0TH INTERNATIONAL SYMPOSIUM, LJUBLJANA, 9. DECEMBER 2023 PEER REVIEWED PROCEEDINGS, PART II EDITED BY: VERONIKA KRALJ-IGLIČ, YELENA ISTILEULOVA AND ANNA ROMOLO FACULTY OF HEALTH SCIENCES, UNIVERSITY OF LJUBLJANA

Yelena Istileulova

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex condition arising in susceptible people, predominantly following viral infection, but also other stressful events. The susceptibility factors discussed here are both genetic and environmental although not well understood. While the dysfunctional physiology in ME/CFS is becoming clearer, understanding has been hampered by different combinations of symptoms in each affected person. A common core set of mainly neurological symptoms forms the modern clinical case definition, in the absence of an accessible molecular diagnostic test. This landscape has prompted interest in whether ME/CFS patients can be classified into a particular phenotype/subtype that might assist better management of their illness and suggest preferred therapeutic options. Currently, the same promising drugs, nutraceuticals, or behavioral therapies available can be beneficial, have no effect, or be detrimental to each individual patient. We have...

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a poorly understood, complex, multisystem disorder, with severe fatigue not alleviated by rest, and other symptoms, which lead to substantial reductions in functional activity and quality of life. Due to the unclear aetiology, treatment of patients is complicated, but one of the initial problems is the insufficient diagnostic process. The increase in the number of undiagnosed ME/CFS patients became specifically relevant in the light of the COVID-19 pandemic. The aim of this research was to investigate the issues of undiagnosed potential ME/CFS patients, with a hypothetical forecast of the expansion of post-viral CFS as a consequence of COVID-19 and its burden on society. Methods: The theoretical research was founded on the estimation of classic factors presumably affecting the diagnostic scope of ME/CFS and their ascription to Latvian circumstances, as well as a literature review to assess the potential inter...

The aim of this study was to describe the clinical evolution during 6 months of follow-up of adults recovered from COVID-19. We tried to determine how many met the definition of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). A total of 130 patients (51.0 ± 14 years, 34.6% female) were enrolled. Symptoms were common, participants reported a median number of 9 (IQR 5–14) symptoms. Fatigue was the most common symptom (61/130; 46.9%). Patients with fatigue were older 53.9 ± 13.5 years compared with 48.5 ± 13.3 years in those without fatigue (p = 0.02) and had a longer length of hospital stay, 17 ± 14 days vs. 13 ± 10 days (p = 0.04). There was no difference in other comorbidities between patients with fatigue and those without it, and no association between COVID-19 severity and fatigue. After multivariate adjustment of all baseline clinical features, only age 40 to 50 years old was positively associated with fatigue, OR 2.5 (95% CI 1.05–6.05) p = 0.03. In our survey, only...

10TH INTERNATIONAL SYMPOSIUM, LJUBLJANA, 9. DECEMBER 2023 PEER REVIEWED PROCEEDINGS, PART II EDITED BY: VERONIKA KRALJ-IGLIČ, YELENA ISTILEULOVA AND ANNA ROMOLO FACULTY OF HEALTH SCIENCES, UNIVERSITY OF LJUBLJANA Proceedings of 10 th Socratic Lectures 2024 Socratic Lectures 10th International Symposium, Ljubljana, December 9, 2023 Peer Reviewed Proceedings, Part II Edited by Veronika Kralj-Iglič,Yelena Istileulova and Anna Romolo Reviewers: Boštjan Kocjančič, Vladimira Erjavec, Yelena Istileulova Published by: University of Ljubljana Press For the publisher: Gregor Majdič, the Rector of University of Ljubljana Issued by: University of Ljubljana, Faculty of Health Sciences For the issuer: Martina Oder, the Dean of Faculty of Health Science, UL Design: Anna Romolo Gallery Marguerite de Saint Champs: Oil on canvass by Roberto Braida, Italy. With permission of Gallery 'Casa d'Arte San Lorenzo' – San Miniato, Pisa, Italy. Image on the front page: Drago Videmšek First digital edition. Publication is available online in PDF format at: https://www.zf.uni-lj.si/images/stories/datoteke/Zalozba/Sokratska_10_II.pdf http://ebooks.uni-lj.si DOI: 10.55295/PSL.2024.II Publication is free of charge. Ljubljana, 2024 This work is available under a Creative Commons Attribution 4.0 International ___________________ Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID 191823619 ISBN 978-961-297-316-2 (PDF) I Proceedings of 10 th Socratic Lectures 2024 The members of the Organizing Committee of 10th Socratic Lectures: Mariam Chkhikvishvili, Drago Dolinar, Alima Dostiyarova, Mitja Drab, Vladimira Erjavec, Tjaša Griessler Bulc, Tomaž Gyergyek, Aleš Iglič, Yelena Istileulova, Monika Jenko, Gvantsa Jichoshvili, Boštjan Kocjančič, Samo Kralj, Veronika Kralj-Iglič, Larisa Melia, Alenka Nemec Svete, Makhanov Nursultan, Vesna Osojnik, Gabriella Pocsfalvi, Irena Pulko, Anna Romolo, Gitta Schlosser, Špela Tadel Kocjančič, Polonca Trebše, Renata Vauhnik Program of the Symposium Socratic Lectures, December 9, 2023, 10:30 – 14:00 (Ljubljana time) 10.30 Welcome to participants (Veronika Kralj-Iglič, University of Ljubljana) https://uni-ljsi.zoom.us/j/98919724250 10.35 -11.15 Plenary lecture: Bernd Giebel, Institute for Transfusion Medicine University Hospital Essen, Germany: Clinical Potential of MSC-EVs and Translational Challenges Scientific sections Section 1: Human medicine, organized by Špela Tadel Kocjančič First group: 11.30 - 11.50 Tsanava K: Ongoing Toxic epidermal necrolysis (TEN) complicated with acute kidney injury (AKI) 11.50 - 12.10 Malidze D: Effect of Colchicine on atrial fibrillation onset prevention in patients with coronary artery diseases 12.10 - 12.25 Kolar M: Geometrical specificities of femora fractured secondary to total hip replacement 12.25 - 12.45 Tadel Kocjančič Š: COVID-19 and extracorporeal membrane oxygenation (ECMO) 12.45 - 13.00 Ipavec M: Experience with smart above knee prosthesis 13.00 - 13.15 Amon M; Kresal F: Therapeutic potential of hypoxia 13.15 - 13.30 Kovačič P: Decoding the facial expressions of cats: Insights through scientific illustration Section 2 : Human medicine orgnaized by Larisa Melia Second group: 11.30 - 11.50 Bensal R: Enterohemorrhagic E-coli leading to haemolytic uremic syndrome- Case study and review 11.50 - 12.10 Khelaia A: Metabolic endotoxemia and male infertility 12.10 - 12.25 Sulukhia R, Melia L, Davidova N, Pkhaladze L: Pregnancy complications in patients with endometriosis 12.25 - 12.45 Mantskava M: Blood rheology properties in patients with acute lymphoblastic leukemia 12.45 - 13.00 Khatuna K: Sebaceous nevus in children: clinical-dermoscopic differences 13.00 - 13.15 Jabua M, Gognadze T: Jprner's syndrome caused by ultrasound guided supraclavicular nerve block (Aversi Clinic) 13.15 - 13.30 Vojkovič R: Epidemiology of periprosthetic proximal femur fractures II Proceedings of 10 th Socratic Lectures 2024 Section 3: Veterinary medicine First group: organized by Mariam Chkhikvishvili 11.30 - 11.50 Mamukelashvili N, Kalandia E, Georgian Mountain Dog – Kartuli Nagazi 11.50 - 12.10 Mamatsashvili G, Kereselidze M, Beruashvili M, Mikadze K: Justification of the ways of landscape-epizootological-ecological monitoring in order to determine the risk of a possible or expected outbreak of anthrax disease and to minimize the risk 12.10 - 12.30 Nebieridze S, Kereselidze M, Beruashvili M, Zibzibadze M: Food safety problems in Georgia 12.30 - 12.50 Arko M et al.: Extracellular particles from bovine and equine milk 12.50 - 13.10 Chkhikvishvili M, Milashvili N, Omarashvili N: Benefits of flank spay in cats – compared to midline spay in cats - compared to midline spay 13.10 - 13.30 Plavšič Z, Bee-Wellness: Apitherapy and the pursuit of vitality Section 4: Veterinary medicine Second group: organized by Vladimira Erjavec 11.30 – 11.50 Šimundić M: Urinary tract infection in dog and cat 11.50 - 12.10 Dučić N: Feline tooth resorption - Experiences from the clinic of veterinary dentistry at University of Sarajevo 12.10 - 12.30 Vejzović A: Exotic animals vascular system characteristics and its application in clinical practice 12.30 - 12.50 Beletić A: Glycosylation research in bovines-the significance and recent updates 12.50 - 13.10 Vasić Vilić J: Apitherapy - our experience 13.10 - 13.30 Erjavec V, Lukanc B: Retrospective review of 27 cases of congenital portosystemic shunt in dogs from 2015 to 2023 Section 5: Physiotherapy organized by Renata Vauhnik 11.30 - 11.45 Ošlak A, Effects of therapeutic approaches in treating varus malalignment of the knee joint 11.45 - 12.00 Vrbinc NK, The Impact of sleep deprivation on physical activity 12.00 - 12.15 Urbančič Š, Physiotherapy approach for treating lateral epicondilalgia 12.15 -12.30 12.30 - 12.45 Štuhec M, Physiotherapy approach for treating De Quervain tenosynovitis during and after pregnancy Močilar M, Telerehabilitation to improve balance and mobility in patients post- stroke 12.45 - 13. 00 Battalian T, Respiratory management of Acute Respiratory Distress Syndrome (ARDS) in the ICU from early diagnosis: narrative review 13.00 - 13.15 Hemery K, Adherence of musculoskeletal patients to home exercise programmes 13.15 - 13.30 Vauhnik R, Zuil Escobar JC, Martinez Cepa C, Functional approach to musculoskeletal injuries in physiotherapy: A COIL project in Physiotherapy III Proceedings of 10 th Socratic Lectures 2024 Section 6: Prosthetics organized by Monika Jenko, Drago Dolinar, Boštjan Kocjančič 11.30 - 12.00 Russo A, New trends in knee prosthesis alignment 12.00 - 12.15 Kocjančič E, Hip arthrosis and endoprosthesis 12.15 - 12.30 Merčun A, Trabecular tital hip arthroplasty - glimpse of past 12.30 - 12.45 Hojker M, Implant failure in metal-on-metal hip endoprosthesis 12.45 - 13.00 Kocjančič B, Oxidized zirconium in hip prosthesis 13.00 - 13.15 Dolinar D, Study of surface and biointerface phenomena of implant materials and biosystems 13.15 - 13.30 Jenko M, The mechanisms of premature fracture in modular-neck stems made of CoCrMo/Ti6Al4V and Ti6Al4V/Ti6Al4V Alloy Section 7: Green transition organized by Tjaša Griessler Bulc, Sylwester Rzoska 11.30 - 11.50 Rzoska S, When physics meets life: high pressure processing for food and pharmaceuticals 11.50 - 12.10 Zaskavska K, Salun M, Strategies for resilience in a dynamic world. From VUCA to BANI 12.10 - 12.30 Lavtižar V, Circular Economy: Designing a new, greener world 12.30 - 12.50 Cepec E, Griessler Bulc T, Istenič D, Uncovering algae biomass potentials: from wastewater to biostimulants 12.50 - 13.10 Klemenčič L, Istenič D, Griessler Bulc T, Challenges of the efficient algae harvesting 13.10 - 13.30 Mežnar E, Štuhec