9th COLLOQUIUM OF GENETICS Book of Abstracts September 29th 2023 Piran GENETIC SOCIETY OF SLOVENIA IN COLLABORATION WITH THE SLOVENIAN SOCIETY OF HUMAN GENETICS Scientific committee Matej Butala, Biotechnical Faculty, University of Ljubljana Maja Čemažar, Institute of Oncology Ljubljana Damjan Glavač, Faculty of Medicine, University of Ljubljana Simon Horvat, Biotechnical Faculty, University of Ljubljana Jernej Jakše, Biotechnical Faculty, University of Ljubljana Jernej Ogorevc, Biotechnical Faculty, University of Ljubljana Ester Stajič, Biotechnical Faculty, University of Ljubljana Marjanca Starčič Erjavec, Biotechnical Faculty, University of Ljubljana Organization committee Maja Čemažar, Institute of Oncology Ljubljana Simona Kranjc Brezar, Institute of Oncology Ljubljana Boštjan Markelc, Institute of Oncology Ljubljana Andreja Ramšak, National Institute of Biology Sponsors Omega d.o.o. Mediline d.o.o. Merck d.o.o. Plastika Skaza d.o.o. Roche d.o.o. VWR International Gmbh 9th COLLOQUIUM OF GENETICS Book of abstracts Editors Simona Kranjc Brezar, Maja Čemažar, Boštjan Markelc Reviewers Matej Butala, Maja Čemažar, Damjan Glavač, Simon Horvat, Jernej Jakše, Jernej Ogorevc, Marjanca Starčič Erjavec, Ester Stajič Technical editor Simona Kranjc Brezar Design Simona Kranjc Brezar Publisher Genetic Society of Slovenia in collaboration with the Slovenian Society of Human Genetics Date of publishing September 29th 2023, Ljubljana Webpage: https://sgd.si/docs/Colloquium-9th-2023.pdf Published as e-book (.pdf) Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID 165570051 ISBN 978-961-93545-8-2 (Slovensko genetsko društvo, PDF) 9th COLLOQUIUM OF GENETICS Book of abstracts September 29th 2023 National Institute of Biology Marine Biology Station Piran GENETIC SOCIETY OF SLOVENIA IN COLLABORATION WITH THE SLOVENIAN SOCIETY OF HUMAN GENETICS 1 TABLE OF CONTENTS Program 3 List of Short Lectures 6 List of Posters 8 Opening and Closing Lectures 10 Abstracts of Short Lectures 13 Abstracts of Posters 35 Author Index 62 Sponsors 66 2 PROGRAM 8:00 - 9:00 REGISTRATION 9:00 - 9:10 OPENING OF THE 9th COLLOQUIUM OF GENETICS Maja Čemažar 9:10 - 10:40 MICROBIAL GENETICS, ANIMAL GENETICS SHORT LECTURES Chairs: Matej Butala, Jernej Ogorevc 9:10 - 9:25 Genetic variation in polyadenylation signals of differentially expressed mRNA isoforms in mouse selection lines for fatness and leanness Špela Mikec 9:25 - 9:40 Archaeogenetic analysis of Late Pleistocene and Holocene Bison fossils from the Slovenia and Hungary Lars Zver 9:40 - 9:55 Identifying introgression between domestic ( Capra hircus) and Alpine ibex (C. ibex): a case study from Slovenian part of Alps Neža Pogorevc 9:55 - 10:10 Hybridization and population genetic structure of European wildcats from Dinaric Alps and Pannonian Basin Felicita Urzi 10:10 - 10:25 The conjugation frequency of the pOX38:Cm conjugative plasmid in in vitro models of human intestine and in an anaerobic chamber Aleksander Lesar 10:25 - 10:40 A small bacteriophage protein determines the hierarchy over co-residential jumbo phage in Bacillus thuringiensis serovar israelensis Anja Pavlin 3 PROGRAM 10:40 - 11:15 OPENING LECTURE Chair: Maja Čemažar 10:45 - 11:15 How a biased genetic code creates order from proteins’ disorder? Jernej Ule 11:15 - 11:45 COFFEE BREAK AND POSTER VIEWING – PART I 11:45 - 13:15 PLANT GENETICS, HUMAN GENTICS SHORT LECTURES Chairs: Damjan Glavač, Jernej Jakše, Maja Čemažar, Ester Stajič 11:45 - 12:00 Epigenetic insights into viroid pathogenesis from genome-wide DNA methylation analysis of CBCVd-infected hop plants ( Humulus lupulus var. 'Celeia') Andrej Sečnik 12:00 - 12:15 Genome-wide association study and population structure of different hemp ( Cannabis sativa L.) genotypes Marjeta Eržen 12:15 - 12:30 Irradiation and gene electrotransfer of plasmid DNA encoding chemokines CCL5 and CCL17 induce immunomodulatory effects in murine tumors Tim Božič 12:30 - 12:45 HPV-positive mouse model of oral squamous cell carcinoma: development and transcriptome analysis Živa Modic 12:45 - 13:00 Molecular and clinical characteristics of Slovenian patients with small cell carcinoma of the ovary, hypercalcemic type Ana Blatnik 4 PROGRAM 13:00 - 13:15 Isolate-specific differences in macrophage response to group B Streptococcus Larisa Janžič 13:15 - 14:30 LUNCH AND POSTER VIEWING – PART II 14:30 - 15:00 CLOSING LECTURE Chair: Jernej Jakše 14:30 - 15:00 Coding and non-coding RNA in dormant cells Jernej Murn 15:00 - 15:30 GENERAL ASSEMBLY OF SGD PRIZE ANNOUNCEMENTS AND CLOSING OF THE 9th COLLOQUIUM OF GENETICS 5 LIST OF SHORT LECTURES l1 Genetic variation in polyadenylation signals of differentially expressed mRNA isoforms in mouse selection lines for fatness and leanness Špela Mikec 14 l2 Archaeogenetic analysis of Late Pleistocene and Holocene Bison fossils from the Slovenia and Hungary Lars Zver 16 l3 Identifying introgression between domestic ( Capra hircus) and Alpine ibex ( C. ibex): a case study from Slovenian part of Alps Neža Pogorevc 18 l4 Hybridization and population genetic structure of European wildcats from Dinaric Alps and Pannonian Basin Felicita Urzi 20 l5 The conjugation frequency of the pOX38:Cm conjugative plasmid in in vitro models of human intestine and in an anaerobic chamber Aleksander Lesar 22 l6 A small bacteriophage protein determines the hierarchy over co-residential jumbo phage in Bacillus thuringiensis serovar israelensis Anja Pavlin 24 l7 Epigenetic insights into viroid pathogenesis from genome-wide DNA methylation analysis of CBCVd-infected hop plants ( Humulus lupulus var. 'Celeia') Andrej Sečnik 25 l8 Genome-wide association study and population structure of different hemp ( Cannabis sativa L.) genotypes Marjeta Eržen 26 6 LIST OF SHORT LECTURES l9 Irradiation and gene electrotransfer of plasmid DNA encoding chemokines CCL5 and CCL17 induce immunomodulatory effects in murine tumors Tim Božič 28 l10 HPV-positive mouse model of oral squamous cell carcinoma: development and transcriptome analysis Živa Modic 30 l11 Molecular and clinical characteristics of Slovenian patients with small cell carcinoma of the ovary, hypercalcemic type Ana Blatnik 31 l12 Isolate-specific differences in macrophage response to group B Streptococcus Larisa Janžič 33 7 LIST OF POSTERS p1 Engineered M13 filamentous bacteriophage as a novel antigen delivery system for vaccination in the treatment of malignant melanoma Nuša Brišar 36 p2 Identification of viruses in cv. malvasia ( Vitis vinifera L.) and effect of thermotherapy on their elimination and differential expression of genes involved in the RNA silencing pathway Hana Flajnik 38 p3 Virulence and resistance genotypes of ESBL-producing E. coli recovered from lower respiratory tract between 2018–2022) in relation to biofilm formation Katja Hrovat 40 p4 Development of qPCR and dPCR approach for detection of CRISPR/Cas9-induced mutations in cabbage protoplasts Žiga Javornik 42 p5 Identification and functional characterization of pathogenic genetic variants causing human monogenic conditions with rna-transcript analysis Matevž Jus 44 p6 Effective gene therapy with combination of IL-2 and IL-12 in two different murine tumour models Tilen Komel 46 p7 Whole transcriptome expression array analysis of human colon fibroblasts culture treated with Helichrysum italicum supports its use in traditional medicine Katja Kramberger 48 p8 Development of 3D spheroids from murine cell line K7M2 as models for osteosarcoma tumors Saša Kupčič 50 8 LIST OF POSTERS p9 Development of an artificial viroid inoculation method for hop tissue cultures Patricija Lap 52 p10 Gene upregulation in specific stages of CAR T cell-mediated cytotoxicity: a quantitative real-time PCR approach to enhance monitoring of cytotoxic efficacy and therapy progression Lucija Levstek 53 p11 Upregulation of cytosolic pattern recognition receptors after gene electrotransfer of plasmid encoding IL-12 Ajda Medved 55 p12 Digitalisation of biodiversity Gaja Ramić 57 p13 An amplitude-based multiplex ddPCR assay for diagnosis of hereditary α-tryptasemia Manca Svetina 58 p14 Irradiation results in molecular and transcriptional shifts of tumor endothelial cells Iva Šantek 60 p15 Bioinformatics analysis of RNA-seq data of colorectal cancer and liver metastases elucidates genes associated with metastasizing of colorectal cancer Kristian Urh 61 9 OPENING AND CLOSING LECTURES 10 HOW A BIASED GENETIC CODE CREATES ORDER FROM PROTEINS’ DISORDER Rupert Faraway1,2,#, Neve Costello Heaven1,2,*, Holly Digby1,2,*, Oscar G. ol Wilkins1,2,3, Anob M. Chakrabarti1,2,4, Ira A. Iosub1,2, Lea Knez1,7, Stefan L. Ameres5, Clemens Plaschka6, Jernej Ule1,2,3,8,# 1The Francis Crick Institute, London, UK 2UK Dementia Research Institute at King's College London, London, UK 3UCL, UCL Queen Square Institute of Neurology, Department of Neuromuscular Diseases, London, UK 4University College London, UCL Respiratory, Division of Medicine, London, UK 5University of Vienna, Max Perutz Labs, Vienna BioCenter, Vienna, Austria 6Research Institute of Molecular Pathology, Vienna BioCenter, Vienna, Austria 7Ludwig-Maximilians-Universität München, Munich, Germany 8National Institute of Chemistry, Ljubljana, Slovenia E-mail: rupert.faraway@gmail.com, jernej.ule@kcl.ac.uk; #corresponding authors; * These authors contributed equally to this work Protein dosage is regulated to maintain cellular homeostasis and health. The dosage of proteins containing disordered low complexity domains (LCDs) must be particularly well-controlled, yet no mechanism to maintain their mutual homeostasis has been identified. Here we report a mutual homeostatic mechanism that controls the concentration of such proteins, termed 'interstasis', in which proteins with similar LCDs co-regulate their combined dosage through mRNA-mediated negative feedback. We focused on the mechanism that exploits the fundamental multivalency of GA-rich RNA regions that encode charged LCDs, including those with arginine-enriched mixed charge domains (R-MCDs). Modest variations in the abundance of an R-MCD protein change the properties of nuclear speckles, a protein-RNA condensate, selectively trapping multivalent GA-rich mRNAs to promote their nuclear retention. This interstasis depends on the inherent characteristics of the genetic code, and on codon biases that are most pronounced in amniotes, which enhance the multivalency of GA-rich regions encoding the charged LCDs. The threshold of interstasis is modulated by CLK kinases, which affect the nuclear speckle localisation of proteins such as TRA2B, key binder of GA-rich RNAs. Notably, many classes of LCDs are encoded by RNA regions containing multivalency-enhancing codon biases, each preferentially bound by specific proteins, suggesting that interstasis might co-regulate many classes of functionally related LCD-containing proteins through dose-sensitivity of various types of protein-RNA condensates. 11 CODING AND NON-CODING RNA IN DORMANT CELLS Jernej Murn cl University of California, Department of Biochemistry, Riverside, California E-mail: murnj@ucr.edu Specific signals can interrupt relentless cell proliferation by inducing quiescence, a dormant cell state characterized by a reversible exit from cell cycle that can be sustained over long periods of time. Whereas quiescence is likely to have emerged early in evolution to facilitate survival of micro-organisms during periods of limitation, it has since then spread widely in phylogeny to suit specialized purposes in diverse biological contexts. Indeed, quiescence is known to play essential roles in tissue differentiation, resistance to stress, aging, and longevity of organisms, while its disruptions underlie cancer. However, understanding quiescence is limited by lack of efficient, broad-spectrum inducing conditions. In this lecture, I will discuss our recent finding that rapid depletion of two highly conserved ribozymes, RNases P and MRP, induces a dormant, quasi-quiescent state in commonly studied mammalian cells in culture. I will focus on altered processing of the most abundant coding and noncoding RNA species and how this may contribute to the induction and reversibility of cell dormancy. 