, respectively, compared to the parent drug. Moreover, a study of the bacterial antibiofilm activity revealed that these complexes were able to prevent approximately 90 % of biofilm formation at their low concentrations. Also, both complexes have shown more pronounced anti-Candida activity than econazole itself, being less toxic on the human normal fibroblast cell line MRC-5. The total amount of ergosterol was reduced in the presence of the subinhibitory concentrations of Ag1 and Ag2 complexes, which was also confirmed by a molecular docking study with two isomers of cytochrome P450 sterol 14α-demethylase, CYP51B and CYP130, as target receptors

Amino Acid Substitutions in Bacteriocin Lactolisterin BU Reveal Functional Domains Involved in Biological Activity Against Staphylococcus aureus

The emergence of multidrug-resistant pathogens has driven the development of novel antimicrobial peptides (AMPs) as therapeutic alternatives. Lactolisterin LBU (LBU) is a bacteriocin with promising activity against Gram-positive bacteria, including Staphylococcus aureus. In this study, we designed and evaluated a panel of amino acid variants of LBU to investigate domain–activity relationships and improve activity. Peptides were commercially synthesized, and their effect was evaluated for minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), hemolytic activity, cytotoxicity, in vivo toxicity, and virulence modulation. AlphaFold3 structural prediction of LBU revealed a four-helix topology with amphipathic and hydrophobic segments. Helical wheel projections identified helices I and IV as amphipathic, suggesting their potential involvement in membrane interaction and activity. Glycine-to-alanine substitutions at helix I markedly increased antimicrobial activity but altered toxicity profiles. In contrast, changes at helix junctions and kinks reduced antimicrobial activity. We also showed differential regulation of virulence genes upon sub-MIC treatment. Overall, rational substitution enabled identification of residues critical for activity and toxicity, providing insights into therapeutic tuning of lactolisterin-based peptides

CIRCULATING PLASMA LONG NON-CODING RNA GAS5 AS NON-INVASIVE BIOMARKER IN MULTIPLE MYELOMA PATIENTS

Background: Growth arrest specific 5 (GAS5) is a long non-coding RNA that has been reported as a prognostic biomarker in numerous malignancies. Clinical and prognostic significance of circulating plasma GAS5 expression in patients with multiple myeloma (MM) is unknown so far. Aims: The aim is to analyze the expression level of plasma circulating GAS5 in patients with MM at presentation and in the relapse of the disease, also to investigate its association with clinical characteristics, overall response rate (ORR), progression free survival (PFS) and overall survival (OS). Methods: This is a prospective research which includes patients with MM who were diagnosed and treated in Clinical-hospital center (CHC) Bezanijska kosa and CHC Zemun from November 2021 to October 2024. Plasma samples were collected from 72 MM patients at diagnosis and also from 6 patients at relapse. Relative quantification analysis of GAS5 expression level was performed by RQ-PCR methodology with GAPDH gene as endogenous control and using comparative ddCt method with healthy controls as calibrator. Results: This group included 38 (52.7%) males and 34 (47.3%) females; median age was 69 years (range 44-90). ISS score 1, 2 and 3 was present in 11 (15%), 17 (24%) and 44 (61%) patients; respectively. High-risk cytogenetic aberrations were present in 24 (34%) patients. Favorable therapeutic response was registered in 61 (84.7%) patients. Mortality rate was 29.1% (21 patients). Median expression of GAS5 in de novo MM patients was 0.810 (range 0.012-6.516), which was not significantly lower in comparison to expression level among healthy controls (median 1.001, range 0.660-2.435) (p=0.069). No difference was shown in expression levels of GAS5 in relapse compared to the level detected at presentation (median 0.580, range 0.021-1.830) (p=0.818). We used ROC curve analysis to determine cut-off value for the GAS5 expression and the most predictive value was 1.068 (AUC=0.6, Sensitivity=72.1%, Specificity=63.6%, p=0.034). Using this value, patients were divided into low and high GAS5 expression group (GAS5low and GAS5high). In our cohort of patients, 67% (48/72) were in GAS5low group. No correlation was found between the level of GAS5 expression and clinical characteristics. Also, the level of GAS5 expression was not associated with any cytogenetic risk group, while gain of 1q21 was more frequent in GAS5highpatients (p=0.05). Patients in GAS5low group had significantly better ORR compared to GAS5high patients (p=0.021). Moreover, survival analysis showed that GAS5lowpatients had longer OS (16.6 vs. 9 months) (Long-Rank 4.317, p=0.038), while no significant difference was shown in PFS between GAS5lowand GAS5high patients (Log-Rank 3.122, p=0.077). Summary/Conclusion: Our study showed that the level of plasma circulating GAS5 was reduced in de novo patients compared to healthy controls, but with no statistical difference. Our results imply that low GAS5 expression level could predict better ORR and OS in patients with MM.30th Congress of the European Hematology Association EHA2024 Annual Congress Edition June 202