A, Istenič D, Godič Torkar K, Occurrence of antimicrobial resistance in bacteria of faecal origin at different stages of treatment in two municipal waste water treatment plants Section 8: New world organized by Polonca Trebše 11.30 - 11.50 Uridia R, Kereselidze M, Barbakadze N, Karkashadze N, Tserodze N, Aflatoxin-inhibiting Adsorptive Nanocomposites Based on Natural Raw Material - Lignin 11.50 - 12.10 Nenadović M, Kržišnik K, Trebše P, Bavcon Kralj M, Effect of time, pH, alcohol and sugar content on nicotine release from pouches available on Slovene market 12.10 - 12.30 Kaporov A, Zore A, Maniecki T, Trebše P, Synthesis and application of Co-MOF compounds based on 2-methylimidazole and 2,5-dihydroxyterephthalic acid 12.30 - 12.50 Roganović A, Istileulova Y, Lithium-ion battery project: ESTEAM perspectives 12.50 - 13.10 Turnšek A, Kresal F, Physiotherapy and treatment of neuropathic pain 13.10 - 13.30 Haque MdM, The effects of energy on protein misfolding and aggregation Section 9: Therapeutic plasma organized by Veronika Kralj-Iglič and Gitta Schlosser 11.30 - 11.45 Steiner N, Effect of autologous therapeutic plasma on wound healing after operation in the ear 11.45 - 12.00 Schlosser G, Mass spectrometry of samples with extracellular particles IV Proceedings of 10 th Socratic Lectures 2024 12.00 - 12.15 Michelini S, Quantification of growth and inflammatory factors in platelet- and EV–rich plasma (PVRP): an update 12.15 - 12.30 Starzonek S, Following erythrocyte sedimentation by optical methods 12.30 - 12.45 Penič S, Sensor for sedimentation of erythrocytes 12.45 - 13.00 Berry M, Validation of Interferometric Light Microscopy for assessment of extracellular vesicles in plasma: Preparing the path for future clinical practices 13.00 - 13.15 Paliska N, Operating sirens - The highest notes in Mozart's vocal works 13.15 - 13.30 Ulokina O, Hommage to Sergej Rachmaninov Section 10: Farm extracellular vesicles organized by Gabriella Pocsfalvi 11.30 - 10.50 Moubarak M, Tomato extracellular vesicles 11.50 - 11.10 Robledo G, Microfluidics for extracellular vesicles 12.10 - 12.30 Schabussova I, Bacterial extracellular vesicles 12.30 - 12.50 Cillo F, Extracellular vesicles – virus interplay 12.50 - 13.10 Vaino S, In vitro analysis of farm extracellular vesicles 13.10 - 13.30 Kwang-Pyo K, Mass spectrometry – based extracellular vesicles analyses Section 11: Sustainable use of polymers organized by Irena Pulko 11.30 - 11.40 Pulko I, Bioplastics and sustainable development 11.40 - 11.50 Viltužnik B, Industry 4.0 and sustainable development 11.50 - 12.00 Oberleitner A, Controlled modification of biopolymers and their versatile applications 12.00 - 12.10 Bolka S, Nardin B, Pešl T, Rozman T, The effect of size and surface treatment of nucleating agents on PA6 morphology studied by Flash DSC 12.10 - 12.20 Verbič A, Golja B, Likozar B, Novak U, Unveiling PFAS-free solutions for Hydrophobic and Oleophobic textile coatings 12.20 - 12.30 Hriberšek M, Kulovec S, Design Optimization and Fatigue Evaluation of Wood Composite Gears 12.30 - 12.40 Bolka S, Nardin B, Pešl T, Rozman T, The Use of Different Fractions of Waste Thermosets for Thermoplastic composites 12.40 - 12.45 Kusič D, Sustainable use of polymers using the AMT2P e-platform in the field of robotics, 3D printing and injection molding 12.45 - 12.50 Kusič D, CIRCI – The application of circular economy principles in industry processes 12.50 - 12.55 Kusič D, Optimisation of Production by 3DP 12.55 - 13.00 Kusič D, Kusič D, Presentation of the AIS measuring system for polymer injection control V Proceedings of 10 th Socratic Lectures 2024 13.00 - 13.10 Sever Škapin A, Mušič B, Weathering effect of the cellulose acetate microplastic from discarded cigarette butts 13.10 - 13.20 Horvat B, Mušič B, Green Transition in Building and Civil Engineering Industry, Alkali-Activated Materials and Alkali-Activated Foams Section 12: Colloids and membranes organized by Aleš Iglič 11.30 - 11.50 Raj Kumar S, Coiling of cellular protrusions around fibers 11.50 - 12.10 Cordoyiannis G, Bar L, Losada-Pereza P, Experimental studies of biomimetic membranes by means of quartz crystal microbalance 12.10 - 12.30 Mesarec L, Orientational ordering of curved membrane proteins attached to curved membrane surfaces 12.30 - 12.50 Benčina M, Stainless steel in biomedical aplications 12.50 - 13.10 Rawat N, Nanostructured TiO2 for selective biological response 13.10 - 13.30 Birk L, Use of gaseous plasma for dental applications Section 13: Physics of matter organized by Tomaž Gyergyek 11.30 - 12.10 Matej Daniel, Schrodinger equation 12.10 – 12.20 Žulj T, Dark matter 12.20 – 12.30 Panov P, Use of electromyography in neuroprosthesis control 12.30 - 12.40 Navarette Armisen R, Rankine cycle, steam turbines and optimization of its efficiency 12.40 - 12.50 Garcia Moya L, Maxwell's equations 12.50 - 13.00 Vitkovski F, Nanoelectronics 13.00 - 13.10 Chikh N, Effect of a lightning strike on an airplane 13.10 - 13.20 Pinilla Doblas E, The Doppler effect 13.20 - 13.30 Barriola Andraga N, Carnot cycle Section 14: Topology and particles organized by Samo Kralj 11.30 - 12.00 Dobovišek A, Principle of maximal entropy 12.00 - 12.30 Markovič R, Biological systems and networks 12.30 - 13.00 Krajnc T, Goričan G, Čokor E, Bucik D, Kralj S, Particle description of nature 13.00 - 13.10 Savujec M, Caloric effects in liquid crystals 13.10 - 13.20 Hoebl A, Quasi-particles in liquid crystals 13.20 - 13.30 Zid M, Topological charge conservation VI Proceedings of 10 th Socratic Lectures 2024 Section 15: Digital Transformation organized by Makhanov Nursultan 11.30 -11.45 Makhanov N, AI in Medicine: Federeted Learning and Medical Imaging 11.45 -12.00 Nurseitov A, Innovative Approaches to Language Learning through the Lens of Use Artificial Intelligence 12.00 - 12.15 Protasenko O, Digital education in the development of the company’s human capital 12.15 - 12.30 Dybach I, Features of the formation of cross- cultural communication skills in it business 12.30-12.45 Vlasenko T, Nemashkalo K, Shapovalova I, Digital transformations: trends and perspectives worldwide 12.45-13.00 Salun M, Zaslavska K, Strategies for resilience in a dynamic world: from VUCA to BANI 13.00 - 13.15 Haborets O, Kushkovyi A, OSINT Technologies: Applications and Challenges in the Digital Age 13.15 -13.30 Lunhol O, Torhalo P, Artificial Intelligence in Law Enforcement: current state and development prospects Section 16: Science meets Business, Innovation and Art organized by Yelena Istileulova 11.30 - 11.50 Kublashvili B, Maglakelidze T, Transformative trends: A paradigm shift to student-oriented learning in higher education 11.50 - 12.10 Tokushev D, Corporate social responsibility as the key factor of sustainable development of business 12.10 - 12.30 Ovin R, Geder M, Development gaps at public universities in the region – the case of Slovenia 12.30 - 12.50 Cizelj B, Challenges of modernising university curricula in Europe 12.50 - 13.10 Dostiyarova A, Start-up simulation in change management; students experience 13.10 - 13.30 Istileulova Y, Innovations, arts and artificial intelligence: Innovative teaching and learning through STEAM approach (SMS method) Section 17. How to organized by Mitja Drab 11.30 - 12.00 Kregar A, How to encode the date of Younger Dryas comet impact on the stone pillars of Gobekli Tepe 12.00 - 12.15 Drab M, How to shape cells using membrane inclusions 12.15 - 12.30 Turk J, How to model small length scale deformations of injection-molded parts 12.30 - 12.45 Jan M, How to think about the nature of Descartes' ideas 12.45 - 13.00 Sojecka AA, How to think about Universal patterns of global population change: from doomsday day growth to the reversed criticality 13.00 - 13.15 Kostanjevec J, How to say the right thing? Remarks on expresivity and thinkability in recent debates on category theory 13.15 - 13.30 Marin A, How to hide: Camouflage from ultraviolet to infrared VII Proceedings of 10 th Socratic Lectures 2024 13.35-13.55 Honorary lecture for all participants Marjana Rupnik, European Commission, Language Department, Linguistic Professions in the European Union Institutions and Artificial Inteligence 13.55-14.00 Cultural program Agata Angelika Sojecka - harp, Borys Urbanowicz - viola C. Saint-Saens - The Swan G. Faure - Apres un reve 14.00 Closing of the symposium https://uni-lj-si.zoom.us/j/98919724250 VIII Proceedings of 10 th Socratic Lectures 2024 Editorial Proceedings of the 10th Socratic Lectures is divided into two parts as we obtained 47 contributions that were after review accepted for publication; the Part I is composed of 30 papers and the Part II is composed of 17 papers and 10 posters that were presented at the Symposium. This volume presents Part II. The 10th Socratic lectures consisted of three events: an Organ recital of Roberta Schmid from Naples, Italy at the Church of Assumption, Tromostovje on Friday, 8.12. 2023, the Scientific symposium on Saturday, 9.12.2023, online and a social - cultural event on Monday, 11.12.2023 the accompanying social event was held at the Academy of Music, Palace Kazina. The event included classical and contemporary music performed by musicians and by scientists. We have embedded Socratic lectures into the so-called Z-STEAM project (STEAM means Science, Technology, Engineering, Arts and Mathematics). Welcome to the future activities within Z-STEAM project. We welcome contributions that support life-friendly methods and collaboration in all fields of knowledge and creation. Contact kraljiglic@gmail.com or annaromolo@gmail.com Veronika Kralj-Iglič, Yelena Istileulova and Anna Romolo IX Proceedings of 10 th Socratic Lectures 2024 CONTENTS PAPERS 1. Zupanc Timon, Milovanović Branislav: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome – Etiology, Pathophysiology, Diagnosis and Treatment …………………………………………………………..……...1 2. Battalian Théo: Respiratory management of Acute Respiratory Distress Syn-drome (ARDS) in the Intensive Care Unit from Early Diag-nosis: Narrative Review ………………..………………………………………8 3. Erjavec Vladimira, Lukanc Barbara: Retrospective Review of 27 Cases of Congenital Portosystemic Shunt in Dogs from 2015 to 2023 ………………………………………………………………………………………...39 4. Erjavec Vladimira, Celinšek Barbara, Jelovčan Petra, Krofič Žel Martina, Lisjak Anja, Milevoj Nina, Nemec Svete Alenka, Pavlica Matic, Pogorevc Estera, Praper Ksenija, Ravnik Verbič Urška, Suhadolc Scholten Sara, Šilc Ema, Turk Živa, Verk Barbara, Lukanc Barbara: Data of 27 Cases of Congenital Portosystemic Shunt in Dogs from 2015 to 2023 …..………………………………………………………………….. 48 5. Vejzović Anel, Tandir Faruk, Hadžiomerović Nedžad, Avdić Rizah, Dučić Nejra: Exotic Animals Vascular Syste Characteristics and Its Application in Clinical Practice ……………………………………………..51 6. Romolo Anna, Kralj-Iglič Veronika: Milestones in Research of Small Cellular Particles. Membrane Modeling and Theoretical Description in Connection with In vitro and Ex vivo processes…………….