12 ABSTRACTS OF SHORT LECTURES 13 GENETIC VARIATION IN POLYADENYLATION SIGNALS OF DIFFERENTIALLY EXPRESSED mRNA ISOFORMS IN MOUSE SELECTION LINES FOR FATNESS AND LEANNESS l1 Špela Mikec1, Zhihua Jiang2, Simon Horvat1, Tanja Kunej1 1University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Domžale, Slovenia 2Washington State University, Department of Animal Sciences, Pullman, WA, USA E-mail: spela.mikec@bf.uni-lj.si Alternative polyadenylation (APA) regulates genes by creating distinct 3'-ends of transcripts by selecting different polyadenylation (PA) sites along genes, thereby influencing transcription termination and poly(A) tail addition [1]. Using the Whole-Transcriptome Termini Site Sequencing (WTTS-seq) method [2], we determined PA sites in the hypothalamus of the Fat and Lean selection mouse lines, models for polygenic obesity and healthy leanness [3]. A total of 29,091 PA sites were expressed in both lines in the hypothalamus, a major brain region regulating energy balance. They were located within 13,837 genes (86.9 %), predominantly protein-coding (93.6 %). There were 472 differentially expressed PA (DE-PA) sites in the Fat compared to the Lean line, located within 357 genes (log2FC ≥ |1.5|, p < 0.05). Due to APA, adj 5,733 genes (41.4%) had multiple PA sites that could lead to mRNA isoforms. After additional filtering, we obtained 4,753 genes with more reliable PA sites producing mRNA isoforms. One hundred forty-eight of these genes had at least one DE-PA site, with 13 having multiple. Gene Ontology enrichment of 148 genes highlighted processes important for neuronal communication, including behavior, cognition, and synaptic signaling, suggesting altered brain activity in both lines. Additionally, these genes influence intracellular protein transport and organization, indicating potential changes in cellular structure and protein distribution in the Fat and Lean mice. Moreover, six genes with mRNA isoforms and DE-PA sites had SNPs within polyadenylation signals (PAS); and PAS-SNPs. Each line had three PAS-SNPs within three distinct genes: Nrsn2 (rs27369860), Ric3 (rs36754429), and Rpl14 (rs263963399) genes in the Fat line; Hlf (rs229072835), Taf1a (rs32656801), and Ints11 (rs227466545) genes in the Lean line, the latter aligning with our previous study [4]. PAS-SNPs altered the canonical AATAAA PAS motif, possibly resulting in decreased expression of these DE-PA sites, suggesting that PAS-SNPs might affect their expression. Future functional studies are needed to explore the role of PAS-SNPs and mRNA isoforms due to APA in polygenic obesity, extend the research to other metabolic tissues, and evaluate APA isoforms as novel potential therapeutic targets for obesity management. 14 References: 1. Tian B, Manley JL. Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol 2016;18(1):18–30. 2. Zhou X, Li R, Michal JJ, Wu XL, Liu Z, Zhao H, et al. Accurate profiling of gene expression and alternative polyadenylation with whole transcriptome termini site sequencing (WTTS-Seq). Genetics 2016;203(2):683–97. l1 3. Mikec Š, Horvat S, Wang H, Michal J, Kunej T, Jiang Z. Differential alternative polyadenylation response to high-fat diet between polygenic obese and healthy lean mice. Biochem Biophys Res Commun 2023;666:83–91. 4. Šimon M, Mikec Š, Morton NM, Atanur SS, Konc J, Horvat S, et al. Genome-wide screening for genetic variants in polyadenylation signal (PAS) sites in mouse selection lines for fatness and leanness. Mamm Genome 2023;34(1):12–31. 15 ARCHAEOGENETIC ANALYSIS OF LATE PLEISTOCENE AND HOLOCENE BISON FOSSILS FROM SLOVENIA AND HUNGARY Lars Zver1, Borut Toškan2, Elena Bužan3,4 l2 1University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia 2Research Centre of the Slovenian Academy of Sciences and Arts, Institute of Archaeology, Ljubljana, Slovenia 3University of Primorska, Faculty of Mathematics, Natural Sciences and Information Technologies, Department of Biodiversity, Koper, Slovenia 4Faculty of Environmental Protection, Velenje, Slovenia E-mail: lars_zver@yahoo.de During the last 130,000 years, two species of Bison were traditionally acknowledged to be present in Europe: the steppe bison ( Bison priscus) and the European bison or wisent ( Bison bonasus) [1]. The steppe bison appeared already during the Middle Pleistocene and occupied an area ranging from Western Europe to North America [1,2]. It became extinct in Europe at the end of the Late Pleistocene [3], while in some areas, it survived into the Early [4] or Middle Holocene [5]. The morphologically distinct osteological remains of the extant European bison first appear in the fossil record around the start of the Holocene [6]. Phylogenetic inferences based on mitochondrial DNA prove the existence of three clades in the last 50,000 years: Bp, which includes the steppe bison, and Bb1 and Bb2, which are related to the European bison [1,7-9]. Although several dozen Late Pleistocene and Holocene bison remains have been discovered in Slovenia and Hungary, genetic analysis has been mostly performed on (sub)fossils from Western Europe, Poland, the Caucasus, and Siberia. Therefore, our study aimed to analyse the ancient DNA (aDNA) from teeth/bones found in these two regions in order to assign the specimens to the existing clades. For this purpose, we extracted the DNA from 8 samples from the second half of the Late Pleistocene from Slovenia, and 2 Holocene samples from Hungary, morphologically attributed to Bison sp. , in a specialised aDNA laboratory. After using an amplicon-based approach with a maximum amplicon length of 198 base pairs, targeting the 12S, 16S, ND2, COI, ND4, and cytochrome b regions of the mitochondrial DNA, we sequenced the amplicons on the Ion Torrent S5 next-generation sequencing system. We processed the sequencing results using an in-house developed bioinformatics pipeline and constructed phylogenetic trees using the BEAST software. We successfully amplified and sequenced aDNA from the samples, followed by the assembly of 2,195 bp long concatenated consensus sequences of the mitochondrial DNA. Our results show that most of the Late Pleistocene Bison from the studied areas belong to the Bp clade, while the one close to the Pleistocene/Holocene transition and the Holocene specimens belong to the Bb2 clade. We did not detect the presence of the extinct Bb1 clade. Our research can, therefore contribute new insights into the paleogeography of 16 Late Pleistocene and Holocene bison in Central Europe. l2 Fig. 1: Phylogenetic tree, constructed with BEAST. The tree shows the relationship between the clades: Bb1 (green) and Bb2 (blue) are closely related to each other, and the Bp clade (red) represents the steppe bison. Our samples are marked with a purple square. References: 1. Massilani D, Guimaraes S, Brugal J-P, Bennett EA, Tokarska M, Arbogast R-M, et al. Past climate changes, population dynamics and the origin of Bison in Europe. BMC Biol 2016;14(1):93. 2. Markova AK, Puzachenko AY, van Kolfschoten T, Kosintsev PA, Kuznetsova T V., Tikhonov AN, et al. Changes in the Eurasian distribution of the musk ox (Ovibos moschatus) and the extinct bison (Bison priscus) during the last 50 ka BP. Quat Int 2015;378:99–110. 3. Kurtén B. Pleistocene mammals of Europe. AldineTransaction; 1968. 317 p. 4. Kirillova I V., Zanina OG, Kosintsev PA, Kul’kova MA, Lapteva EG, Trofimova SS, et al. The first finding of a frozen Holocene bison (Bison priscus Bojanus, 1827) carcass in Chukotka. Dokl Biol Sci 2013; 452:296-99. 5. Froese D, Stiller M, Heintzman PD, Reyes A V., Zazula GD, Soares AER, et al. Fossil and genomic evidence constrains the timing of bison arrival in North America. Proc Natl Acad Sci 2017;114(13):3457–62. 6. Verkaar ELC, Nijman IJ, Beeke M, Hanekamp E, Lenstra JA. Maternal and paternal lineages in cross-breeding bovine species. Has wisent a hybrid origin? Mol Biol Evol. 2004;21(7):1165–70. 7. Soubrier J, Gower G, Chen K, Richards SM, Llamas B, Mitchell KJ, et al. Early cave art and ancient DNA record the origin of European bison. Nat Commun 2016;7:1–7. 8. Grange T, Brugal JP, Flori L, Gautier M, Uzunidis A, Geigl EM. The evolution and population diversity of bison in Pleistocene and Holocene Eurasia: Sex matters. Diversity 2018;10(3):1–25. 9. Wang K, Lenstra JA, Liu L, Hu Q, Ma T, Qiu Q, et al. Incomplete lineage sorting rather than hybridization explains the inconsistent phylogeny of the wisent. Commun Biol 2018; 1:169. 17 IDENTIFYING INTROGRESSION BETWEEN DOMESTIC GOAT ( CAPRA HIRCUS) AND ALPINE IBEX ( C. IBEX): A CASE STUDY FROM SLOVENIAN PART OF ALPS l3 Neža Pogorevc1, Maulik Upadhyay1, Mojca Simčič2, Simon Horvat2, Ivica Medugorac1 1LMU Munich, Faculty of Veterinary Medicine, Population Genomics Group, Munich, Germany 2University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Ljubljana, Slovenia E-mail: neza.pogorevc@gen.vetmed.uni-muenchen.de Domestic goats and Alpine ibexes share a habitat on mountain pastures. The spatial overlap occasionally results in hybrid offspring, as reported in Italy, France, Switzerland, and Austria [1]. In addition, we encountered three hybrids in the Slovenian part of the Alps. Confirmation of their identity and survival potential raises the question about the actual extent of such interspecific hybridisations and the possibility of ancient or more recent introgression events since the hybrids seem to be fertile. To address this, we analysed whole genome sequences from more than 160 animals belonging to domestic goat breeds and wild goat species from Africa, Asia, and Europe. We performed clustering based on the identity by state distances [2] and examined the genomic structure of the populations [3]. Our analyses confirmed that the hybrids have ancestries from both populations, the Slovenian Drežnica goat breed and the Alpine ibex. Furthermore, D-statistics [4] were used to detect and quantify gene flow between the sampled populations. The D-statistic test considers a scheme with four taxonomic units: two sister taxa (P1 and P2), of which P2 is a candidate for gene flow with an external group (P3), and an outgroup (O) that is used to determine ancestral alleles. Significant values were found in comparisons between European ibex species (Alpine and Iberian) and domestic goats. The similarity of values among domestic goats with both ibex species suggested that introgression was ancient. This inference was further supported by identifying introgressed genome sequences using the program IBDmix [5]. Despite the discovered hybrids, we did not detect any signs of recent introgression in the existing Drežnica goat and Alpine ibex populations. As the latter could represent a threat to both endangered populations, our analysis is the first step towards developing genomic tools for monitoring and coexistence of both populations in Slovenia and other countries. 18 References: 1. Moroni B, Brambilla A, Rossi L, Meneguz PG, Bassano B, Tizzani P. Hybridization between Alpine Ibex and Domestic Goat in the Alps: A Sporadic and Localized Phenomenon? Animals 2022;12(6):751. 2. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MAR, Bender D, et al. PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses. Am J Hum Genet 2007;81(3):559–75. l3 3. Alexander DH, Novembre J, Lange K. Fast model-based estimation of ancestry in unrelated individuals. Genome Res 2009;19(9):1655–64. 4. Malinsky M, Matschiner M, Svardal H. Dsuite - Fast D-statistics and related admixture evidence from VCF files. Mol Ecol Resour 2021;21(2):584–95. 5. Chen L, Wolf AB, Fu W, Li L, Akey JM. Identifying and Interpreting Apparent Neanderthal Ancestry in African Individuals. Cell 2020 ;180(4). 19 HYBRIDIZATION AND POPULATION GENETIC STRUCTURE OF EUROPEAN WILDCATS FROM THE DINARIC ALPS AND PANNONIAN BASIN l4 Felicita Urzi1, Marco Sollitto1, Nikica Šprem2, Hubert Potočnik3, Duško Ćirović4, Elena Bužan1,5* 1Faculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, Koper, Slovenia 2Faculty of Agriculture, Department of Fisheries, Apiculture, Wildlife Management and Special Zoology, University of Zagreb, Zagreb, Croatia 3 Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia 4Faculty of Biology, University of Belgrade, Belgrade, Serbia 5Environmental Protection College, Velenje, Slovenia E-mail: Felicita.urzi@upr.si Fragmentation and habitat loss have contributed significantly to the demographic decline of European wildcat (Felis silvestris silvestris) populations, and hybridization with domestic cats (F. catus) poses a threat to the loss of genetic purity of the species. Here, we explore the genetic structure, demographic history, and population differentiation based on whole genome data of the endangered European wildcat in the intersection of the Dinaric Alps and the Pannonian Basin in Slovenia, Croatia, and Serbia, with the particular focus on historical and recent introgression between wild (sub)species and domestic cats. We whole-genome sequenced 32 wildcats. Twenty-two of the individuals included in the study were identified as pure wildcats (69%) and ten as hybrids (31%) between wildcats and domestic cats by microsatellite markers (Urzi et al. 2021*). We have included 17 samples downloaded from GenBank in the analysis. These samples belong to the following taxa: F. catus, F. s. silvestris and F. s. lybica. Using microsatellite markers, we found that wildcat populations were divided into two genetic clusters, largely consistent with a geographic division into a genetically diverse northern group and a genetically eroded south-eastern group. The WGS data also revealed a statistically significant? divergence of populations in two clusters, but the introgression pattern differs from the microsatellite analysis. In Serbia, we found that half of the Serbian "hybrids" (as indicated by microsatellite markers) resulted from introgression from Felis silvestris lybica. This may reflect a historical introgression that persists in the area and may result from a historical contact zone between F. s. lybica and F. s. silvestris in the region. We found population differentiation of European wildcats, including recent persecution-driven population divergence. The low level of domestic introgression 20 found in this study indicates a substantial level of “resistance” of this elusive species towards major anthropogenic impacts, such as the omnipresence of domestic cats as well as substantial habitat fragmentation. In Serbia, we likely found introgression that is a consequence of historic contact zone, but a more detailed study of selective pressures is needed. l4 21 THE CONJUGATION FREQUENCY OF THE pOX38:Cm CONJUGATIVE PLASMID IN IN VITRO MODELS OF HUMAN INTESTINE AND IN AN ANAEROBIC CHAMBER l5 Aleksander Lesar1,2, Jessica Verhoeven2, Sanne Verbrugen2, Tomaž Acetto1, Koen Venema2, Marjanca Starčič Erjavec1 1University of Ljubljana, Biotechnical Faculty, Department of Microbiology, Ljubljana, Slovenia, 2University of Maastricht, Campus Venlo, Department of Human Biology, Venlo, Netherlands E-mail: aleksander.lesar@gmail.com Bacterial antimicrobial resistance (AMR) has emerged as one of the leading public health threats of the 21st century [1]. AMR can occur by mutations or by acquisition of antibiotic resistance genes (ARGs) via horizontal gene transfer (HGT), the latter is considered to be the most important factor in the AMR spread. Of the three canonical mechanisms of HGT, conjugation is thought to have the greatest influence on the dissemination of ARGs [2]. The human gut microbiome is a known 'melting pot' for conjugation, with ARG transfer in this environment widely documented [3]. The aim of this study was to determine the conjugation frequency of the plasmid pOX38:Cm from the Escherichia coli N4i strain, the probiotic Nissle 1917 strain with gentamicin resistance, (=MSE259) [4] to the DL82, a uropathogenic E. coli, under different conditions in TNO intestinal models (TIM-1 and TIM-2) and in anaerobic atmosphere. The TIM-1 is a multi-compartment model of the upper gastrointestinal tract with four compartments mimicking conditions in the stomach and three parts of the small intestine, while the TIM-2 mimics the colon containing the human microbiota. The effect of MSE259 and DL82 on human microbiota present in TIM-2 was also studied by sequencing. In addition, similar conjugation experiments were performed in an anaerobic chamber. The obtained conjugation frequencies were compared with the those obtained in mating assays performed aerobically in LB liquid medium. In TIM-1 pOX38:Cm conjugation was successful, albeit the conjugation frequency was lower compared to the one obtained aerobically in LB liquid medium. From assays in TIM-2 conjugation frequency could not be calculated, as no transconjugants were detected, which could be due to lower conjugation frequency under anaerobic conditions, lower viability of the MSE259 or DL82 in TIM-2 and presence of other microbiota. Addition of MSE259 in TIM-2 provoked a change in the structure of microbiota: a significant increase of 5 taxa and a decrease of 10 taxa was observed. In order to exclude an effect of the peristaltic movement, which is simulated in the TNO intestinal models, mating assays with mixing of the mating mixture were performed. The mixing did not have any significant effect on conjugation frequency. Further, to exclude the effect of lower pH, mating assays in liquid LB with lower pH were performed. A decrease in conjugation frequency in such medium 22 was observed, but not as much as in the TIM-1 model. Mating assays performed in TIM-1 medium (a special solution with salts and no nutrients) aerobically in test tube, did not affect conjugation frequency. From mating assays performed in liquid LB in anaerobic chamber conjugation frequencies were calculated, a significant decrease in conjugation frequency was observed, when compared to the conjugation frequency l5 obtained aerobically in LB liquid medium. References: 1. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022;399(10325):629-655. 2. von Wintersdorff CJ, Penders J, van Niekerk JM, Mills ND, Majumder S, van Alphen LB, Savelkoul PH, Wolffs PF. Dissemination of Antimicrobial Resistance in Microbial Ecosystems through Horizontal Gene Transfer. Front Microbiol 2016;7:173. 3. McInnes RS, McCallum GE, Lamberte LE, van Schaik W. Horizontal transfer of antibiotic resistance genes in the human gut microbiome. Curr Opin Microbiol 2020;53:35-43. 4. Starčič Erjavec M, Petkovšek Ž, Kuznetsova MV, Maslennikova IL, Žgur-Bertok D. Strain ŽP - the first bacterial conjugation-based "kill"-"anti-kill" antimicrobial system. Plasmid 2015;82:28-34. 23 A SMALL BACTERIOPHAGE PROTEIN DETERMINES THE HIERARCHY OVER CO-RESIDENTIAL JUMBO PHAGE IN BACILLUS THURINGIENSIS SEROVAR ISRAELENSIS l6 Anja Pavlin University of Ljubljana, Biotechnical Faculty, Department of Biology, Ljubljana, Slovenia E-mail: anja.pavlin@bf.uni-lj.si Bacillus thuringiensis serovar israelensis is the most widely used biopesticide against insects, including vectors of animal and human diseases. Among several extrachromosomal elements, this endospore-forming entomopathogen harbors two bacteriophages: a linear DNA replicon named GIL01 that does not integrate into the chromosome during lysogeny and a circular-jumbo prophage known as pBtic235. Here, we show that GIL01 hinders the induction of cohabiting prophage pBtic235. The GIL01-encoded small protein, gp7, which interacts with the host LexA repressor, is a global transcription regulator and delays the induction of pBtic235 after DNA damage to allow GIL01 to selectively produce its own progeny. In a complex with host LexA in stressed cells, gp7 down-regulates the expression of more than 250 host and pBtic235 genes, many of which are involved in the cellular functions of genome maintenance, cell-wall transport, and membrane and protein stability. We show that gp7 homologs that are found exclusively in bacteriophages act in a similar fashion to enhance LexA’s binding to DNA, while likely also affecting host gene expression. Our results provide evidence that GIL01 influences both its host and its co-resident bacteriophages. 24 EPIGENETIC INSIGHTS INTO VIROID PATHOGENESIS FROM GENOME-WIDE DNA METHYLATION ANALYSIS OF CBCVd- INFECTED HOP PLANTS ( HUMULUS LUPULUS VAR. 'CELEIA') l7 Andrej Sečnik1, Nataša Štajner1, Sebastjan Radišek2, Urban Kunej1, Jernej Jakše1 1 University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Ljubljana, Slovenia 2Slovenian Institute of Hop Research and Brewing, Plant Protection Department, Žalec, Slovenia E-mail: andrej.secnik@bf.uni-lj.si Viroids are small, single-stranded RNA molecules, that exploit host factors for propagation, causing severe diseases in agricultural crops. Notably, recent outbreaks such as the Citrus bark cracking viroid (CBCVd) in hop plants in Slovenia, Germany, and Brazil, emphasize the urgency to study the underlying mechanisms underscoring viroid pathogenesis and host response to infection. Prevailing research delineates the intricate interplay between viroid RNA and host RNAi factors, orchestrating nuanced changes in gene expression, metabolic pathways, and the observable phenotype. Further investigation has discerned viroid-induced alterations in DNA methylation patterns via the RNA-directed DNA methylation pathway. Previous study has indicated consistent whole-genome DNA methylation levels between CBCVd-infected and healthy hop plants. Our current work, however, employs a comprehensive bisulfite sequencing approach, illuminating subtle yet significant shifts in specific parts of the DNA methylation landscape of the hop genome consequent to CBCVd infection. In addition, our study analysed important epigenetic marks of CBCVd-infected hop plants, providing novel evidence for host DNA methylation's role in defence against viroids. We found that genes linked to pathogen interaction pathways, such as MAPK signalling and LRR, exhibited hypomethylation, potentially enhancing host defence. Conversely, key RNA transcription genes like POL II, POL IV, and POL V displayed hypermethylation, highlighting DNA methylation's defensive significance. Additionally, our efforts were centred on establishing a vital connection between DNA methylation and pre-existing transcriptomic data, allowing us to gain a more comprehensive insight into plants' defence against viroids. Interestingly, the mediator subunit MED7a was upregulated and hypomethylated, suggesting impaired RNA transcription and emphasizing mediator complexes' role in viroid-infected plants. 25 GENOME-WIDE ASSOCIATION STUDY AND POPULATION STRUCTURE OF DIFFERENT HEMP ( CANNABIS SATIVA L.) GENOTYPES l8 Marjeta Eržen1, Andreja Čerenak1, Jernej Jakše2 1Slovenian Institute of Hop Research and Brewing, Žalec, Slovenia 2University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Ljubljana, Slovenia E-mail: marjeta.erzen@ihps.si Hemp ( Cannabis sativa L.) is one of the oldest cultivated plants in the world, with a multipurpose range of uses. It is a dioecious annual plant [1]. Due to wind pollination, heterozygous plants frequently occur within varieties, leading to appearance of different phenotypes, which can manifest at both the chemical and genetic levels. Based on visual traits, different phenotypes were identified within three different hemp varieties (Carmagnola selected, Tiborszallasi, and Finola selection). DNA of selected phenotypes was isolated and shotgun NGS libraries were designed using an in-house protocol. Libraries were hybridized with 4537 capture probes for target loci enrichment. After enrichment, samples were loaded on the Ion v3 chip and sequenced using the Ion Proton Sequencer. Two chips were used for 171 samples. Raw UBAM reads were imported into CLC Genomic Workbench 22. Reads were trimmed for low quality regions and mapped to the reference genome cs10. SNP calling was performed using the Genome Analysis Toolkit (GATK). Population structure analysis was conducted using ADMIXTURE 1.3.0 [2]. Cross-validation (CV) error was calculated for each group. Genome-wide association studies (GWAS) were conducted using Tassel 5 software [3]. Genotyping data were filtered, resulting in 3670 SNP positions out of 4537. These data were combined with three recorded phenotype traits (CBD, THC, and anthocyanin coloration of leaf petiole). Kinship analysis was performed to minimize false positives. GWAS analysis was done using MLM model, incorporating filtered and combined genotype and phenotype data, along with kinship matrix and PCA results. GWAS results were visualized using Manhattan plots with p-values in RStudio and the 'qqman' package [4]. False discovery rate (FDR) was determined using the 'qvalue' package at thresholds of 0.01, 0.001, or 0.0001. According to ADMIXTURE, genotype data were divided into 5 groups with a minimum CV value of 0.44940. The Carmagnola selected variety was homogeneous with no distinct groups, while Tiborszallasi and Finola selections were divided into two groups. GWAS revealed 14 significant SNPs above the FDR threshold associated with THC. Among them, two were on chromosome 1, one on chromosome 2, two on chromosome 4, one on chromosome 6, two on chromosome 7, four on chromosome 8, and one on chromosome 10, which was also the most significant SNP with a p-value of 0.000135 and an R² of 0.3474. For CBD, one significant SNP was associated with 26 chromosome 10. The p-value was 0.0000795, the marker's effect on the trait was 0.1479, and R² was 0.04245. Anthocyanin coloration of leaf petiole was associated with three statistically significant SNPs, all on chromosome 8. The most significant SNP had a p-value of 0.000504, a marker effect of 0.05, and an R² of 0.35167. Based on significant SNPs, genes located at defined positions were determined for each l8 phenotypic trait. The GWAS analysis results serves as a valuable tool for understanding the mechanisms and functions of genes correlated with specific hemp traits. References: 1. Hillig KW. Genetic evidence for speciation in Cannabis (Cannabaceae). Genet Resour Crop Evol 2005: 52:161–80. 2. ADMIXTURE 1.3.0. 2023. https://dalexander.github.io/admixture (accessed May 10, 2023). 3. Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES. TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics 2007: 2633–35. 4. Turner S. qqman: an R package for visualizing GWAS results using Q-Q and manhattan plots 2018; https://doi.org/10.1101/005165. 