………......59 7. Nenadović Milena, Kržišnik Katja, Trebše Polonca, Bavcon Kralj Mojca: Effect of Time, pH, Alcohol and Sugar Content on Nicotine Release from Pouches Available on Slovene Market ……………………………..70 8. Rozman Iza, Štern Alja, Žegura Bojana: . New Approaches for Testing the (Geno)Toxic Activity of Nanoparticles In Vitro…………………………………………………………………………………………..………... 77 9. Nardin Blaž, Pešl Teja, Rozman Tamara, Bolka Silvester: The Effect of Size and Surface Treatment of Nucleating Agents on Polyamide 6 Morphology Studied by Flash Differential Scanning Calorimetry ……………………………………………………………………………………………………..………… 87 10. Nardin Blaž, Pešl Teja, Rozman Tamara, Bolka Silvester: The Use of Different Fractions of Waste Thermosets for Ther-moplastic Composites ……..……………………………………………………………………..99 11. Žulj Tim : Dark Energy and Dark Matter………………………………………………………………………...…... 113 12. Lunhol Olha, Torhalo Pavlo: Artificial Intelligence in Law Enforcement: Current State and Development Prospects …………………………………………………………………………………….…………...120 13. Tokushev Damir: Corporate Social Responsibility as a Key Factor for Develop-ment of Sustainable Business ………………………………………………………………………………………………………………..….126 14. Cizelj Boris: Challenges of Modernising European Universities …………--……………………………………... 131 15. Istileulova Yelena: STEAM Approach: SMS (Stories Based on Music about Scientists) on Artificial Inteligence Created by Jacob Bruce (1669–1735) …..……………………………………………………….…………138 16. Lap Patricija, Lipovšek Anja, Jeran Marko: The Slovenian Model of Scientific Research Work of Secondary School Students and their Participation in Competitions as Motivation for Career Choice……………………………………………………………………………………………………….………….….148 17. Kovačič Pšenica: Cat Facial Action Coding System (CatFACS) and Scientific Il-lustration……………………...159 X Proceedings of 10 th Socratic Lectures 2024 POSTERS 18. Árva Zsolt, Barbulova Ani, Fiume Immacolata, Maneea Moubarak Maneea, Pócsfalvi1 Gabriella: A multiplex GC-MS/MS method for the quantitative analysis of active ingredients in Ginkgo biloba derivatives………………………………………………………………………………………..………………………...P 1 19. Iglesias Batalla Alvaro, Defior Pons Aina, Mur Maruša, Schofs Laureano, Kralj Iglič Veronika, Ponikvar Svet Maja, Tramšek Melita, Tavčar Gašper, Jeran Marko : Production and morphological properties of water based cellular particles containing biologically active molecules from European spruce (Picea abies)needle homogenates by experimental and theoretical approaches ……… …………………...P 2 20. Handanagić Amar, Karaeminogullar Oguz : Minimally invasive surgery : end oscopic spine surgery………...P 3 21. Kovačič Pšenica: Decoding the facial expressions of cats: Insights through scientific illustration…………….....P 4 22. Pečavar Nežmah Patrik, Erdani Kreft Mateja, Jeran Marko: Sodium disufonate salt as a water soluble fluorescence active fluorescein analogue and its activity in labelling urothelial urinary bladder cancer cells……………………………………………………………………………………………………….P 5 23. Roganović Aleksandra, Istileulova Yelena: Lithium ion battery project: ESTEAM perspectives………………..P 6 24. Romolo Anna, Iglič Aleš, Griessler-Bulc Tjaša, Kralj-Iglič Veronika: Hybridosomes from Dunaliella tertioletca and Phaedactylum tricornutum sludge………………………………………………………………………..P 7 25. Rozman Iza, Alja Štern Alja, Žegura Bojana: New Approaches for Testing the (Geno)Toxic Activity of Nanoparticles In Vitro…………………………………………………………………………………………………P 8 26. Ruparčič Matija, Dolinar Marko: New Approaches for Testing the (Geno)Toxic Activity of Nanoparticles In Vitro……………………………………………………………………………………………………P 9 27. Vrabec Katja, Goričar Blaž, Božič Darja, Mavri Ana, Raspor Andrej, Novak Valentina, Petrović Koshmak Ivana, Leskovec Maja: Characterization of EVs Subpopulations From CIMmultus ® EV Using PATfix ® System …………………………………………………………………………………………….P 10 XI Proceedings of 10 th Socratic Lectures 2024 1 of 165 Myalgic Encephalomyelitis/Chronic Fatigue Syndrome – Etiology, Pathophysiology, Diagnosis and Treatment Zupanc Timon1,*, Milovanović Branislav 1 1. University of Belgrade, Faculty of Medicine * Correspondence: Timon Zupanc; zupanc.timon@gmail.com Citation: Zupanc T, Milovanović B. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome – Etiology, Pathophysiology, Diagnosis and Treatment. Proceedings of Socratic Lectures. 2024, 10, 1-6. Abstract: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex condition characterized by a broad spectrum of overlapping symptoms and manifesting in multiple systems of the body. Patients experience symptoms such as profound fatigue, postexertional malaise (PEM), unrefreshing sleep, and cognitive impairments. It affects millions worldwide, yet much is still unknown about its etiology and pathophysiology. The condition's onset is frequently linked to infectious triggers, including viral infections, suggesting a dysregulated immune response as a central component. Diagnosing ME/CFS poses significant challenges due to many unspecific symptoms that overlap with various other conditions. Current treatment strategies focus primarily on symptomatic relief and lifestyle modifications to manage disease impact. The COVID-19 pandemic has further spotlighted ME/CFS, drawing parallels between long COVID and ME/CFS symptomatology and underscoring the urgent need for comprehensive research. https://doi.org/10.55295/PSL.2024.II1 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). Keywords: Myalgic Encephalomyelitis, Chronic Fatigue Syndrome, Infectious trigger, Post-Exertional Malaise, COVID-19, Post-COVID syndrome Proceedings of 10 th Socratic Lectures 2024 2 of 165 1. Introduction Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex and disabling, multi-systemic chronic disease affecting millions of people around the world. Despite its increasing prevalence and debilitating nature, it is still largely unknown to both the public and many medical professionals. Understanding of its etiology and pathophysiology is limited, diagnostic criteria inconsistent, and diagnostic and treatment guidelines inadequate and outdated (Committee on the Diagnostic Criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, 2015). The disease was initially defined only as a “persistent or relapsing, debilitating fatigue of new onset and unknown origin” (Holmes et al., 1988). In recent years it has been established that it is a disease characterized by a broad spectrum of overlapping symptoms manifesting in multiple systems of the body. These symptoms are often widespread, and shared by many other conditions, making ME/CFS difficult to diagnose. Marked variability in presentation, severity, course, and duration of disease in patients as well as lack of a definitive diagnostic test or biomarker, has resulted in controversy and lack of consensus around its diagnosis, with over a dozen different clinical definitions being used. The inconsistency in diagnostic criteria, along with lack of interest and funding, made it difficult to conduct conclusive research and studies that would help us better understand this disease (Committee on the Diagnostic Criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, 2015; Bested and Marshall, 2015). 2. Etiology and pathophysiology The onset of disease appears to be precipitated by both genetic and environmental factors (Bested and Marshall, 2015). Very often there is a triggering event present at the start of the illness, usually a prolonged infection. Infectious triggers connected to ME/CFS include: Epstein-Barr virus (EBV), Cytomegalovirus, Varicella-Zoster virus, Borrelia burgdorferi (Lyme disease), Coxiella burnetii (Q fever), parvovirus B19, Coxsackie B virus, Chlamydia pneumoniae, mycoplasma, and SARS-CoV-2. Patients may also experience reactivation of previously latent infections (eg. Herpes viruses) (Frémont et al., 2009). Several studies found high titers of specific EBV antibodies in ME/CFS patients as well as the presence of a specific IgG that was not detected in healthy individuals with previous EBV infection (Loebel et al., 2017). The onset of ME/CFS could be explained as an EBV infection-triggered immune system dysregulation. One of the most consistent findings in ME/CFS patients is poor cytotoxic activity of natural killer (NK) cells (Bateman et al., 2021). NK cells are the body’s first line of defense and play a crucial role in surveillance against tumor cells and infections, preventing latent viruses from reactivating. A study of cytokine networks has found significant alterations in the cytokine profiles of ME/CFS patients, which showed differences in the relationships between the cytokines when compared to healthy controls (Broderick et al., 2010). A common hypothesis states that an abnormal antiviral immune response to the original state of microbial inflammation triggers a state of low-grade systemic inflammation. There is decreased ability to fight active or latent infections due to decreased NK cell function as well as chronic immune activation even in the absence of microbial infection, characterized by consistently elevated levels of proinflammatory cytokines and oxidative stress. This constant proinflammatory state of the body may bring about changes in different systems of the body, explaining the wide range of symptoms of ME/CFS. Another mechanism heavily contributing to the severe lack of energy seen in patients is the impairment of aerobic respiration. Mitochondria are a major producer of reactive oxygen species (ROS) in the cell which also makes them extremely susceptible to damage by oxidative stress. Increased levels of proinflammatory cytokines and increased oxidative stress damage mitochondrial DNA and membrane and interfere with the process of oxidative phosphorylation, resulting in decreased ATP production by mitochondria (Bested and Marshall, 2015). 