27 IRRADIATION AND GENE ELECTROTRANSFER OF PLASMID DNA ENCODING CHEMOKINES CCL5 AND CCL17 INDUCE IMMUNOMODULATORY EFFECTS IN MURINE TUMORS l9 Tim Božič1, Iva Šantek1,2, Simona Kranjc Brezar1,2, Gregor Serša1,3, Maja Čemažar1,4, Boštjan Markelc1 1Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia 2University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 3 University of Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia 4 University of Primorska, Faculty of Health Sciences, Izola, Slovenia E-mail: tbozic@onko-i.si Chemokines regulate immune cell migration. The degree and the type of immune cells in the tumor affects disease progression and correlates with the efficacy and outcome of immunotherapies. Similarly, beneficial immunomodulatory effects were also observed after irradiation. Therefore, we sought to investigate gene electrotransfer (GET) of proinflammatory chemokines CCL5 or CCL17 in combination with irradiation, as a potential therapeutic strategy for cancer therapy. Tumor models were chosen to correspond to an inflamed (CT26 murine colon cancer) or immunosuppressive (4T1 murine breast cancer) immunophenotype. First, chemotactic properties of investigated chemokines were examined in vitro. Next, the potential of chemokines to induce the extravasation of fluorescently labelled splenocytes was determined using intravital microscopy of tumors in dorsal window chamber model (DWC). The antitumor effectiveness of combined therapy utilizing GET of chemokines and two irradiation regimes (single dose of 10 Gy and fractionated dose of 3× 5 Gy) was then determined in vivo. Lastly, qRT-PCR was used to evaluate gene expression of several cytokines in tumors after the therapies, while changes in the abundance of CD4+, CD8+ cells and vasculature (CD31+ cells) were determined with immunofluorescent staining. Both chemokines CCL5 and CCL17 induced the migration of murine macrophages RAW264.7 in vitro. Similarly, in both CT26 and 4T1 tumors growing in DWC, GET of chemokines showed increased retention of splenocytes compared to control. CT26 tumor growth delay after combined therapy of GET of chemokines and both irradiation regimes was significantly longer compared to control and led to tumor cures. In the case of 4T1 tumors, only GET of chemokines combined with irradiation led to a pronounced tumor growth delay but without tumor cures. Gene expression analysis showed increased expression of both chemokines after corresponding therapies. Moreover, increased expression of CXCL9 and CXCL10, two potent chemoattractants of cytotoxic CD8+ T lymphocytes, was determined in tumors after 28 most of the combined therapies. Immunofluorescence showed increased numbers of CD4+ and CD8+ T lymphocytes in tumors after GET of chemokines, however their numbers decreased whenever irradiation was used. Our results show that combined therapy elicits an antitumor immune response in inflamed tumors CT26 and to some extent in immunosuppressive tumors 4T1, indicating the potential of chemokines in l9 cancer immunotherapy. 29 HPV-POSITIVE MOUSE MODEL OF ORAL SQUAMOUS CELL CARCINOMA: DEVELOPMENT AND TRANSCRIPTOME ANALYSIS Živa Modic1,2, Maja Čemažar1,3, Boštjan Markelc1, Simona Kranjc Brezar1,2, l10 Gregor Serša1,4, Tanja Jesenko1,2 1Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia 2University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 3University of Primorska, Faculty of Health Sciences, Izola, Slovenia 4University of Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia E-mail: zmodic@onko-i.si Head and neck cancers are a heterogeneous group of tumors that includes oral squamous cell carcinoma (OSCC). One of the risk factors for the development of some anatomical subtypes of OSCC is persistent infection with high-risk human papillomavirus (HPV) strains, such as HPV-16. Clinical data indicate, that some patients with HPV-positive OSCC have a better prognosis and respond better to various treatment modalities, including radiotherapy and immunotherapy. To evaluate the differences between HPV positive and HPV negative OSCC and therapies based on adaptive immune response, immunocompetent mouse models are essential. Nevertheless, HPV-positive mouse model of OSCC are still scarce due to the species specificity of HPV. Therefore, the aim of our study was to establish and characterize a mouse model of HPV-positive OSCC. We established two monoclonal MOC1-HPV cell lines (MOC1-HPV K1 and MOC1-HPV K3) by transduction of murine OSCC cell line MOC1 with LXSN16E6E7 retrovirus, which encodes the HPV-16 E6 and E7 open reading frames. We confirmed a stable expression of HPV-16 E6 and E7 in both monoclonal MOC1-HPV cell lines on mRNA and protein level with quantitative PCR and immunofluorescent staining, respectively. In vitro characterization demonstrated differences in cell morphology and cell migration capacity, where MOC1-HPV K3 cells migrated significantly slower in the wound healing assay compared to the parental or MOC1-HPV K1 cell line. In vivo, we characterized the tumor microenvironment (TME) of the newly established tumor models by immunofluorescent staining of blood vessels (CD31), hypoxic areas (EF5), and proliferation (EdU). MOC1-HPV K1 tumors were less hypoxic, with a higher proportion of proliferating cells compared to MOC1 and MOC1-HPV K3 tumors. Both in vitro and in vivo results correlate with transcriptome analysis results of the three cell lines, where the 20 most enriched gene ontology “biological process” terms in MOC1-HPV K1 cell line include terms related to angiogenesis and blood vessel formation, as well as cell migration and motility. In conclusion, we established a mouse model of HPV-positive OSCC that can be used to study basic characteristics as well as tumor microenvironment and immune responses to different therapies of HPV-positive OSCC. 30 MOLECULAR AND CLINICAL CHARACTERISTICS OF SLOVENIAN PATIENTS WITH SMALL CELL CARCINOMA OF THE OVARY, HYPERCALCEMIC TYPE l11 Ana Blatnik1, Vita Šetrajčič Dragoš2, Olga Blatnik3, Vida Stegel2, Gašper Klančar2, Srdjan Novaković2, Primož Drev3, Tina Žagar4, Ksenija Strojnik5, Sebastjan Merlo6, Erik Škof7, Mirjana Pavlova Bojadžiski7 1Institute of Oncology Ljubljana, Department of Clinical Cancer Genetics, Ljubljana, Slovenia 2Institute of Oncology Ljubljana, Department of Molecular Diagnostics, Ljubljana, Slovenia 3Institute of Oncology Ljubljana, Department of Pathology, Ljubljana, Slovenia 4Institute of Oncology Ljubljana, Epidemiology and Cancer Registry Sector, Ljubljana, Slovenia 5Institute of Oncology Ljubljana, Department of Clinical Cancer Genetics, Ljubljana, Slovenia 6Institute of Oncology Ljubljana, Division of Surgery, Ljubljana, Slovenia 7Institute of Oncology Ljubljana, Division of Medical Oncology, Ljubljana, Slovenia E-mail: ablatnik@onko-i.si Description Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is rare and usually affects young women with median age at diagnosis of 241. It has a poor prognosis with long-term survival of 30% in early-stage disease [1]. Germline and somatic variants in SMARCA4 gene, which encodes a catalytic subunit of the SWI/ SNF chromatin-remodelling complex, are associated with SCCOHT. Penetrance of SMARCA4 germline pathogenic variants (PV) and incidence of SCCOHT is currently unknown. The aim of our study was to identify all cases of SCCOHT in Slovenia from 1991 to 2021 and present their genetic testing results, histopathological and clinical characteristics. We also aimed to estimate the incidence of SCCOHT and assess the penetrance of germline SMARCA4 PVs in Slovenian families. First, we analysed medical records and data from the Slovenian Cancer Registry. In patients suspected of having SCCOHT, histopathological review with immunohistochemical staining for SMARCA4/BRG1 was undertaken to confirm the diagnosis. In cases of confirmed SCCOHT, both germline and somatic genetic analyses using next-generation sequencing were performed. We identified 7 patients who developed SCCOHT aged 21-41 in a population of two million during a 30-year period and estimate the minimal incidence of SCCOHT to be 0.12/million/year [2]. Two cases were sporadic, with presumably biallelic SMARCA4 variants in their tumours not seen on germline testing. In one patient, a SMARCA4 PV and loss-of heterozygosity was identified in tumours tissue, but germline testing was unfeasible. In four cases from two different families, two novel germline loss-of-function variants in SMARCA4, c.1423_1429delTACCTCA p.(Tyr475Ilefs*24) and c.3216-1G>T were identified. Based on pedigree analysis, 31 they appear to be associated with relatively high penetrance. In all tested tumours, tumour mutational burden was low. Five tumours consisted of small cells with scant cytoplasm. In two cases, there was a predominance of large cells with eosinophilic cytoplasm, i.e. large cell variant of SCCOHT. Patients included in our study were diagnosed with stage FIGO IA-III disease and died due to SCCOHT within 36 months. l11 In one case, temporary disease stagnation was achieved using immunotherapy. In conclusion, our study presents all known cases of SCCOHT diagnosed in Slovenia between 1991 and 2021. We offer a first estimate of SCCOHT incidence in the non-paediatric population, but expect studies in larger populations will lead to more accurate assessments. References: 1. Tischkowitz M, Huang S, Banerjee S, Hague J, Hendricks WPD, Huntsman DG, et al. Small-cell carcinoma of the ovary, hypercalcemic type–genetics, new treatment targets, and current management guidelines. Clin Cancer Res 2020;26(15):3908–17. 2. Blatnik A, Dragoš VŠ, Blatnik O, Stegel V, Klančar G, Novaković S, et al. A population-based study of patients with small cell carcinoma of the ovary, hypercalcemic type, encompassing a 30-year period. Arch Pathol Lab Med 2023; doi:10.5858/arpa.2022-0297-oa. 32 ISOLATE-SPECIFIC DIFFERENCES IN MACROPHAGE RESPONSE TO GROUP B STREPTOCOCCUS Larisa Janžič, Lucija Levstek, Sara Petrin, Alojz Ihan, Andreja Nataša Kopitar l12 University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, Department of Cell Immunology, Ljubljana, Slovenia E-mail: larisa.janzic@mf.uni-lj.si Although Streptococcus agalactiae (Group B Streptococcus, GBS) is primarily an opportunistic pathogen that colonizes the gastrointestinal and genitourinary tracts of 15-30 % of healthy individuals, it remains a major cause of invasive disease in neonates [1]. The latter can occur in the first week of life and usually manifest as sepsis or pneumonia (i.e., early-onset-disease), or at 1 week to 3 months of age, which usually manifests as meningitis (i.e., late onset disease) [2]. To date, 10 different serotypes (Ia, Ib, II – IX) have been described, distinguished by their virulence and the immune response they elicit [3]. Since intrapartum antibiotic prophylaxis can only prevent early-onset, but not late-onset disease and infections in the elderly and immunocompromised individuals, further studies are needed to investigate GBS-associated pathogenesis. Because adaptive immunity is underdeveloped in neonates, cells of innate immunity play a critical role in protection against pathogens [4]. Of the latter, macrophages are particularly important as they are involved in pathogen recognition, phagocytosis, and elimination [5]. Because disease severity depends on both the GBS isolate and the immune status of the individual, we investigated possible isolate-specific differences in the immune response of THP-1 macrophages to 12 different previously genotyped GBS isolates [6]. Significant isolate-specific differences were observed in phagocytic uptake and expression of macrophage polarization markers [7]. Furthermore, by measuring inflammatory and anti-inflammatory cytokines and chemokines at the protein level, as well as expression of genes involved in antimicrobial activity and inflammation at different time points, we observed that different isolates have different potential to become invasive or remain colonizing. In addition, by measuring IL-1B, IL-18, and caspases 1 and 3 at the mRNA and protein levels, LDH secretion, and caspase-1 activity, we demonstrated that some isolates were significantly more cytotoxic to macrophages and induced pyroptosis. By measuring glycolysis and oxidative phosphorylation using Seahorse extracellular flux analyzer and analyzing the expression of glycolytic genes, we have shown that different GBS isolates activate macrophage metabolism differently and that differences in metabolism lead to differences in macrophage effector functions [7]. 33 References: 1. Armistead B, Oler E, Adams Waldorf K, Rajagopal L. The Double Life of Group B Streptococcus: Asymptomatic Colonizer and Potent Pathogen. J Mol Biol 2019;431(16):2914-31. 2. Korir ML, Manning SD, Davies HD. Intrinsic Maturational Neonatal Immune Deficiencies and Susceptibility to Group B Streptococcus Infection. Clin Microbiol Rev 2017;30(4):973-89. l12 3. Pietrocola G, Arciola CR, Rindi S, Montanaro L, Speziale P. Streptococcus agalactiae Non-Pilus, Cell Wall-Anchored Proteins: Involvement in Colonization and Pathogenesis and Potential as Vaccine Candidates. Front Immunol 2018;9:602. 4. Kumar SK, Bhat BV. Distinct mechanisms of the newborn innate immunity. Immunol Lett 2016;173:42-54. 5. Zhang L, Wang CC. Inflammatory response of macrophages in infection. Hepatobiliary Pancreat Dis Int 2014;13(2):138-52. 6. Perme T, Golparian D, Bombek Ihan M, Rojnik A, Lucovnik M, Kornhauser Cerar L, et al. Genomic and phenotypic characterisation of invasive neonatal and colonising group B Streptococcus isolates from Slovenia, 2001-2018. BMC Infect Dis. 2020;20(1):958. 7. Janzic L, Repas J, Pavlin M, Zemljic-Jokhadar S, Ihan A, Kopitar AN. Macrophage polarization during Streptococcus agalactiae infection is isolate specific. Front Microbiol 2023;14:1186087. 34 ABSTRACTS OF POSTERS 35 ENGINEERED M13 FILAMENTOUS BACTERIOPHAGE AS A NOVEL ANTIGEN DELIVERY SYSTEM FOR VACCINATION IN THE TREATMENT OF MALIGNANT MELANOMA Nuša Brišar1,2, Katja Šuster3, Simona Kranjc Brezar2,4, Andrej Cör3,2 1University of Primorska, Faculty of Health Sciences, Izola, Slovenia 2University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia p1 3Valdoltra Orthopaedic Hospital, Ankaran, Slovenia 4Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia E-mail: nusa.brisar@fvz.upr.si Bacteriophages are prokaryotic viruses that are the most abundant organisms on Earth and can be found in virtually every environment, including the human body [1]. With many intriguing characteristics, bacteriophages have become a favorable tool in bioengineering. In the field of cancer immunotherapy, bacteriophages have been exploited to create powerful vaccine platforms designed to overcome the tolerogenic tumour microenvironment while inducing specific immune responses [2,3]. Our study aimed to create a novel vaccine for malignant melanoma treatment by constructing non-lytic M13 filamentous bacteriophages expressing MAGE-A1 161-169 tumour peptides as fusion proteins with pIIII minor and pVIII major coat proteins. Using the phage display technology, recombinant bacteriophages were released from Escherichia coli, after transformation with recombinant phagemids pComb3XSS-MAGE-A1 or pComb8-MAGE-A1 followed by super-infection with VCSM13 helper bacteriophages. Phage-displayed tumour peptides were confirmed by proteomic analysis. C57BL/6 mice were vaccinated intraperitoneal, receiving 1012 pfu of genetically engineered bacteriophages per mouse three times at two-week intervals, with PBS and wild-type M13 bacteriophage used as controls. One week after each vaccination, sera were collected and analysed by ELISA. One week following the second vaccine boost, splenocytes were harvested to assess T-cell cytotoxicity by co-culture assay. Phage-displayed tumour peptides were confirmed by nano-LC-MS/MS (protein pVIII-MAGE-A1) and Western blot (protein pIII-MAGE-A1). We have shown that administration of the M13 filamentous phage vaccine to mice is safe, with no evidence of side effects. Despite variations in display valencies, both pIII and pVIII display techniques effectively stimulated humoral responses, leading to increasing anti-phage and anti-MAGE-A1 antibodies which exhibited an increase from the first to the third phage dose. Moreover, the elicited anti-MAGE-A1 antibodies demonstrated the capacity to bind to naturally expressed epitopes on B16F10 tumor cells in vitro. Splenocytes from recombinant bacteriophage-vaccinated mice also demonstrated enhanced induction of CD8+ T cell cytotoxicity against B16F10 cells compared to control groups. 36 To summarize, vaccination with engineered M13 filamentous bacteriophages displaying the MAGE-A1 tumor peptide is safe and triggers adaptive immune responses. We believe that an M13 filamentous bacteriophage-based vaccine holds significant promise for the treatment of malignant melanoma. References: 1. Hess KL, Jewell CM. Phage display as a tool for vaccine and immunotherapy development. Bioeng Transl Med 2020;5(1). 2. Dong X, Pan P, Ye JJ, Zhang QL, Zhang XZ. Hybrid M13 bacteriophage-based vaccine platform for personalized cancer immunotherapy. Biomaterials 2022;289:121763. p1 3. Chang C, Guo W, Yu X, Guo C, Zhou N, Guo X, etc. Engineered M13 phage as a novel therapeutic bionanomaterial for clinical applications: From tissue regeneration to cancer therapy. Mater Today Bio 2023;20:100612. 37 IDENTIFICATION OF VIRUSES IN CV. MALVASIA ( VITIS VINIFERA L.) AND EFFECT OF THERMOTHERAPY ON THEIR ELIMINATION AND DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN THE RNA SILENCING PATHWAY Hana Flajnik1, Vanja Miljanić1, Andreja Škvarč2, Nataša Štajner1 1University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Ljubljana, Slovenia p2 2Chamber of Agriculture and Forestry of Slovenia, Agriculture and Forestry Institute Nova Gorica, Nova Gorica, Slovenia E-mail: hana.flajnik@gmail.com Grapevine ( Vitis vinifera L.) is one of the most widely used fruit crops and it is important for its economic properties. However, it can harbour more than 86 viruses and viroids [1], which cause yield reduction, alterations in chemical and sensorial quality of grape and wine, developmental and morphological malformations in vine organs, and affect the length of vine life [2]. In Slovenia, according to the Official Gazette of the RS N°93/05 and 101/20 all propagated vine material must undergo mandatory testing on viruses ArMV, GFLV, RpRSV, TBRV, GVA, GVB, GRSPaV, GLRaV-1/-3 and GFkV (only for rootstocks). Testing is recommended on GLRaV-2 and GLRaV-4–9, as well as for emerging virus GPGV. In our study, we tested 17 vines of cv. Malvasia on 14 viruses with obligatory and recommended testing with RT-PCR and RT-qPCR. Six viruses were identified (GRSPaV, GFLV, GFkV, GLRaV-2, GLRaV-3, GPGV). The most prevalent viruses were GRSPaV and GFLV, followed by GPGV. In addition, due to its symptomatology and the physiological characteristics of the tested Malvasia variety, we hypothesised that grapevine asteroid mosaic-associated virus (GAMaV), which causes peripheral vesiculation of the chloroplasts, could also be associated with a star-like structure at the distal end berry rupture point, but it was only detected in two vines using RT-PCR and in five vines using RT-qPCR. In the second part of our study, we conducted thermotherapy in combination with shoot tip culture in order to study the elimination efficiency of detected viruses. Thermotherapy is often used as a successful sanitation method for obtaining virus-free planting material due to inhibition of virus replication or virus RNA degradation [3]. It is also associated with an antiviral immune defense mechanism, RNA silencing [4,5]. A 100 % elimination rate was observed for viruses GFLV, GFkV, and GLRaV-2, the hardest to eliminate was GRSPaV, for which successful elimination was obtained only for one vine. In the third part of our study, we monitored the effect of high temperature on the differential expression of Dicer-like, Argonaute and RNA-dependent RNA-polymerase genes in grapevine, which could contribute to an improved response to viral infection. Differences in expression were observed with RT-qPCR for all genes considered (DCL1/2/3, AGO1/2a/4, and 38 RDR2), with significant differences for all genes except DCL3. The biggest expression differences between treated and untreated samples with high temperature were observed for the genes DCL1 and DCL2. We suggest further studies in the field of RNA silencing in grapevine, as our results indicate significant changes in the expression profiles of core RNAi proteins. References: 1. Fuchs M. Grapevine viruses: a multitude of diverse species with simple but overall poorly adopted management solutions in the vineyard. J Plant Pathol 2020;102:643–53. 2. Miljanić V, Rusjan D, Škvarč A, Chatelet P, Štajner N. elimination of eight viruses and two viroids p2 from preclonal candidates of six grapevine varieties ( Vitis vinifera L.) through in vivo thermotherapy and in vitro meristem tip micrografting. Plants 2022;11:1064. 3. Cooper VC, Walkey DGA. Thermal inactivation of cherry leaf roll virus in tissue cultures of Nicotiana rustica raised from seeds and meristem-tips. Ann Appl Biol 1978;88:273–78. 4. Liu J, Zhang X, Yang Y, Hong N, Wang G, Wang A, et al. Characterization of virus-derived small interfering RNAs in Apple stem grooving virus-infected in vitro-cultured Pyrus pyrifolia shoot tips in response to high temperature treatment. Virol J 2016;13:166. 5. Kim Y, Kim YJ, Paek K-H. Temperature-specific vsiRNA confers RNAi-mediated viral resistance at elevated temperature in Capsicum annuum. J Exp Bot 2021;72:1432–48. 39 VIRULENCE AND RESISTANCE GENOTYPES OF ESBL-PRODUCING E. COLI RECOVERED FROM LOWER RESPIRATORY TRACT BETWEEN 2018–2022) IN RELATION TO BIOFILM FORMATION Katja Hrovat1, Jerneja Čremožnik Zupančič1, Katja Seme2, Jerneja Ambrožič Avguštin1 1University of Ljubljana, Biotechnical Faculty, Department of Biology, Ljubljana, Slovenia p3 2University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, Ljubljana, Slovenia E-mail: katja.hrovat@bf.uni-lj.si Extraintestinal pathogenic Escherichia coli (ExPEC) can cause a variety of different infections for which treatment options are limited, primarily due to the increasing prevalence and spectrum of antimicrobial resistance. Of particular concern are extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC), especially the pandemic E. coli sequence type 131 clone (ST131). This study aims to investigate the biofilm formation ability and its correlation with genotypes of clinical ESBL-EC isolates from lower respiratory tract (LRT) samples. A total of 116 ESBL-EC, isolated from LRT, were assigned to phylogroups, sequence type, and clonal groups. Isolates were also screened for virulence-associated (VAGs) and antimicrobial resistance genes, as well as for class 1, 2, and 3 integrons. Furthermore, we examined the biofilm formation ability by culturing the bacterial isolates for 24 and 48 hours in a 96-well Calgary Biofilm Device (CBD) in two different growth media. The biofilms formed were stained with 1% w/v crystal violet and spectrophotometrically quantified. The resistance profile of the 116 ESBL-EC isolates showed a high prevalence of group 1 bla (75.9%) and a dominance of phylogenetic group B2 (59.5%), which was also CTX-M significantly associated with group ST131. After 24 hours of incubation, strong biofilm formation was observed in 18.1% and 51.7% of ESBL-EC isolates in LB and MG with 0.02% glucose, respectively. Spearman’s rho correlation matrix showed a statistically significant positive correlation between strong biofilm formation in the MG medium after 24 hours of incubation and sequence type 131, phylogenetic group B2, bla group 9 genes, the biocide resistance gene emrE and the VAGs afa/dra, fluA, CTX-M fyuA, iha, iroN, irp2, kpsMTII, sat, usp. When isolates were grown in the nutrient-rich Luria–Bertani (LB) media, we found statistically significant positive correlations with phylogenetic group C and with virulence genes papC and papGII. Our results suggest that ST131 ESBL-producing Escherichia coli, a multidrug-resistant human extraintestinal clonal group, has the ability to form biofilms, especially under 40 nutrient deprived conditions, and that biofilm production is associated with certain virulence factors. Our results suggest a possible reason for the successful global spread of ST131, particularly in clinical settings. References: 1. Ebrahimi MT, Hedayati MA, Pirlar RF, Mortazavi N, Nazari M, Ahmadi A, et al. Investigation of the biofilm formation in extra-intestinal pathogenic Escherichia coli ST131 strains and its correlation with the presence of fimH, afa, and kpsMSTII genes. J Appl Genet 2023;64(2):367-73. 2. Sarkar S, Vagenas D, Schembri MA, Totsika M. Biofilm formation by multidrug resistant Escherichia coli ST131 is dependent on type 1 fimbriae and assay conditions. Pathog Dis 2016;74(3):ftw013. p3 41 DEVELOPMENT OF qPCR AND dPCR APPROACH FOR DETECTION OF CRISPR/Cas9-INDUCED MUTATIONS IN CABBAGE PROTOPLASTS Žiga Javornik, Nataša Štajner, Ester Stajič University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Ljubljana, Slovenia p4 E-mail: zj0901@student.uni-lj.si Targeted genome editing with CRISPR/Cas9 is nowadays the most common method for creating new plant varieties because it is efficient, simple, and faster than conventional breeding. The invention of new methods for rapid and sensitive detection of mutations in genome-edited plants is crucial to shorten the time to obtain modified plants. At present, there are several methods for detecting mutations after CRISPR/Cas9 genome editing, such as qPCR, Sanger sequencing, HRMA, T7 endonuclease, etc., but they are less suitable for detecting mutations in polyploid plants or when we have low editing efficiency. On the other hand, next-generation sequencing (NGS) has high sensitivity but is cost-efficient when larger number of samples are analyzed. Therefore, we decided to develop a new, fast, and efficient method using qPCR and dPCR to detect mutations after genome editing using CRISPR/Cas9. For our samples, we used transformed protoplasts of three distinct cabbage cultivars (Huzaro F1, Reball F1, Rebecca F1), all of which had different mutation ratios in the CENH3 gene revealed by NGS. In our experiments, we used a TaqMan duplexed assay with a non-mutated reference gene ( PDS) which allowed us to quantify mutations in our samples. However, before the dPCR method could be used to determine the percentage of mutations in edited samples, it had to be optimized. In the first part of the experiment, designed probes for C ENH3 and PDS genes were tested using qPCR. The same method was also used to determine primer concentrations to achieve optimal amplification of both genes in the duplex reaction. Since we did not obtain optimal amplification of both genes, new primers for PDS gene were designed which gave us shorter amplicons and required amplification efficiency. After the optimization, the assay was used in dPCR to check whether this method is suitable for detection of mutations. We were able to detect the mutated samples and distinguish them from the wild-type samples. The percentage of mutations was calculated by dividing positive partitions of CENH3 with positive partitions of PDS gene. The range of mutations in our samples were between 2.8% - 26.4%. The same samples were