3. Symptoms and manifestations ME/CFS manifests itself in multiple systems of the body. Patients usually exhibit a cluster of signs and symptoms. Some develop all symptoms shortly after the onset, while others develop additional symptoms over time (Committee on the Diagnostic Criteria for Myalgic Proceedings of 10 th Socratic Lectures 2024 3 of 165 Encephalomyelitis/Chronic Fatigue Syndrome, 2015). Fatigue is a broad and common symptom that presents in many different diseases. However, the fatigue experienced by ME/CFS patients is more profound, intense, debilitating and longer-lasting than in other diseases. It is also not a consequence of ongoing exertion and is not alleviated by rest (Bateman et al., 2021). Post-exertional malaise (PEM) refers to the worsening or exacerbation of disease symptoms following physical or mental exertion that was normally tolerated before disease onset. It is the hallmark symptom of ME/CFS, distinct from symptoms of other chronic diseases (Committee on the Diagnostic Criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, 2015). PEM differs from normal exercise intolerance in that it has a delayed onset, an abnormally prolonged recovery period. It usually sets in 12-48 hours after activity and may last anywhere from a few days to many weeks (Jason et al., 2021). Most patients with ME/CFS experience sleep dysfunct ion. The most common sleep-related symptom that patients report is unrefreshing or nonrestorative sleep, often describing it as “feeling as tired upon waking as before going to bed” (Bateman et al., 2021). Patients also suffer from symptoms of neurocognitive dysfunction. When asked to summarize their problems, they usually characterize them as so-called “brain fog” (Bested and Marshall, 2015). This includes symptoms such as confusion, absent-mindedness, difficulty concentrating, disorientation, inability t o multitask, and problems with short-term memory (FDA, 2013). In ME/CFS both the sympathetic and parasympathetic parts of the autonomic nervous system (ANS) and the equilibrium between them may be disturbed, manifesting in dysautonomia (Van Cauwenbergh et al., 2014). The most prevalent manifestation of dysautonomia, present in up to 97% of patients is orthostatic intolerance (OI) (Committee on the Diagnostic Criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, 2015). OI is a group of clinical syndromes in which symptoms worsen upon assuming and maintaining a standing position and are alleviated by lying down. This includes symptoms such as fatigue, lightheadedness, dizziness, syncope, nausea, headaches, heart palpitations, and visual disturbances. Specific subtypes of OI found in ME/CFS are: postural orthostatic tachycardia syndrome (POTS), neurally mediated hypotension (NMH) and delayed postural hypotension. Other common symptoms include: chronic pain (fibromyalgia), gastrointestinal symptoms, food and chemical hypersensitivities, and psychiatric symptoms (Bested and Marshall, 2015). 4. Prognosis Prognosis in ME/CFS is generally considered to be fairly poor. Full recovery is rare with only 5% of patients returning to pre-morbid levels of functioning (Cairns and Hotopf, 2005). About 40% report substantial improvement in their condition, allowing them to resume daily activities, but still functioning at a lower level than pre-disease (Cairns and Hotopf, 2005). Degree of functional impairment ranges from mild to severe. Around 25% of patients are mildly impaired, capable of participating in daily activities at a reduced level. About 50% suffer from a moderate form of disease, are severely limited in their daily activities, and usually incapable of work. Severe form of disease is present in 25% of patients, who spend their days mostly bedbound, requiring assistance to perform basic activities (Committee on the Diagnostic Criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, 2015). 5. Diagnosis The foundation for suspected ME/CFS is a presentation of new-onset fatigue accompanied by a substantial reduction in functional activity that persists for 6 months, along with additional symptoms. A diagnosis of ME/CFS is made if diagnostic criteria are met following an appropriate history, physical examination, and diagnostic testing. Table 1 contains the data on, and comparisons between four of the most used ME/CFS diagnostic criteria in the 21st century (Committee on the Diagnostic Criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, 2015; Fukuda et al., 1994; Carruthers et al., 2003; National Institute for Health and Care Excellence, 2021). Proceedings of 10 th Socratic Lectures 2024 4 of 165 Table 1. Comparison between different diagnostic criteria for ME/CFS. Criteria 1994 Fukunda Required 50% decrease 6 months Required New onset Functional impairment Minimal duration Fatigue Motor-sensory disturbances Cognitive problems (CP) Pain Sleep disturbances PEM Flu-like symptoms Infection susceptibility Food sensitivities Gastrointestinal problems Genitourinary problems Orthostatic intolerance (OI) Respiratory problems Cardiovascular problems Temperature intolerance Thermostatic instability 4 symptoms required from any of these 5 categories 2003 CCC Required Substantial 6 months Required 2 symptoms from either Required Required Required 1 symptom from these 3 categories 1 symptom from these 3 categories 1 symptom from these 4 categories 2015 IOM Required Substantial 6 months Required 2021 NICE Required Significant 3 months Required CP or OI Required Required Required Required Required CP or OI Table 2. Differential diagnoses of ME/CFS. Neurological disorders: multiple sclerosis, vitamin B12 deficiency, spinal stenosis, craniocervical instability Gastrointestinal disorders: celiac disease, food allergy/intolerances, inflammatory bowel disease, small intestinal bacterial overgrowth Infectious diseases: HIV, tick borne diseases, hepatitis B/C, giardia, West Nile virus, Q-fever, Epstein-Barr virus, parvovirus 19 Endocrine disorders: primary Rheumatological disorders: systemic adrenal insufficiency, hyperlupus erythematosus, rheumatoid arthritis, cortisolism, hyperor hypopolymyositis, polymyalgia rheumatica thyroidism Sleep disorders: sleep apnea, Psychiatric disorders: anxiety, depression, narcolepsy bipolar disorder Other: Gulf War illness, cancer, adverse medication effects, severe obesity, COPD Cardiovascular disorders: cardiomyopathy, coronary disease, valve disease, arrhythmias Hematological disorders: anemia (iron deficiency, other forms), iron overload Table 3. Comorbidities of ME/CFS. Neurological disorders: sensory hypersensitivities, migraine headaches, peripheral neuro-pathy, small fiber neuropathy Endocrine disorders: hypothyroidism, HPA axis dysregulation, metabolic syndrome Gastrointestinal disorders: food allergy/intolerances, gut motility issues, celiac disease, irritable bowel syndrome, small intestinal bacterial overgrowth Rheumatological disorders: fibromyalgia, Ehlers-Danlos syndrome, temporomandibular joint dysfunction, Sic casyndrome (dry eyes/mouth) Sleep disorders: sleep apnea, restless Psychiatric disorders: secondary anxiety, leg syndrome secondary depression Other: nutritional deficiencies, vitamin B12 and D deficiencies, obesity Immunological disorders: new or worsened allergies, mast cell activation syndrome, multiple chemical sensitivities, immunodeficiency Autonomic dysfunction: postural orthostatic tachycardia syndrome (POTS), neurally mediated hypotension (NMH), orthostatic hypotension Gynecological disorders: endometriosis, premenstrual syndrome ME/CFS is often a diagnosis of exclusion. Patients present with a wide array of symptoms, many of which are nonspecific and can be commonly observed in other chronic or acute conditions. When making a diagnosis of ME/CFS these diseases need to be excluded as the primary cause of the patient’s problems. Many diseases can also co-exist with ME/CFS and precipitate or potentiate some of its symptoms (Bested and Marshall, 2015). Diagnosis of these comorbid conditions does not exclude diagnosis of ME/CFS but can help guide Proceedings of 10 th Socratic Lectures 2024 5 of 165 treatment and improve the patient's health, function, and quality of life. Tables 2 and Table 3 detail many of the potential differential diagnoses and comorbidities of ME/CFS as outlined in the 2020 US ME/CFS Clinician Coalition Recommendations (US ME/CFS Clinician Coalition, 2020). 6. Treatment Currently, there is no treatment that would entirely cure ME/CFS. The disease can be managed and controlled through symptomatic treatment, non-pharmacological therapy, nutritional supplementation as well as alternative approaches (ME/CFS treatment recommendations, US ME/CFS Clinician Coalition, 2021). The goals of ME/CFS treatment are the improvement of current symptoms, functioning, and quality of life, prevention of symptom worsening, and helping patients understand and cope with the impact of the disease (International Association of CFS/ME, 2014). A drug that would interfere with the pathophysiological processes of ME/CFS has not been identified and approved for use in ME/CFS. In 2021 the first official recommendations for pharmacological therapy of ME/CFS, which focus on symptomatic treatment were outlined and released by the US ME/CFS Clinician Coalition (ME/CFS treatment recommendations, US ME/CFS Clinician Coalition, 2021). Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently prescribed to manage muscle pain and headaches. Tricyclic antidepressants are usually given to improve sleep quality. Fludrocortisone and fluid expansion therapy have proven to be effective in managing orthostatic intolerance. One of the most effective nonpharmacological methods for the management of disease is to teach patients about physical and cognitive “pacing”. Pacing is an individualized approach centered around the adaptation of the patient’s lifestyle based on their functional capabilities by balancing rest and activity, with the goal of conserving energy and avoiding flare-ups of postexertional malaise. Once the patient establishes a baseline level of functioning through pacing, gradual increases in activity can be applied. The use of alternative approaches such as meditation, massage therapy, acupuncture, and chiropractic treatments is not well documented in literature, but may improve the general well-being of patients (International Association of CFS/ME, 2014). 7. COVID-19 and future Over the last two years, the COVID-19 pandemic has resulted in 530 million cases and 6 million deaths worldwide (Worldometer, 2021). While the devastating acute effects of COVID-19 have been well documented, recently the research community’s interest has turned towards the chronic sequelae of the disease (Wong and Weitzer, 2021). Around 87% of patients affected by COVID-19 continue to experience at least one symptom two months after disease onset and up to 30% of people continue to experience at least one symptom, six months after onset (Carfì et al., 2020). This chronic phase of COVID-19 has been given names such as “long COVID” or “post-COVID syndrome. The most frequently reported symptoms six months after onset were fatigue, post-exertional malaise, and cognitive dysfunction (“brain fog”) (Wong and Weitzer, 2021). Considering the characteristic pattern of symptoms along with the post-viral onset, parallels can be drawn to ME/CFS and suggest that post-COVID syndrome could be a very similar disease. 8. Conclusion In conclusion, ME/CFS represents a a growing global issue for patients, healthcare professionals and economies. The advent of the COVID-19 pandemic has not only amplified the global burden of chronic, post-viral syndromes but also highlighted the similarities between long COVID and ME/CFS and the need for new research into the etiology, pathophysiology, diagnosis, and treatment strategies of these diseases. Embracing a multidisciplinary approach to research and patient care will be key in finding a path towards identifying disease triggers, understanding its mechanism refining diagnostic criteria and exploring effective treatments. Proceedings of 10 th Socratic Lectures 2024 6 of 165 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Bateman L, Bested AC, Bonilla HF, Chheda BV, et al. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Essentials of Diagnosis and Management. Mayo Clin Proc. 2021; 96:2861-2878. DOI: 10.1016/j.mayocp.2021.07.004 Bested AC, Marshall LM. Review of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: an evidence-based approach to diagnosis and management by clinicians. Reviews on Environmental Health, 2015; 30: 223-249. DOI: 10.1515/reveh-2015-0026 Broderick G, Fuite J, Kreitz A, Vernon SD, et al. "A formal analysis of cytokine networks in chronic fatigue syndrome." Brain Behav Immun. 2010; 24: 1209-1217. DOI: 10.1016/j.bbi.2010.04.012 Cairns R, Hotopf M. "A systematic review describing the prognosis of chronic fatigue syndrome." Occup Med (Lond). 2005; 55:20-31. DOI: 10.1093/occmed/kqi013 Carfì A, Bernabei R, Landi F. "Persistent symptoms in patients after acute COVID-19." JAMA. 2020; 324: 603-605. DOI: 10.1001/jama.2020.12603 Carruthers BM, Jain AK, De Meirleir KL, Peterson D, Klimas NG, et al. Myalgic encephalomyelitis/chronic fatigue syndrome: clinical working case definition, diagnostic and treatments protocols. J Chronic Fatigue Synd 2003; 11: 7–115. Committee on the Diagnostic Criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome; Board on the Health of Select Populations; Institute of Medicine. "Beyond Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Redefining an Illness." Washington (DC): National Academies Press (US); 2015 Feb 10. Available from: https://www.ncbi.nlm.nih.gov/books/NBK274235/ doi: 10.17226/19012 European Parliament, June 10, 2020. "European Parliament resolution on additional funding for biomedical research on Myalgic Encephalomyelitis." Available from: https://www.europarl.europa.eu/doceo/document/B-92020-0186_EN.html FDA (Food and Drug Administration). "The voice of the patient: Chronic fatigue syndrome and myalgic encephalomyelitis." Bethesda, MD: Center for Drug Evaluation and Research (CDER), FDA; 2013. Available from: https://www.fda.gov/media/86879/download Frémont M, Metzger K, Rady H, Hulstaert J, De Meirleir K. Detection of herpesviruses and parvovirus B19 in gastric and intestinal mucosa of chronic fatigue syndrome patients. In Vivo. 2009; 23: 209-213. Fukuda K, Straus SE, Hickie I, Sharpe MC, et al. The chronic fatigue syndrome: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann Intern Med. 1994; 121: 953-959. DOI: 10.7326/0003-4819-121-12-199412150-00009 Holmes G, Kaplan J, Gantz N, Komaroff AL, et al. Chronic fatigue syndrome: A working case definition. Annals of Internal Medicine. 1988; 108: 387–389. DOI: 10.7326/0003-4819-108-3-387 International Association of CFS/ME. Chronic fatigue syndrome/myalgic encephalomyelitis: a primer for clinical practitioners. July 2014. Available from: https://www.iacfsme.org/assets/pdf/Primer_Post_2014_conference/ Loebel M, Eckey M, Sotzny F, Hahn E, et al. Serological profiling of the EBV immune response in Chronic Fatigue Syndrome using a peptide microarray. PLoS One. 2017; 12: e0179124. DOI: 10.1371/journal.pone.0179124 National Institute for Health and Care Excellence. Myalgic encephalomyelitis (or encephalopathy)/chronic fatigue syndrome: diagnosis and management. NICE guideline [NG206]. Available from: https://www.nice.org.uk/guidance/ng206 ME/CFS treatment recommendations, US ME/CFS Clinician Coalition. Version 1. February 20, 2021. Available from:https://batemanhornecenter.org/wp-content/uploads/filebase/Treatment-Recs-MECFS-Clinician-CoalitionV1-Feb.-2021.pdf US ME/CFS Clinician Coalition. "Diagnosing and Treating Myalgic Encephalomyelitis/Chronic Fatigue syndrome (ME/CFS)," Version 2. July 2020. Available from: https://mecfscliniciancoalition.org/wpcontent/uploads/2021/06/MECFS-Clinician-Coalition-Diagnosis-and-Treatment-Handout-V2.pdf Van Cauwenbergh D, Nijs J, Kos D, Van Weijnen L, Struyf F, Meeus M. Malfunctioning of the autonomic nervous system in patients with chronic fatigue syndrome: a systematic literature review. Eur J Clin Invest. 2014; 44: 516-526. DOI: 10.1111/eci.12256 Wong TL, Weitzer DJ. Long COVID and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)—A Systemic Review and Comparison of Clinical Presentation and Symptomatology. Medicina. 2021; 57:418. DOI: 10.3390/medicina57050418 Proceedings of 10 th Socratic Lectures 2024 8 of 165 Respiratory management of Acute Respiratory Distress Syndrome (ARDS) in the Intensive Care Unit from Early Diagno-sis: Narrative Review Battalian Théo1* 1. Universidad CEU San Pablo, Madrid, Spain * Correspondence: Théo Battalian (theobattalian@gmail.com) Citation: Battalian T. Respiratory management of Acute Respiratory Distress Syndrome (ARDS) in the Intensive Care Unit from Early Diagnosis: Narrative Review. Proceedings of Socratic Lectures. 2024, 10, 8-37. https://doi.org/10.55295/PSL.2024.II2 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Abstract: This study aims to determine the optimal ventilatory technique for different levels of acute respiratory distress syndrome (ARDS) severity to reduce the need for endotracheal intubation (ETI) in the ICU. Secondary objectives include identifying predictors of failure of non-invasive ventilation (NIV), assessing the role of physiotherapists in the ICU across countries, and identifying areas for further investigation. A review of articles from 2013 to 2023 involving adults with ARDS in the ICU was conducted. The PEDro scale assessed article quality, and exclusion criteria included specific aetiologies of acute respiratory failure, extreme obesity, and pregnancy, as well as case series and case study designs. Data were extracted from PUBMED, EBSCO, and ScienceDirect, and a validated questionnaire was administered to physiotherapists in Chile, Spain, and France. Eight studies (number of patients n = 883) were included, and a questionnaire was completed by 22 physiotherapists (6 from France, 6 from Spain, 10 from Chile). Findings highlighted the management of high flow nasal cannula (HFNC) and NIV in different patient populations. Helmet NIV had an 18.2% failure rate and better outcomes than facemask interface. NIV non-response correlated with increased mortality rates. Cox analysis identified predictors of NIV failure. Caution is advised in NIV use, considering predictors of failure, and HFNC may be beneficial in mild ARDS. The majority of included studies had small sample sizes, limiting generalizability. The questionnaire results were constrained by a small sample size and lack of meta analysis. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Keywords: Acute respiratory distress syndrome, high flow nasal cannula, non-invasive ventilation, endotracheal intubation, predictors of failure, physiotherapy. Proceedings of 10 th Socratic Lectures 2024 9 of 165 1. Introduction ARDS is a syndrome characterized by acute respiratory failure due to increased permeability of the alveolar-capillary membrane, leading to non-cardiogenic pulmonary oedema, hypoxemia, and respiratory system compliance impairment. (Thompson et al., 2017; Grasselli et al., 2023). The pathophysiology of ARDS is complex and involves various inflammatory mediators and cellular mechanisms. The initial insult to the lungs can be caused by pulmonary or extrapulmonary factors. Pulmonary ARDS is often caused by direct injury to the lungs, such as pneumonia, inhalation of toxic gases or fumes, aspiration of gastric contents, non – protective ventilation (i.e., Ventilator induced Lung Injury) or drowning (Saguil and Fargo, 2020). In these cases, the inflammatory response is initiated within the lungs, leading to damage to the alveolar-capillary membrane and resulting in pulmonary oedema and hypoxemia. Extrapulmonary ARDS, on the other hand, is caused by systemic inflammation due to a variety of factors such as sepsis, trauma, pancreatitis, or transfusion-related acute lung injury (Kassirian et al., 2020). One of the primary treatments for ARDS is invasive mechanical ventilation (Fan et al., 2017; Tasaka et al., 2022) which involves the use of a ventilator to provide mechanical support to the lungs. The use of invasive ventilation in ARDS has been a topic of extensive research and debate in recent years, with the goal of optimizing patient outcomes and minimizing complications. On the other hand, non-invasive therapies such as NIV and HFNC have been used more extensively in the treatment of ARDS. NIV is a respiratory support technique that aims to deliver positive pressure to the lungs without the need for an endotracheal tube. It is typically delivered through a mask, nasal prongs or a helmet and the positive pressure helps to improve oxygenation and reduce carbon dioxide retention (Peter et al., 2006; Thompson et al., 2017). There is already strong level of evidence for NIV effectiveness in case of exacerbation of Chronic Obstructive Pulmonary Disease (COPD) (Rochwerg et al., 2017) and for cardiogenic oedema (Peter et al., 2006). HFNC is a type of respiratory support that delivers heated and humidified air or oxygen to the patient through a nasal cannula at a flow rate of up to 60 litters per minute (Tasaka et al., 2022). The high flow rates and humidification make it a comfortable and well-tolerated alternative to conventional oxygen therapy or NIV. The use of HFNC is based on the principle that high flow rates and humidification can improve gas exchange, reduce work of breathing, and Positive End Expiratory Pressure (PEEP) to prevent atelectasis. HFNC can be used in a variety of clinical settings, including acute respiratory failure, post-extubation support, pre-oxygenation, and bronchiolitis in infants (Frat et al., 2015; Tasaka et al., 2022). It is known that invasive ventilation through endotracheal intubation can lead to complications such as barotrauma, ventilator-associated pneumonia, and hemodynamic instability (Saguil and Fargo, 2020; Coleman and Aldrich, 2021). In the realm of professions to treat it, ARDS requires many of them. It is important to note that medical and nursing care are usually the ones described when considering the approach. Physical therapy is a cornerstone in the re-education of patients, starting in the ICU. Little is known about the implication of physiotherapists in interventions regarding