also analyzed by qPCR, where in general the percentage of mutations was higher (0.65% - 46.9%). Detection of mutations with qPCR is faster, requires fewer reagents and a lower amount of initial DNA. We also compared the results of dPCR and qPCR with results of NGS method and concluded 42 that they are comparable. Reference: 1. Peng C, Zheng M, Ding L, Chen X, Wang X, Feng X, et al. Accurate Detection and Evaluation of the Gene-Editing Frequency in Plants Using Droplet Digital PCR. Front Plant Sci 2020; 11:610790. p4 43 IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF PATHOGENIC GENETIC VARIANTS CAUSING HUMAN MONOGENIC CONDITIONS WITH RNA-TRANSCRIPT ANALYSIS Matevž Jus1, Alenka Hodžoć2, Aleš Maver2, Borut Peterlin2 1University Medical Centre Maribor, Clinical Institute of Genetic Diagnostics, Maribor, Slovenia 2University Medical Centre Ljubljana, Clinical Institute of Genomic Medicine, Ljubljana, p5 Slovenia E-mail: matevz.jus@ukc-mb.si Next-generation sequencing represents an important step in diagnostics of monogenic conditions, as it allows the analysis of the majority of the human genome, including non-coding regions. However, characterization of the pathogenicity of variants identified by these approaches and their functional assessment remain a challenge. The aim of this research was analysis of cases with undiagnosed genetic conditions that harbor genetic variants of uncertain clinical significance, and cases in which conventional genetic approaches identified no causal variants. We sought to establish that by analyzing RNA transcripts, we can improve the diagnosis of pathological variants situated in non-coding regions, thereby augmenting the insights obtained from exome and genomic sequencing approaches. Through targeted sequencing of gene transcripts, we clarified both the pathological variants present, as well as the underlying mechanisms that led to their appearance and resulted in the clinical picture. Based on the functional data, we reassessed the pathogenicity of variants according to ACMG and ACGS standards and we found that the clinical classification of pathogenicity has improved in every case. In four cases we confirmed exon skipping, in three cases a frameshift mutation, and one case of each intron retention and novel splice site introduction. The findings demonstrate a noteworthy enhancement in sensitivity through RNA transcript analysis methods. Focusing on specific regions and increasing sequencing depth offers advantages in terms of data volume and investigation accessibility. In accordance with the 2022 study by Wai et al. [1], which focused on the advantages of targeted sequencing of (shorter) RNA-transcripts over classical RNA-seq, we also confirmed the higher accuracy of targeted sequencing compared to whole-genome sequencing, considering that we clarified pathogenicity in most cases. In terms of economics and efficiency, this approach proves more cost-effective than whole genome sequencing. It's a crucial aspect in clinical settings, as it's user-friendly and doesn't necessitate high-performance sequencers. These methods hold significant applicability. They allow for detecting residual isoform expression that might otherwise degrade in the nonsense-mediated decay process. Furthermore, they enable identification of a broad spectrum of exon splicing mechanisms, a feat unattainable with previous-generation methods. In contrast to 44 concerns raised by de Klerk and 't Hoen in 2015 [2] about the limitations of Illumina technology based on short read length mapping, our study yielded precise and consistent data with high coverage. In the case of an unexplained clinical picture, we are only left with the sequencing of wider regions for which we assume the presence of pathological variants, which prolongs the diagnostic process, but at the same time does not allow us to clarify the genopathy in the subject. References: 1. Wai HA, Constable M, Drewes C, Davies IC, Svobodova E, Dempsey E, et al. Short amplicon reverse transcription-polymerase chain reaction detects aberrant splicing in genes with low expression p5 in blood missed by ribonucleic acid sequencing analysis for clinical diagnosis. Hum Mutat 2022;43(7):963–70. 2. de Klerk E, ’t Hoen PAC. Alternative mRNA transcription, processing, and translation: insights from RNA sequencing. Trends Genet. 2015;31(3):128–39. 45 EFFECTIVE GENE THERAPY WITH COMBINATION OF IL-2 AND IL-12 IN TWO DIFFERENT MURINE TUMOUR MODELS Tilen Komel1, Maša Omerzel1, Urška Kamenšek1, Katarina Žnidar1, Urša Lampreht Tratar1, Simona Kranjc Brezar1, Klemen Dolinar2, Sergej Pirkmajer2, Emanuela Signori3, Mariangela De Robertis4, Gregor Serša1,5, Maja Čemažar1,6 1Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia p6 2Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3National Research Council-Institute of Translational Pharmacology, Rome, Italy 4University of Bari, Department of Biosciences, Biotechnology and Biopharmaceutics, Bari, Italy 5University of Ljubljana, Faculty of Health sciences, Ljubljana, Slovenia 6University of Primorska, Faculty of Health sciences, Izola, Slovenia E-mail: tkomel@onko-i.si Immunotherapy has become an important approach in the treatment of cancer. One example is the use of cytokines such as interleukin 2 (IL-2) and 12 (IL-12), which stimulate immune cells directly in the tumour. In this way, the tumour microenvironment could be altered by recruiting and activating tumour-specific immune cells, leading to tumour eradication [1,2]. The application of the above cytokines was primarily in the form of recombinant proteins, but is nowadays being replaced by the gene therapy approach [3]. Electroporation represents a safe and effective method of introducing genetic material (gene electrotransfer) into the cell [4]. The aim of our study was to compare the antitumor effects of gene electrotransfer of plasmids encoding IL-2 (pIL-2) and IL-12 (pIL-12) to B16F10 and CT26 tumour models in vivo. Tumour growth delay was measured and the concentrations of different immunostimulatory cytokines in tumour and serum samples were determined. Moreover, histology analysis was also performed in tumour samples to assess different immune cell populations inside the tumour and also to determine the extent of tumour vascularization. Tumour growth delay was observed in all therapeutic groups, the highest delay was observed in group with gene electrotransfer of both plasmids (combination group). Furthermore, complete tumour regression was observed only in the groups with gene electrotransfer of pIL-12 alone and in combination group. In the latter, long-term anti-tumour immunity after tumour rechallenge was also observed. After gene electrotransfer, increased expression of different cytokines with potent anti-tumour activity was detected, mainly in combination group. In addition, immune infiltration in tumour was the highest in combination group and more pronounced in B16F10 in comparison to CT26 tumour model. The antiangiogenic effect of the therapy was observed in the group with geneelectrotransfer of pIL-12 alone and in 46 combination group in both tumour models. To conclude, we demonstrated the anti-tumour effectiveness of gene electrotransfer of IL-2 and IL-12 in murine B16F10 melanoma and CT26 colon carcinoma. References: 1. Floros T, Tarhini AA. Anticancer Cytokines: Biology and Clinical Effects of IFN-α2, IL-2, IL-15, IL-21, and IL-12. Semin Oncol 2015;42(4):539 –48. 2. Lasek W, Zagożdżon R, Jakobisiak M. Interleukin 12: still a promising candidate for tumor immunotherapy? Cancer Immunol Immunother 2014;63(5):419-35. 3. Berraondo P, Sanmamed MF, Ochoa MC, Etxeberria I, Aznar MA, Pérez-Gracia JL, et al. Cytokines in p6 clinical cancer immunotherapy. Br J Cancer 2019;120:6–15. 4. Sokołowska E, Błachnio-Zabielska AU. A Critical Review of Electroporation as A Plasmid Delivery System in Mouse Skeletal Muscle. Int J Mol Sci 2019;20(11):2776. 47 WHOLE TRANSCRIPTOME EXPRESSION ARRAY ANALYSIS OF HUMAN COLON FIBROBLASTS CULTURE TREATED WITH HELICHRYSUM ITALICUM SUPPORTS ITS USE IN TRADITIONAL MEDICINE Katja Kramberger1, Darja Barlič-Maganja1, Zala Jenko Pražnikar1, Tadeja Režen2, Damjana Rozman2, Jure Pražnikar3, Saša Kenig1 p7 1University of Primorska, Faculty of Health Sciences, Department of Nutritional counselling – dietetics, Izola, Slovenia 2University of Ljubljana, Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, Centre for Functional Genomics and Bio-Chips, Ljubljana, Slovenia 3University of Primorska, Faculty of Mathematics, Natural Sciences and Information Technologies, Department of Applied Natural Sciences, Koper, Slovenia E-mail: katja.kramberger@fvz.upr.si Helichrysum italicum (HI) is a Mediterranean plant with well-reported use in traditional medicine for a wide range of applications, including digestive and liver disorders, intestinal parasitic infections, wound healing, stomach ache and asthma [1]. However, little is known about the global mechanism behind its pleiotropic activity. The aim of our study was to explain the mechanism behind the previously demonstrated effects of HI and to justify its use in traditional medicine. A microarray-based transcriptome analysis was used to discover the global transcriptional alterations in primary colon fibroblasts (CCD112CoN) after exposure to HI infusion for 6 h and 24 h. Microarray results were verified by quantitative real-time PCR. In addition, functional in vitro assays were performed to confirm the functional annotation of the differentially expressed genes. Transcriptome analysis identified a total of 217 differentially expressed genes compared to non-treated cells, of which only 8 were common to both treatments. Gene ontology term enrichment analysis showed that 24 hour treatment with HI infusion altered the expression of genes involved in cytoskeletal rearrangement and cell growth, whereas pathway enrichment analysis further revealed the importance of interleukin signalling and transcriptional regulation by TP53. For the 6 hour treatment only the process of hemostasis appeared in the results of both enrichment analyses. In functional assays, HI infusion increased the migration potential of Caco-2 cells and conditioned media of CCD112CoN cells decreased blood clotting and prothrombin time, whereas HI infusion alone did not affect blood coagulation. With the careful evaluation of the role of individual genes, especially SERPING1, ARHGAP1, IL33, and CDKN1A, represented in the enriched pathways and processes, we propose the main mode of HI action, which is wound healing. In addition to its 48 indirect prevention of diseases resulting from the impaired barrier integrity, HI also effects inflammatory and metabolic processes directly, by regulating genes such as LRPPRC, LIPA, ABCA12, PRKAR1A, and ANXA6 [2]. References: 1. Antunes Viegas D, Palmeira-de-Oliveira A, Salgueiro L, Martinez-de-Oliveira J, Palmeira-de-Oliveira R. Helichrysum Italicum: From Traditional Use to Scientific Data. J Ethnopharmacol 2014;151:54–65. 2. Kramberger K, Barlič-Maganja D, Jenko Pražnikar Z, Režen T, Rozman D, Pražnikar J, Kenig S. Whole transcriptome expression array analysis of human colon fibroblasts culture treated with Helichrysum italicum supports its use in traditional medicine. J Ethnopharmacol 2022;296(3):1-10. p7 49 DEVELOPMENT OF 3D SPHEROIDS FROM MURINE CELL LINE K7M2 AS MODELS FOR OSTEOSARCOMA TUMORS Saša Kupčič1,2, Urša Lampreht Tratar1, Metka Novak3, Barbara Breznik3, Gregor Serša1,4, Maja Čemažar1,5 1Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia 2University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia p8 3National Institute of Biology, Ljubljana, Slovenia 4University of Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia 5University of Primorska, Faculty of Health Sciences, Izola, Slovenia E-mail: skupcic@onko-i.si Osteosarcoma (OS) is a common primary bone cancer that mostly affects adolescent children between ages 10 to 14 and has a poor prognosis in case of metastatic disease. Current established therapy includes surgical amputation with adjuvant standard chemotherapy [1,2]. Due to no important advances in treatment for the last 50 years there is a need for advancement. One novel therapy, that could be used for the treatment of OS, is electrochemotherapy (ECT). ECT is a local tumor ablation therapy that combines a physical delivery method; electroporation, with cytotoxic drugs, such as bleomycin and cisplatin. It has already proved effective in treating tumors of different histological types, including bone metastasis of various primary tumors [3,4]. However, there is still a lack of information on its suitability to be employed as a treatment for osteosarcoma tumors. Effects of ECT on osteosarcoma tumors can be studied on different preclinical models, from 2D cell cultures to homologous animal models. For that purpose, we developed 3D spheroids from murine cell line K7M2. Spheroids of different sizes (5000 and 10 000 cells per spheroid) were incubated for 1 or 2 weeks in a rotary incubator Clinostar® (CelVivo, Inc, Chevy Chase, MA, USA), specifically suited for 3D cell models. Spheroids were then electroporated at 2 different voltage to distance ratios. We measured the proportions of apoptotic and necrotic cells in spheroids and determined cytotoxicity at multiple time points after electroporation. Smaller spheroids, formed from 5000 cells and growing for 1 week, turned out to be a promising research tool, as they have a lower proportion of apoptotic and necrotic cells than other groups. However, additional molecular and genetic testing will be applied to further confirm, how suitable spheroids are as a model for evaluation of ECT effectiveness in treatment of OS tumors. 