patients with ARDS in the ICU. Even less is known about the differences that could exist in this same level of implication according to different countries. Therefore, this narrative review proposes to determine - according to the grade of severity of the pathology - which ventilatory technique should be adopted from the early diagnostics, to avoid as much as possible ETI as a primary objective. Additionally, the best non-invasive ventilation support interface discernment will be the secondary objective. To start understanding physiotherapist’s role in the ICU according to the country will be the primary objective of the questionnaire that has been produced during this study. Proceedings of 10 th Socratic Lectures 2024 10 of 165 2. Methods For the narrative review, the search was done from October 2022 until December 2022 in the following databases: PubMed, EBSCO, Science Direct. The identification process retrieved a total of 296 articles (13 from PubMed, 135 from EBSCO and 148 from Science Direct). The software Mendeley was used as a reference tool. The following data in addition to the reasons of exclusion in the assessment of eligibility are represented in Figure 1. The updated preferred reporting items for systematic reviews and meta-analyses (PRISMA) has been used to report the following study. The eligibility criteria encompass individuals aged 18 or older diagnosed with acute respiratory distress syndrome (ARDS), with consideration given to articles published between 2013 and 2023. Inclusion requires adherence to a PEDro scale score of 22 or 23 greater than 5/10 for randomized controlled trials and a focus on the intensive care unit (ICU) population. Conversely, exclusion criteria encompass individuals with other pathologies leading to acute respiratory failure, hypercapnic conditions such as Chronic Obstructive Pulmonary Disease (COPD), clinically extremely obese patients (BMI > 35 kg/m2), or those who are pregnant. Additionally, case series and case studies are excluded from consideration. The 2012 Berlin classification being the updated version of the AECC (American European Consensus Conference) for the diagnosis of ARDS is used by each article. Include Screening Identification Identification of studies via databases and registers Records identified from: (n = 296) PubMed (n = 13) Science Direct (n = 148) EBSCO (n = 135) Records removed before screening: Duplicate records removed (n = 12) Records screened. (n = 284) Records excluded (n = 265) Reports assessed for eligibility. (n = 19) Reports excluded: No access to PDF (n = 4) Review articles (n = 3) COPD population (n = 1) Study protocol (n = 1) Studies included in review. (n = 10) Reports of included studies (n = 10) Figure 1. PRISMA Flow chart. Proceedings of 10 th Socratic Lectures 2024 11 of 165 A questionnaire on the topic of the role of physiotherapists in ICU wards on the management of ARDS and the decision-making process has been conducted. This questionnaire has been answered by physiotherapists who work or used to work in ICUs from France, Spain, and Chile. The primary objective of the later was to determine whether the role of the professional was drastically different between countries, secondarily to have a state of the current practice, and lastly to determine if the COVID 19 pandemic changed the approach. The answers got collected from October 2022 to February 2023. The confection of the questionnaire is based on the current literature and guidelines about the management of ARDS (Arcelo et al., 1998; Peter et al., 2006; Ranieri et al., 2012; Slutsky and Ranieri, 2013; Fan et al., 2017; Munshi et al., 2017; Rezoagli et al., 2017; Thompson et al., 2017; Dries, 2019; Saguil and Fargo, 2020; Grasselli et al., 2020; Grieco et al., 2020; Coleman and Aldrich, 2021; Tasaka et al., 2022) and has been validated in October 2022 by a panel of one experimented teacher in cardiorespiratory physiotherapy of the CEU San Pablo, and the two head of intensive physiotherapy of the Clínica Dávila Vespucio de Santiago de Chile. It has been created with the platform Google Forms, in three languages (Spanish, French and English). Chilean and Spanish answers were collected on two distinct versions. The questionnaire (see Appendix 1) is divided in four sections for a total of 30 questions: Section 1: Personal information – 7 questions; Section 2: Diagnosis of the pathology – 4 questions; Section 3: Management of the pathology – 13 questions; Section 4: Physiotherapist involvement – 6 questions. Overall, the questionnaire got a total of 22 answers (n = 10 from Chile, n = 6 from France, n = 6 from Spain). 3. Results Based on the questionnaire, interpretations can be drawn. While 100% of French physiotherapists found the Berlin classification relevant, 33.3% of Spanish physiotherapists and 40% of Chilean physiotherapists did not. 60% of patients in Chile received between 3 – 5 sessions of physiotherapy per day, but nor Spanish neither French patient exceeded 3 sessions. Respectively 50% of physiotherapists in Spain and Chile did not include the exclusion of cardiogenic oedema as a diagnosis criterion, while only 26.7 of physiotherapists in France did not. 60% of Chilean physiotherapists believed that COVID 19 pandemics changed the diagnostic method while only 33.3% of Spanish and 16.7% of French physiotherapists did not. All the participants used protective factors when setting up mechanical ventilation. However, only 50% of Spanish physiotherapists considered the driving pressure, while 100% of Chilean and French professionals did. Additionally, only 83,3% of French physiotherapists but 100% of Spanish and Chilean physiotherapists considered tidal volume between 6 – 8 ml/kg as a protective factor. From Chilean practice, 70% considered they were completely in charge of HFNC (5/5 points), and 60% considered themselves completely in charge on NIV (5/5 points). Both of those parameters are dropping to 33% for French physiotherapists, and 16% for Spanish ones. 50% of French physiotherapists considered their role in the daily gestation of ventilation parameters as a 3/5, when the majority (70%) of Chilean professionals graded it between 3 and 4/5, and 50% of Spanish graded it 0/5. 100% of Spanish physiotherapists graded 0/5 on their implication in ETI manoeuvre, 66% graded 0/5 and 33 graded 1/5 in France, 40% graded 2/5 and 30% graded 3/5 in Chile. 66% of Spanish physiotherapists graded between 0 and 1/5 of involvement in the decision-making process for evolution between treatment strategies, 70% in Chile graded between 2 and 3/5, 33% in France graded 4/5, and 33% as a 2/5. Lastly, 100% of Chilean physiotherapists but only 33% of French and 66% of Spanish physiotherapists considered that their role in those points were more important after the pandemic. This review is including 7 observational studies ((Frat et al., 2015; Messika et al., 2015; Sehgal et al., 2015; Chawla et al., 2016; Bellani et al., 2017; Menzella et al., 2021; Yaroshetskiy et al., 2022) and one randomized control trial (Patel et al., 2016). They are classified in three categories according to the technique of their investigation. The first Proceedings of 10 th Socratic Lectures 2024 12 of 165 category regards the use of HFNC as a treatment method and includes two articles (Frat et al., 2015; Messika et al., 2015). The second one regards NIV and includes 4 articles (Sehgal et al., 2015; Patel et al., 2016; Bellani et al., 2017; Menzella et al., 2021). Lastly, the third category regards predictors of NIV failure and includes 2 articles (Chawla et al., 2016; Yaroshetskiy et al., 2022). We decided to separate group 2 and 3 since group 2’s results are enhancing the focus on the facts and group 3’s results are focused on drawing conclusions about the failure predictors of NIV. Firstly, we will present the results of the articles, in order to analyse those in a critical way. 3.1. Group 1: Publications on treatment with High Flow Nasal Canula (HFNC) The summary of the characteristics along with the study design, the intervention, and a brief view of the results of the publications in Group 1 can be found in Table 1. 3.1.1. Article characteristics Two studies evaluated the effect of HFNC on ARDS patients. Both publications dated from 2015. The population of interest in each of the article is based on the Berlin classification from 2012 and is scoping patients with approved diagnosis of ARDS, that are above 18 years old and are admitted to the ICU. However, Messika et al. (2015) are not taking into consideration in their diagnosis the parameter that requires a PEEP of 5 cm/H2O on NIV since the treated population would be with HFNC. The sample size of the studies was 23 (Frat et al., 2015) and 45 (Messika et al., 2015). Table 1: The summary of the characteristics, the study design, the intervention and a brief view of the results of the Publications on treatment with High Flow Nasal Canula (HFNC) (Group 1). Population characteristics Frat et al. (2015) - n = 23 within a year - ARDS patients from Berlin classification - Male gender 78% - Median age 61 years - SAPS II 36 points - Pneumonia 64% - Severity (mild 34%, moderate 61%, severe 4%) Messika et al. (2015) - n = 45 within a year - ARDS from Berlin classification without considering the NIV criterion. - Male gender 49% - Median age 57.9 years - SAPS II 36 points - Pneumonia 80% - Severity (mild 29%, moderate 38%, severe 33%) Study design Prospective observational Prospective observational single centre Intervention Results 2 hours session of HFNC, then 1 hour session of NIV for a total of 16 hours of HFNC and 8 hours of NIV per day Failure rate of 8/23 subjects (35%) requiring ETI No ETI for intolerance reasons Increase of PaO2 in comparison standard oxygen Increase of PaO2 /FiO2 ratio only in NIV Decrease of RR and HR HFNC better tolerated than NIV, although second session of NIV was better than the first Use of HFNC for first line treatment of ARDS when need >9L/min of oxygen to maintain SpO2 > 92% Failure of 18/45 subjects (40%) requiring secondary ETI Success of 26 subjects only with HFNC and one in combination with NIV Additional organ failure increases HFNC failure rate 3.1.2. Methodology Each study is based on a prospective observational approach, with a (STrengthening the Reporting of OBservational studies in Epidemiology) score of 19 out of 22. On the STROBE score, the lack of study's funding sources and assessment of the generalizability of the study findings, are the items downgrading them to 19 points (Table A2.1 in Appendix 2). Proceedings of 10 th Socratic Lectures 2024 13 of 165 3.1.3. Population characteristics It is quite similar on the following points: median age (61 years (Frat et al., 2015) vs 57.9 years (Messika et al., 2015)) and the Simplified Acute Physiology Score II of 36 in both cases. However, the aetiology is different since population in Messika et al. (2015) were treating cases from pneumonia in 80% of the patients, and Frat et al. (2015) were treating 64% of pneumonia-induced ARDS. The severity of the pathology in the patient group from Messika and al. (2015) is notably homogenous, when the other population is focused to 61% of patients with moderate ARDS. 3.1.4. Results of the therapy Messika et al. (2015) are describing the use of HFNC as first line treatment in 29% of the subjects admitted for acute respiratory failure. Additionally, HFNC was used at all stages of the pathology, from first line treatment to pre-ETI, post-extubation or palliative care. The study of Frat et al. (2015) that is combining HFNC and NIV, points out that PaO2 increased in 20 out of 28 patients included in the study for acute respiratory failure, and that it was even higher when in combination with NIV in comparison with standard oxygen therapy. On the other side, only NIV caused a decrease in the PaO2/FiO2 ratio. Moreover, remaining stable throughout the entire HFNC/NIV sessions, respiratory rate and heart rate significantly decreased after the initiation of the therapy. 3.1.5. Tolerance of the therapy HFNC was better tolerated than NIV, according to a lower score on the visual analogue scale of 16 mm versus 61 mm (P=0.004). However, comfort was better on the second session of NIV at 49 mm on the scale (Frat et al., 2015). 3.1.6. Failure rate In the study of Messika et al. (2015) is about 40% of the patients, and the main reasons for ETI were worsening of hypoxemia in 72%, onset of hemodynamic failure with 22% and onset of neurological failure with 6%. The subjects included in the failure of HFNC group had significantly higher SAPS II scores in comparison to the success group. The failure rate on the combined treatment in the study of Frat et al. (2015) is 35% for ARDS patients and 36% for acute respiratory failure, and the reasons for ETI are worsening of distress in 70% patients, shock for 20% of them, and respiratory arrest for the last 10% 3.1.7. Predictors of failure It is associated to lower PaO2/FiO2 ratio after initiation of HFNC (115.3 vs 145.3 mmHg, P=0.26), SAPS II scores (46 vs 29), and hemodynamic failure in the univariate analysis, but only the SAPS II score in the multivariate analysis according to Messika et al. (2015). In the case of Frat et al. (2015) failure was associated to a breathing frequency of >30 breaths/min at 1 hour after initiation of the first HFNC session with a sensitivity of 94.1% and a specificity of 87.5%. 3.1.8. Mortality rate According to Messika et al. (2015) mortality rate was 50% in patients in whom the treatment failed (p = 0.001 for the failed treatment) and 4% in patients in whom the treatment was successful. Therefore, meaning that mortality rate was much lower in successful patients. Frat et al. (2015) are reporting a mortality rate of 20% for the population in which the treatment failed, but it was not statistically significantly relevant (p = 0.12). Consequently, it does not precisely represent the percentage of deceased patients caused by the failure of the non-invasive treatment. 3.2. Group 2: Publications on treatment with non-invasive ventilation The summary of the characteristics along with the study design, the intervention, and a brief view of the results of the publications in Group 2 can be found in Table 2. Proceedings of 10 th Socratic Lectures 2024 14 of 165 Table 2. The summary of the characteristics, the study design, the intervention and a brief view of the results of the Publications on treatment with non-invasive ventilation (NIV)(Group 2). Population characteristics Bellani et al. (2017) Sehgal et al. (2015) Menzella et al. (2021) Patel et al. (2016) - n = 436 for 4 weeks - ARDS patients from Berlin classification aged > 16 years - Male gender 58.9% - Median age 68 years - non-pulmonary SOFA 3 points - No etiology - Severity (mild 27.3%, moderate 53.2%, severe 19.5%) - n = 41 for one year - ARDS from AECC classification, >18 years - Exclusion of severe ARDS patients - 65.7% women - Median age 30.9 +/- 11.4 years - APACHE II 18 points - Malaria 26.8%, Typhus 19.5% - Severity (mild 56.1%, moderate 43.9%, severe 0%) - n = 79 over a month - COVID 19-ARDS patients - Male gender 56(71%) - Median age 66.5 +/- 11.4 years - SOFA 4.3 points - Charlson comorbidity index 3.4 - Severity (mild 0%, moderate 100%, severe 0%) Study design Prospective observational multicentre international cohort Prospective observational Intervention Failure rate of 131/436 subjects (37.5%) requiring ETI. Greater severity of ARDS associated with an increase of recognition of ARDS and worsening in NIV with any type of interface outcomes including LOS and ICU mortality but not hospital mortality. or ventilatory mode for at least Use of NIV didn’t vary according to the severity 1 or 2 days to be category of ARDS. enrolled as NIV NIV patients had lower PEEP and higher respiratory rate in comparison to IMV. patients NIV failure in case of higher SOFA, lower PaO2 /FiO2 ratio and % of increase of PaCO 2 within first 2 treatment days. NIV using ICU ventilators. First 24 hours, off only for oral intakes, then depending on patient, increase of off periods. Use of NIV on Retrospecpatients with a tive observaPaO2 /FiO2 ratio tional >100 and <200 mmHg - n = 83 for 3 years - ARDS with mask NIV>8 hours, >18yo, berlin criteria - Male gender 54% face mask, Single centre 55% helmet randomized - Median age 60.9 years face clinical trial. mask, 58 helmet - APACHE II 26 points face mask, 25 points helmet. - Pneumonia 36% - Severity (60% PaO 2 /FiO2 < 200 mm Hg) Results After 8 hours of face-mask NIV Control (39/83): continue with face mask on a single limb ventilator. Intervention (44/83): switch to helmet interface with double limb ventilator. Failure of 23/41 subjects (56%) requiring ETI. NIV failure group had significantly higher APACHE II. Duration of ventilation higher in IMV. Significant decrease of respiratory rate in both groups. Predictors of NIV failure: no improvement of PaCO2 /FiO2 within 1 hour and higher baseline APACHE II. Failure of 41/79 subjects (51.9%) requiring ETI in 21(26.6%) and death in 20(25.3%). 18/20 deceased were not eligible for ETI. Failure predictors according to a multivariate COX regression model: SOFA score. No higher mortality rate for failed NIV patients. ETI rate of 61.5% in face mask group, 18.2% in helmet group. PEEP of 8.0 (median) in intervention group VS 5.1 in control. Reduction of tachypnoea from face mask to helmet interface. Helmet group had more ventilator-free days (28 vs 12.5) and less ICU LOS (4.7 vs 7.8 days). Hospital and 90-days mortality significantly lower in helmet group. Independent association of APACHE II with 90days death rate (still lower for helmet group) 3.2.1. Articles characteristics The second part of the review consists of four articles, one randomized control trial (Patel et al., 2016) and three observational studies (Sehgal et al., 2015; Bellani et al., 2017; Menzella et al., 2021). The publication dates are ranging from 2015 to 2021 and they are all investigating the effect of NIV on ARDS patients. Proceedings of 10 th Socratic Lectures 2024 15 of 165 3.2.2. Methodology Two studies are using the Berlin classification as a diagnosis method (Bellani et al. (2017) and Patel et al. (2016), Menzella et al.(2021) are not specifying the diagnosis criteria but are mentioning the treatment of moderate to severe cases. In the case of Sehgal et al. (2015) they are using the now changed classification of the American European Consensus Conference (AECC) (Villar and Kacmarek, 2012) as a diagnostic criterion. Sehgal et al. (2015) are reporting cases of ARDS from tropical diseases such as malaria in 26.8% and typhus in 19.5% of the patients, only 9.7% was pneumonia related. The sample sizes are 436 patients in 4 weeks in Bellani et al. (2017), 41 patients in one year in Sehgal et al. (2015), 79 patients in one month in Menzella et al. (2021), and 83 patients in 3 years in Patel et al. (2016). 3.2.3. Patient’s characteristics Bellani et al. (2017) describe their population as being older, with a lower non-pulmonary SOFA score when comparing to intubated population. Additionally, they report that the NIV population is more prone to suffer from comorbidities such as chronic renal failure, congestive heart failure, and chronic obstructive pulmonary disease compared to intubated patients. Finally, they didn’t find any significant difference about immunocompromised patients between both groups but was still an important number of subjects in each. Patel et al. (2016) are comparing their control and intervention group at baseline, and the findings are related to what Bellani et al. (2017) are stating. It was found that more than half of the overall non-invasive population are immunocompromised by virtue of cancer or transplant, and about one third had an immunocompromised pneumonia. 3.2.4. NIV failure Regarding the outcomes, focusing on the failure of the treatment method, Sehgal et al. (2015) are reporting a rate of 56% of failure, 51.9% of failure for Menzella et al. (2021) with 26.6% requiring ETI, and 25.3% being deceased, 61.5% for face-mask group (control) and most importantly only 18.2% for helmet group (intervention) according to Patel et al. (2016). Lastly, Bellani et al. (2017) are reporting a rate of 37.5% of failure with a bigger sample size than any other of the studies. APACHE II has a high median value in the study of Patel et al. (2016) when looking closer to the same value in the study of Sehgal et al. (2015) (18 vs 25-26 points). Additionally, population in Sehgal et al. (2015) has a median age of 30.9 in comparison to the three other population ranging from 60.9 to 68 years. 3.2.5. Ventilation and patient’s parameters When comparing NIV with invasive ventilation, NIV had significantly lower levels of PEEP, and higher respiratory rates than the second one (Bellani et al., 2017). Moreover, measured tidal volumes and minute ventilation were greater in population treated by NIV. However, the latter and the former were not affected by ARDS severity in patients treated with NIV compared to patients treated with invasive ventilation. Patel et al. (2016) are reporting that patients from their control group had a median sustained PEEP level of 5.1 cm H2O (face mask group) and that their intervention group was at a level of 8.0 cmH2O (helmet group) (p = 0.006 for both values). PEEP titration in the case of face-mask interface was a challenge because patients did not tolerate it well and because of excessive air-leak. Lastly, tachypnoea significantly decreased when patients got randomly assigned from the facemask to the helmet interface (from 27.7 breaths/min to 24.5 breaths/min) (p<0.001). It was pointed out in the study of Bellani et al. (2017) that in their consequent study population, NIV was used almost as frequently in the three severity groups of ARDS, 14.3% for mild, 17.3% for moderate and 13.2% for severe cases of the pathology. 