50 References: 1. Ferrari A, Dirksen D, Bielack SS. Sarcomas of Soft Tissue and Bone. Prog Tumor Res 2016;43:128-41. 2. Bielack SS, Kempf-Bielack B, Delling G, Exner GU, Flege S, Helmke K, Kotz R, Salzer-Kuntschik M, Werner M, Winkelmann M, Zoubek A, Jürgens H, Winkler K. Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols. J Clin Oncol 2002;20(3): 776-90. 3. Mali B, Jarm T, Snoj M, Sersa G, Miklavčič D. Antitumor effectiveness of electrochemotheraPy: A systematic review and meta-analysis. Eur J Surg Oncol 2013;39(1):4-16. 4. Bianchi G, Campanacci L, Ronchetti M, Donati D. Electrochemotherapy in the Treatment of Bone Metastases: A Phase II Trial. World J Surg 2016;40(12):3088-94. p8 51 DEVELOPMENT OF AN ARTIFICIAL VIROID INOCULATION METHOD FOR HOP TISSUE CULTURES Patricija Lap, Jernej Jakše, Helena Volk University of Ljubljana, Faculty of Biotechnology, Department of Agronomy, Ljubljana, Slovenia p9 E-mail: patricija.lap@gmail.com Common hop ( Humulus lupulus L. ) is a herbaceous perennial plant in the botanical family Cannabacae [1]. In Slovenia hop plantations are threatened by various pathogens, including three different viroids: hop latent viroid (HLVd), hop stunt viroid (HSVd) and citrus bark cracking viroid (CBCVd). Viroids are of great economic importance, as they negatively affect the quality of hop cones. In general, the mass of the crop decreases, and the content of plant metabolites, which affect the aroma and taste of beer, also decreases. The presence of viroids negatively affects the rooting of cuttings, chlorosis, slower plant growth and even plant death can be observed [2]. Our goal was to examine a large number of hop cultivars for resistance, tolerance and sensitivity to infection with viroids (HLVd, HSVd and CBCVd), which threaten Slovenian hop farms. During the experiment, 13 different hop cultivars were grown in in-vitro conditions: we prepared the culture medium, subcultivated the plants onto fresh culture media, and monitored the growth of the plants. Viroid transcripts were prepared, the quality of the transcripts validated with the Agilent Bioanalyser 2100 device, and then used for hop plants infection in tissue cultures. The growth and development of infected and uninfected plants was monitored for eight weeks. The success of viroid hop infection was confirmed using the RT-PCR method and agarose gel electrophoresis. Our data showed that using our newly established method of viroid inoculation we have successfully infected all hop cultivars using viroid transcripts. Interestingly, the rate of infection was the highest for HSVd transcripts (100% for all 13 cultivars), followed by CBCVd (100% for 9 cultivars and 75% for 4 cultivars) and the lowest for HLVd (75% for 3 cultivars, 50% for 5 cultivars and 25% for 3 cultivars). Up to date, we did not observe any difference in growth and development of infected and uninfected plants and we did not discover any possible resistance or tolerance in tested varieties. References: 1. Rode J, Zmrzlak M, Kovačevič M. 2002. Hmeljna rastlina. V: Priročnik za hmeljarje. Majer D. (ur). Žalec, IHPS: 21-30. 2. Jakše J, Radišek S, Pokorn T, Matousek J, Javornik B. Deep-sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop. Plant Pathol 2015;64(1): 831−42. 52 GENE UPREGULATION IN SPECIFIC STAGES OF CAR T CELL-MEDIATED CYTOTOXICITY: A QUANTITATIVE REAL-TIME PCR APPROACH TO ENHANCE MONITORING OF CYTOTOXIC EFFICACY AND THERAPY PROGRESSION Lucija Levstek, Larisa Janžič, Alojz Ihan, Andreja Nataša Kopitar University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, p10 Ljubljana, Slovenia E-mail: lucija.levstek@mf.uni-lj.si Chimeric antigen receptor (CAR) T cell therapy holds enormous potential for treating hematologic malignancies. Despite its advantages, it is still used as a second line of therapy mainly because of its severe side effects and/or patient unresponsiveness. To improve the therapeutic efficacy, a better understanding of the complex mechanisms behind effective tumor cell killing is necessary. The aim of this study is to identify upregulated genes at specific stages of the CAR T cell cytotoxic process with which the assessment and monitoring of the cytotoxic activity of CAR T cells and therapy progress with quantitative real-time PCR (qPCR) could be obtained. After infusion into the patient, CAR T cells move through the bloodstream to the tumor sites. This process is enhanced by chemokines and adhesion molecules, with genes such as CXCR4, ICAM1, VCAM1, and SELL [1] possibly playing pivotal roles in ensuring the cells reach their intended destinations. Once in the tumor vicinity, CAR T cells identify specific tumor-associated antigens, a process governed by genes like CBP, Unc119, and IFT20 [2,3]. Upon antigen recognition, the CAR T cell, guided by genes involved in cell adhesion and signaling such as BACH2, EZH2, and TGF-β1 [4-6], attaches to the tumor cell and forms the immunological synapse, a unique junction between the CAR T cell and its target. To exert their cytotoxic effects on tumor cells, CAR T cells undergo a dynamic reorganization. Key cellular organelles, responsible for cytotoxic actions, migrate toward the immunological synapse (Golgi apparatus, mitochondria, etc.). The upregulated genes in these migrations are believed to be e.g., DISC1, GLUT1, MAPRE1, GMAP210, and others [7-9]. Actin filaments, influenced by the upregulation of e.g., BBS2 and ARPC1B [3,10] genes, bolster the structural integrity of the lamellipodium at the synaptic area. Concurrently, the centrosome, guided by genes associated with cytoskeleton organization such as MAP4 and CIP4 [11], aligns with the reconfiguring cytoskeleton. The culmination of these processes is the formation of lytic granules which are transported through the immunological synapse into target cell, where target cell apoptosis is induced. The upregulated genes in these final stages of target cell killing are e.g. HkRP3, SERPINB9, GZMB, GZMA, and PRF1 [6,11,12]. 53 We have identified genes that are potentially upregulated during specific stages of the cytotoxic process, suggesting their potential use as markers for monitoring the cytotoxic efficacy of CAR T cells. The subsequent phase of our research will focus on testing this designated set of genes to evaluate their marker potential. With this knowledge, we would be able to monitor the cytotoxic processes and efficacy of the CAR T cells and consequently improve the course of this complex immunotherapy. References: 1. Sackstein R, Schatton T, Barthel SR. T-lymphocyte homing: an underappreciated yet critical hurdle for successful cancer immunotherapy. Lab Invest 2017;97(6):669-97. p10 2. Zhou Y, Bastian IN, Long MD, Dow M, Li W, Liu T, et al. Activation of NF-κB and p300/CBP potentiates cancer chemoimmunotherapy through induction of MHC-I antigen presentation. Proc Natl Acad Sci USA 2021;118(8). 3. Cassioli C, Onnis A, Finetti F, Capitani N, Brunetti J, Compeer EB, et al. The Bardet-Biedl syndrome complex component BBS1 controls T cell polarity during immune synapse assembly. J Cell Sci 2021;134(16). 4. Roychoudhuri R, Clever D, Li P, Wakabayashi Y, Quinn KM, Klebanoff CA, et al. BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers. Nat Immunol 2016;17(7):851-60. 5. Stairiker CJ, Thomas GD, Salek-Ardakani S. EZH2 as a Regulator of CD8+ T Cell Fate and Function. Front Immunol 2020;11:593203. 6. Jiang P, Gu S, Pan D, Fu J, Sahu A, Hu X, et al. Signatures of T cell dysfunction and exclusion predict cancer immunotherapy response. Nat Med 2018;24(10):1550-58. 7. Poenie MF, Maskalenko NA, Nath S. The dynein-binding protein DISC1 plays important roles in relocating organelles to the immunological synapse during T cell activation. J Immunol 2018;200(1-Supplement):47.5-.5. 8. Cassioli C, Baldari CT. Lymphocyte Polarization During Immune Synapse Assembly: Centrosomal Actin Joins the Game. Front Immunol 2022;13:830835. 9. Zucchetti AE, Bataille L, Carpier JM, Dogniaux S, San Roman-Jouve M, Maurin M, et al. Tethering of vesicles to the Golgi by GMAP210 controls LAT delivery to the immune synapse. Nat Commun 2019;10(1):2864. 10. Randzavola LO, Strege K, Juzans M, Asano Y, Stinchcombe JC, Gawden-Bone CM, et al. Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity. J Clin Invest 2019;129(12):5600-14. 11. Kopf A, Kiermaier E. Dynamic Microtubule Arrays in Leukocytes and Their Role in Cell Migration and Immune Synapse Formation. Front Cell Dev Biol 2021;9:635511. 12. Hidalgo LG, Einecke G, Allanach K, Halloran PF. The transcriptome of human cytotoxic T cells: similarities and disparities among allostimulated CD4(+) CTL, CD8(+) CTL and NK cells. Am J Transplant 2008;8(3):627-36. 54 UPREGULATION OF CYTOSOLIC PATTERN RECOGNITION RECEPTORS AFTER GENE ELECTROTRANSFER OF PLASMID ENCODING IL-12 Ajda Medved1,2, Tanja Jesenko1,2, Maša Omerzel1, Maja Čemažar1,3 1Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia 2University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia p11 3Faculty of Health Sciences, University of Primorska, Izola, Slovenia E-mail: amedved@onko-i.si During gene therapy, therapeutic genes are inserted into cells and recognized by cell defense mechanisms as exogenous nucleic acids and consequently as pathogen-associated molecular patterns (PAMPs) [1]. Exogenous nucleic acids are then recognized by endosomal and cytosolic nucleic acid-specific pattern recognition receptors (PRRs) [2]. Activation of PRRs induces a specific immune response that may lead to antitumor effects [3]. One of the gene therapies that has already been proven to be safe and effective, having good local control and an abscopal effect in the treatment of melanoma skin metastases, is gene electrotransfer (GET) of plasmid DNA coding for cytokine interleukin-12 (IL-12) [4]. Plasmid DNA introduced into cells by GET could activate different cytosolic PRRs, therefore the aim of our study was to evaluate the effect of GET on plasmid DNA coding for IL-12 on PRRs. Parallel electrodes with a 2-mm gap and a clinically used pulse protocol were used for GET; 8 times 1300 V/cm pulses of 100-microsecond duration at a frequency of 5 kHz were applied with CLINIPORATOR®. Plasmid DNA encoding murine IL-12 (1 mg/ml, pmIL12), noncoding backbone plasmid DNA (1 mg/ml, pScramble), and a plasmid coding for a green fluorescent protein (1 mg /ml pEGFP,) were used for GET into B16-F10 melanoma and CT26 colon carcinoma cells. Transfection efficiency was determined 2 days after the electrotransfer of pEGFP using fluorescence microscopy. Cell viability was determined 3 days after treatment using the PrestoBlue™ Cell Viability Reagent. The expression of 15 different PRRs (DNA sensors:DAI, IFI16, IFI204, DDX60, DHX9, DHX36, AIM2, cGAS, STING, DDX41, LRRFIP1, Ku70, and RNA sensors: MDA5, LGP2, RIG-I) was determined using qRT‒PCR 4, 24 and 48 hours after treatment. Clinically used electroporation protocol led to 24 % of transfected B16-F10 cells and only about 6% of transfected CT26 cells. Cell viability was significantly reduced after IL-12 and non-coding DNA plasmid GET. Contrary to our expectation, GET of pmIL12 and noncoding pScramble mainly led to upregulation of RNA binding PRRs. Further studies are needed to elucidate underlying processes for such cellular response. 55 References: 1. Li D, Wu M. Pattern recognition receptors in health and diseases. Sig Transduct Target Ther 2021;6(1):291. 2. Ori D, Murase M, Kawai T. Cytosolic nucleic acid sensors and innate immune regulation. Int Rev Immunol 2017;36(2):74-88. 3. Wu J, Zhijian JC. Innate immune sensing and signaling of cytosolic nucleic acids. Ann Rev Immunol 2014;32:461-88. 4. Cirella A, Luri-Rey C, Augusta Di Trani C, Teijeira A, Olivera I, Bolaños E, et al. Novel strategies exploiting interleukin-12 in cancer immunotherapy. Pharmacol Ther 2022;239:108189. p11 56 DIGITALISATION OF BIODIVERSITY Gaja Ramić1, Andreja Ramšak2 1University of Nova Gorica, School of Environmental Sciences, Nova Gorica, Slovenia 2National Institute of Biology, Marine Biology Station, Piran, Slovenia E-mail: gaja.ramic@gmail.com p12 We are in a time of accelerated biodiversity loss and environmental degradation, and ex situ conservation and archiving of molecular samples are of immense importance as they represent important resources for future research, especially for the study of species under different scenarios (endangered species, non-native species, unpredictable occurrence, voucher specimens, etc.). Specimens must be collected in an appropriate manner, and storage and preservation of biological material must follow the best available practices. Biorepositories must ensure integrity, authenticity, and availability of the associated data to describe the stored samples in detail. Quality checks were performed on archived DNA samples of Mawia benovici, a rare scyphozoan species described from the northern Adriatic Sea [1]. Tissue samples, DNA extraction, and amplification of several genetic markers were performed on 60 individuals in 2013. DNA was extracted using the commercial kit E.Z.N.A. Mollusc DNA Kit (Omega Bio-Tek) or KAPA Express Extract. In 2023, several parameters describing the quality of the archived samples were investigated and evaluated (DNA concentration, fragmentation, purity, DNA integrity) by spectrophotometric measurements (Nanodrop) and automated microfluidic-based electrophoresis (TapeStation System, Agilent). A re-examination of the quality criteria is required to meet the basic criteria for continued storage and to provide guidelines for maintaining and preserving the cnidarian specimens. Currently, the biorepository at the Marine Biology Station in Piran (Slovenia) includes 10 cnidarian species with 4272 records. This contribution is part of the initiative LIFEWATCH-SI (co-financed by the Republic of Slovenia, the Ministry of Education, Science and Sport, and the European Union from the European Regional Development Fund) to promote the digitalisation of biodiversity and the principles of open science. References: 1. Avian M, Ramšak A, Tirelli V, D’ambra I, Malej A. Redescription of Pelagia benovici into a new jellyfish genus, Mawia, gen. nov., and its phylogenetic position within Pelagiidae (Cnidaria: Scyphozoa: Semaeostomeae). Invertebr Syst 2016;30(6):523-46. 57 AN AMPLITUDE-BASED MULTIPLEX DDPCR ASSAY FOR DIAGNOSIS OF HEREDITARY α-TRYPTASEMIA Manca Svetina1, Julij Šelb1,2, Peter Korošec1,3, Matija Rijavec1,4 1University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia 2 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 3 University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia p13 4University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia E-mail: manca.svetina@klinika-golnik.si Hereditary α tryptasemia (HαT) is an autosomal dominant trait characterized by germline multiplication on one single allele in the TPSAB1 gene encoding α-tryptase [1]. The determination of HαT is being discussed as an important biomarker to be included in risk assessment models and future diagnostic algorithms for patients with mastocytosis [2-4]. Patients with HαT are also prone to more severe anaphylaxis [1,5-7]. Therefore, tryptase genotyping should be considered in the clinical evaluation of individuals with a history of, or at risk for, severe anaphylaxis [8]. Due to the complex genetic structure at the human tryptase locus, genetic testing for HαT is presently notably limited and infrequently pursued [9,10]. The aim of this study was to develop, optimize and validate a multiplex droplet digital PCR (ddPCR) assay that can reliably quantify α- and β-tryptase encoding sequences in a single reaction well. Custom primers and probes targeting sequences encoding α- and β-tryptases were reported previously [1]. For the detection of reference copy number invariant locus, additional primers, and probes targeting AP3B1 and AGO1 genes were designed. To optimize the ddPCR conditions and establish an amplitude-based multiplex ddPCR assay, a thermal gradient with varying annealing temperatures, different primers/probe concentrations, and various initial DNA quantities were tested. The multiplex ddPCR performance was compared with separate duplex ddPCRs in 113 DNA samples. An annealing temperature of 60°C, DNA quantity of 55 ng and optimized primer and probe concentrations enabled a clear separation of positive droplets and clear, distinct differentiation of each target set within unique clusters. Results obtained from the multiplex ddPCR were concordant with those achieved with the duplex ddPCRs. Utilizing this multiplex ddPCR assay, in contrast to conducting distinct duplex ddPCRs, presents noteworthy benefits for tryptase genotyping. These advantages encompass a substantial threefold decrease in material costs and considerable time savings. Consequently, this approach exhibits a high degree of suitability and particularly captures interest for routine clinical implementation. 58 References: 1. Lyons JJ, Yu X, Hughes JD, Le QT, Jamil A, Bai Y, et al. Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number. Nat Genet 2016; 48(12):1564–69. 2. Valent P, Hoermann G, Bonadonna P, Hartmann K, Wolfgang R, Broesby-olsen S, et al. The normal range of baseline tryptase should be 1-15 ng/ml and covers healthy individuals with hereditary alpha tryptasemia. J Allergy Clin Immunol Pract 2023; S2213-2198(23)00907-8. 3. Sordi B, Vanderwert F, Crupi F, Gesullo F, Zanotti R, Bonadonna P, et al. Disease correlates and clinical relevance of hereditary α-tryptasemia in patients with systemic mastocytosis. J Allergy Clin Immunol 2023;151(2):485-493.e11. p13 4. Polivka L, Madrange M, Bulai-livideanu C, Barete S, Ballul T, Neuraz A, et al. An elevated prevalence of hereditary alpha-tryptasemia in all mastocytosis subtypes: pathophysiological implications. J Allergy Clin Immunol 2023; S0091-6749(23)01068-0. 5. Lyons JJ, Chovanec J, O’Connell MP, Liu Y, Šelb J, Zanotti R, et al. Heritable risk for severe anaphylaxis associated with increased α-tryptase–encoding germline copy number at TPSAB1. J Allergy Clin Immunol 2021;147(2):622–32. 6. Šelb J, Rijavec M, Kopač P, Lyons JJ, Korošec P. HαT is associated with increased risk for severe Hymenoptera venom-triggered anaphylaxis. J Allergy Clin Immunol 2022;278:4–5. 7. Kačar M, Rijavec M, Šelb J, Korošec P. Clonal mast cell disorders and hereditary α-tryptasemia as risk factors for anaphylaxis. Clin Exp Allergy 2023;53(4):392–404. 8. Glover SC, Carter MC, Korošec P, Bonadonna P, Schwartz LB, Milner JD, et al. Clinical relevance of inherited genetic differences in human tryptases: Hereditary alpha-tryptasemia and beyond. Ann Allergy, Asthma Immunol 2021;127(6):638–47. 9. Lyons JJ. Hereditary Alpha Tryptasemia: Genotyping and Associated Clinical Features. Immunol Allergy Clin North Am 2018;38(3):483–95. 10. Cochran AL, Coop C, Neaves BI, Wood ST. The Curious Case of Elevated Tryptase: Workup and Differential in Family of Four. Cureus 2023;15(4):e38065. 59 RRADIATION RESULTS IN MOLECULAR AND TRANSCRIPTIONAL SHIFTS OF TUMOR ENDOTHELIAL CELLS Iva Šantek1,2, Tim Božič1, Simona Kranjc Brezar1,2, Gregor Serša1,3, Boštjan Markelc1,3 1Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia 2University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia p14 3 University of Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia E-mail: isantek@onko-i.si Aberrant tumor vasculature is characterized by insufficient blood flow and oxygen supply, which leads to the formation of hypoxic regions, resistant to radiotherapy (RT) [1]. Irradiation (IR) kills cancer cells, but also affects the tumor microenvironment, including tumor vasculature. The effect of IR on tumor endothelium is time- and dose-dependent. Importantly, IR can cause tumor endothelial cell (TEC) apoptosis, but, can also lead to vascular remodeling/normalization or activation of endothelial cells (EC), thus possibly reducing hypoxia and enhancing the immune cell infiltration into tumors [2]. However, the impact of changes in tumor vasculature and TECs due to IR on tumor response to RT is yet to be elucidated. Hence, in this research, we aimed to clarify the vascular response to IR focusing on vascular remodeling and activation of ECs. We first irradiated human EA.hy926 and Hulec5a, and murine bEnd.3, 2H11, and SVEC4-10 EC lines with single doses of 0 – 10 Gy, which resulted in the reduction of proliferation and increased EC death, depending on the dose received. Next, we irradiated CT26 and MC38 colon carcinomas grown in Balb/c or C57Bl/6 mice, respectively with a single dose of 15 Gy or the fractionated dose of 5 x 5 Gy, which led to the reduction of tumor growth rate. RNA sequencing analysis of TECs isolated from irradiated tumors showed higher expression of genes ( Cd47, Vcam-1, Vwf, Il6, etc. ) and pathways related to ECs activation and immune response. Finally, frozen tumor sections of irradiated tumors were immunofluorescently stained for endothelial (CD31, MECA-79, and ERG), immune infiltration (CD4 and CD8), and transcription (IRF9) markers. To sum up, IR reduces EC proliferation and survival. Furthermore, a single dose of 15 Gy changes the TEC transcriptional profile and usage of pathways that support TEC activation and augment anti-tumor immune response. References: 1. Siemann DW. The unique characteristics of tumor vasculature and preclinical evidence for its selective disruption by Tumor-Vascular Disrupting Agents. Cancer Treat Rev 2011;37:63–74. 2. Kaeppler JR, Chen J, Buono M, Vermeer J, Kannan P, Cheng W, et al. Endothelial cell death after ionizing radiation does not impair vascular structure in mouse tumor models. EMBO Rep 2022;23(9):e53221. 60 BIOINFORMATICS ANALYSIS OF RNA-seq DATA OF COLORECTAL CANCER AND LIVER METASTASES ELUCIDATES GENES ASSOCIATED WITH METASTASIZING OF COLORECTAL CANCER Kristian Urh, Nina Zidar, Emanuela Boštjančič University of Ljubljana , Faculty of Medicine, Institute of Pathology, Ljubljana, Slovenia p15 E-mail: kristian.urh@mf.uni-lj.si Colorectal cancer (CRC) is a significant cause of cancer-related morbidity worldwide, and patients with advanced CRC have a low five-year survival rate despite new treatment options. Most genetic events associated with tumor development occur early, leading to a need to define biomarkers responsible for malignant transformation and metastasis. To identify differentially expressed genes (DEGs) associated with CRC metastasis, we analyzed RNA-seq data from primary tumors and liver metastases and performed pathway enrichment analysis (PEI) and protein-protein interaction analysis (PPI). By comparing DEGs to those from TCGA data, we identified 668 DEGs, performed PEI and PPI analysis. The identified DEGs contribute to the understanding of gene expression patterns involved in metastasizing of CRC. Genes MMP1 and MMP3, were among the most highly differentially expressed, associated with degradation of the extracellular matrix are interesting candidates for further validation. Additionally, 20 other strongly differentially expressed, and highly significant genes that were only differentially expressed between CRC and liver metastases are candidates for further validation for involvement in development/maintenance of liver metastases. Our results could upon further validation provide new prognostic markers, or targets for treating advanced CRC. 61 AUTHOR INDEX 62 AUTHOR INDEX A Faraway Rupert 11 Acetto Tomaž 22 Flajnik Hana 38 Ambrožič Avguštin Jerneja 40 H Ameres Stefan L. 11 Hodžoć Alenka 44 B Horvat Simon 14 , 18 Barlič-Maganja Darja 48 Hrovat Katja 40 Blatnik Ana 31 I Blatnik Olga 31 Ihan Alojz 33 , 53 Boštjančič Emanuela 61 Iosub Ira A. 11 Božič Tim 28 , 60 J Breznik Barbara 50 Jakše Jernej 25 , 26 , 52 Brišar Nuša 36 Janžič Larisa 33 , 53 Bužan Elena 16 , 20 Javornik Žiga 42 C Jenko Pražnikar Zala 48 Chakrabarti Anob M. 11 Jesenko Tanja 30 , 55 Cör Andrej 36 Jiang Zhihua 14 Costello Heaven Neve 11 Jus Matevž 44 Č K Čemažar Maja 28 , 30 , 46 , 50 , 55 Kamenšek Urška 46 Čerenak Andreja 26 Kenig Saša 48 Čremožnik Zupančič Jerneja 40 Klančar Gašper 31 Ć Knez Lea 11 Ćirović Duško 20 Komel Tilen 46 D Kopitar Andreja Nataša 33 , 53 De Robertis Mariangela 46 Korošec Peter 58 Digby Holly 11 Kramberger Katja 48 Dolinar Klemen 46 Kranjc Brezar Simona 28 , 30 , 36 , 46 , 60 Drev Primož 31 Kunej Tanja 14 E Kunej Urban 25 Eržen Marjeta 26 Kupčič Saša 50 F 63 AUTHOR INDEX L R Lampreht Tratar Urša 46 , 50 Radišek Sebastjan 25 Lap Patricija 52 Ramić Gaja 57 Lesar Aleksander 22 Ramšak Andreja 57 Levstek Lucija 33 , 53 Režen Tadeja 48 M Rijavec Matija 58 Markelc Boštjan 28 , 30 , 60 Rozman Damjana 48 Maver Aleš 44 S Medugorac Ivica 18 Sečnik Andrej 25 Medved Ajda 55 Seme Katja 40 Merlo Sebastjan 31 Serša Gregor 28 , 30 , 46 , 50 , 60 Mikec Špela 14 Signori Emanuela 46 Miljanić Vanja 38 Simčič Mojca 18 Modic Živa 30 Sollitto Marco 20 Murn Jernej 12 Stajič Ester 42 N Starčič Erjavec Marjanca 22 Novak Meta 50 Stegel Vida 31 Novaković Srdjan 31 Strojnik Ksenija 31 O Svetina Manca 58 Omerzel Maša 46 , 55 S P Šantek Iva 28 , 60 Pavlin Anja 24 Šelb Julij 58 Pavlova Bojadžiski Mirjana 31 Šetrajčič Dragoš Vita 31 Peterlin Borut 44 Škof Erik 31 Petrin Sara 33 Škvarč Andreja 38 Pirkmajer Sergej 46 Šprem Nikica 20 Plaschka Clemens 11 Štajner Nataša 25 , 38 , 42 Pogorevc Neža 18 Šuster Katja 36 Potočnik Hubert 20 T Pražnikar Jure 48 Toškan Borut 16 64 AUTHOR INDEX U Ule Jernej 11 Upadhyay Maulik 18 Urh Kristian 61 Urzi Felicita 20 V Venema Koen 22 Verbrugen Sanne 22 Verhoeven Jessica 22 Volk Helena 52 W Wilkins Oscar G. 11 Z Zidar Nina 61 Zver Lars 16 Ž Žagar Tina 31 Žnidar Katarina 46 65 SPONSORS 66 SPONSORS V 25 letih smo sponzorirali ali podprli 57 intervencijskih kliničnih raziskav, ki so gonilo razvoja in napredka v medicini, in s tem je več kot 780 bolnikov dobilo možnost inovativnega zdravljenja. M-SI-00000814 (V2.0) Informacija pripravljena v marcu 2023. 67 SPONSORS 68 SPONSORS 69