3.2.6. Reasons for ETI When coming to the decision to intubate, refractory hypoxemia and tachypnoea caused by the face mask are reported as being the main reasons (Patel et al., 2016, Sehgal et al., 2015). In one case, neurologic failure was described as altered sensorium Proceedings of 10 th Socratic Lectures 2024 16 of 165 and in one case the Richmond Agitation Sedation Scale (RASS) dropped below 8 points. However, when comparing those results with the intervention group of the randomized control trial, the first reason for ETI is neurologic failure and not respiratory failure. This can easily be correlated to the failure rate from t he intervention group with the helmet interface. Lastly, Bellani et al. (2017) are reporting that PaO2/FiO2 ratio was increasing more rapidly in patients treated with invasive mechanical ventilation in comparison to NIV. 3.2.7. Secondary outcomes Regarding the hospital length of stay Bellani et al. (2017) are reporting worsening of this outcome along with ICU mortality according to the severity of the ARDS, although it was also correlated with a higher clinical recognition. According to Patel et al. (2016) the helmet group spent only 4.7 days in the ICU compared to the 7.8 days for the facemask groups, and helmet group had more ventilator free days than the facemask group (28 vs 12.5 days). However, statistically, hospital length of stay was not different. Menzella et al. (2021) reported a longer duration of NIV for successful subjects compared to failed ones and deceased ones (8.7 vs 2.9 vs 6.3 days respectively). In the case of Sehgal et al. (2015) the total ventilation duration was recorded (non-invasive and invasive), with a result of 2.75 days for NIV success, and 5.2 days for failure. Additionally, only regarding the NIV ventilation, the duration was 2.75 days in success patients and 3 hours for failure subjects. However, there was no significant difference in the ICU stay between each group. 3.2.8. Mortality rates According to Menzella et al. (2021) mortality rate was not significantly different between both groups of NIV (failure and success) (43% vs 36%, p=0.61). However, Sehgal et al. (2015) reported that out of the 23 subjects who failed NIV, 19 got deceased (82.6% of NIV failure death), and out of the 18 patients of success, none got deceased. The total percentage of death of the study reached 46.3%. Patel et al. (2016) are reporting that hospital and 90-days mortality was significantly lower in the helmet group compared to the facemask group (48.7% vs 27.3% for hospital, 56.4% vs 34.1% for 90 -days). Additionality they found that APACHE II was independently associated to 90 days death with a confidence interval of 95%. Bellani et al. (2017) are reporting relevant data on the mortality rates. Firstly, between NIV and invasive ventilation, there was no significant difference in ICU and hospital mortality. However, in the case of failed NIV patients, the rates are reaching 42.7% of ICU mortality compared to the 10.6% for the success subjects (p<0.001). After performing a multivariate COX regression model adjusting covariates significantly associated with outcomes; NIV use was independently associated with increased ICU but not hospital mortality with a 95% confidence interval. Another multivariate COX regression analysis was performed on baseline characteristic of NIV group, that showed that chronic heart failure, hematologic or neoplastic disease, chronic liver failure, age, ARDS severity, percentage decrease of PaO2/FiO2 ratio between days 1 and 2, total respiratory rate and non-pulmonary SOFA score were each independently associated to in-hospital death. 3.2.9. Predictors of failure for NIV Sehgal et al. (2015) performed a univariate logistic regression analysis and found out that failure of improvement in the PaO2/FiO2 ratio within an hour of treatment, and a higher baseline APACHE II scores, were associated with failure of NIV. It should be taken into consideration that this analysis in only univariate. Menzella et al. (2021) conducted a univariate and multivariate COX regression model on those parameters associated with NIV failure, and out the two analyses, only SOFA admission score was significantly correlated with failure, leaving PaO2/FiO2 ratio out. This can show that COVID ARDS might behave in a different manner. Bellani et al. (2017) conducted a multivariate COX regression analysis that revealed that higher non pulmonary SOFA score, lower PaO2/FiO2 ratio, and the percentage increase of PaCO2 over the first days of treatment were independently associated to NIV failure within 28 days. The Proceedings of 10 th Socratic Lectures 2024 17 of 165 sample size of this article gives a higher scientific weight, and on the contrary, the small sample size from Sehgal et al. (2015) is dragging its conclusions to a lower level. 3.3 Group 3: Publications on predictors of NIV failure The summary of the characteristics along with the study design, the intervention and a brief view of the results of the publications in Group 3 can be found in Table 3. 3.3.1. Articles characteristics The analysis of two articles was done in the third part of this study. Yaroshetskiy et al. (2022) gathered a population of 80 patients within 6 months of study, and Chawla et al. (2016) gathered a population of 96 patients within 3 years of study. All the patients were treated with NIV, through a full-face mask. Chawla et al. (2016) are using the Berlin classification as a diagnosis method for the patients, knowing that the aetiology of the ARDS is coming from pneumonia in 55.3% of the patients. However, Yaroshetskiy et al. (2022) are evaluating patients with COVID 19-ARDS complying with at least one of the following criteria: fatigue, excessive visible WOB assessed by the Patrick scale (Patrick et al., 1996) (4-5 points), SpO2 < 92%. Precise ARDS classification diagnosis was not reported and only moderate to severe patients were included (Table 3). Table 3. The summary of the characteristics along with the study design, the intervention, and a brief view of the results of the publications in Group 3 Population characteristics Yaroshetskiy et al. (2022) Chawla et al. (2016) - n = 102 within 6 months, 80 with NIV - COVID 19 ARDS with at least one of the following criteria: fatigue, excessive visible WOB assessed by Patrick scale(Patrick et al., 1996) (4-5 points), SpO2<92% - Male gender overall 54(56.3%) - Median age overall group 71.5 - SOFA overall 4 points - Severity only from moderate to severe ARDS - n = 170 within three years, 96 with NIV - ARDS from Berlin classification > 18 years - Male gender overall 61.8% - Median age overall 47.54 years - SOFA overall 8 points - APACHE II overall 17.42 points - Pneumonia 55.3% - Severity overall (mild 34.7%, moderate 41.8%, severe 23.5%) Study design Prospective observational clinical Prospective observational Intervention Results Use of oxygen therapy <15 L/min or CPAP outside ICU as screening. 2 hours NIV trial as entrance test. Then, if tolerated, NIV, if not, ETI Failure rate of 57/80 subjects (71.3%) requiring ETI After 48 hours of NIV, if PaO2 /FiO2 < 112 mmHg, ROX < 5.02, PET CO2 <19.5mmHg and Patrick score >= 2 then failure can be predicted. Respiratory rate can also be considered. NIV failure higher in older and/or more frailer patients, longer COVID duration without NIV NIV through a nonvented full-face mask with and ICU ventilator using a dedicated NIV mode Failure of 42/96 subjects (43.8%) requiring ETI Failure rate higher in severe (83.3%) and moderate (73%) Low PaO2 /FiO2 , septic shock and severity of ARDS as factors associated to failure. Higher mortality in failure of NIV patients. Longer LOS in NIV patients ICU mortality: 30.2% for NIV, 45.9% for IMV 3.3.2. Methodology Yaroshetskiy et al. (2022) is an observational prospective clinical study with a score of 20 out of 22 points on the STROBE scale. Chawla et al. (2016) is a prospective observational study with a STROBE score of 21 out of 22. Study's funding source and an assessment of the generalizability of the study findings is lacking for Yaroshetskiy et al. (2022) and only the assessment of the generalizability of the study findings is lacking for Chawla et al. (2016). Proceedings of 10 th Socratic Lectures 2024 18 of 165 3.3.3. Failure and mortality rates According to Yaroshetskiy et al. (2022), out of 80 patients, 57 (71.3%) failed the treatment. Among those patients, they were all intubated and 3 were put under extracorporeal membrane oxygenation (ECMO). The other ones were not eligible for ECMO because of exclusion criteria (mostly >65 years old). The mortality rate was 100% for this group. According to Chawla et al. (2016) out of 96 patients, 42 (43.8%) failed NIV by requiring invasive mechanical ventilation. Depending on the severity of the ARDS, the failure rate was higher. In moderate ARDS the failure rate was 27/37 (73%) and in severe ARDS it was 5/6 (83.3%) as compared to mild ARDS with 10/53 (18.9%) (p=0.000). Among the patients who failed NIV, 69% (29/42 patients) of them died, with 7/10 (70%) for mild, 18/27 (66.7%) for moderate and 4/5 (80%) for severe ARDS. 3.3.4. NIV failure predictors In the study of Chawla et al. (2016) the univariate analysis showed that admission APACHE II and SOFA scores along with low admission PaO 2/FiO2 ratio, presence of severe sepsis or septic shock or multiorgan dysfunction, presence of confusion, absence of H1N1 influenza A infection, and severity of ARDS were the 9 factors for prediction of NIV failure. In the multivariate analysis however, only low admission PaO2/FiO2 ratio (p = 0.049), presence of septic shock (p = 0.001) and severity of ARDS (p = 0.007) were validated. According to a ROC analysis, Yaroshetskiy et al. (2022) have stated that after 48 hours of treatment the following parameters can be described as predictors of failure of NIV: 1) ROX index <5.02 (p < 0.001), 2) PaO2/FiO2 < 112 mmHg (p < 0.001), 3) PETCO2 < 19.5 mmHg, 4) Patrick scale  2. Lastly, they concluded that the probability of failure was higher in older/frailer patients and in patients with a longer duration of COVID 19 without NIV. 4. Discussion The HFNC is a relatively new technique that has been incorporated into ICUs for good reasons. However, the findings of this review about the use of the latter as an effective treatment option for patients with ARDS are fragile. There are lots of limitations and precautions to consider when interpreting results from those articles. Starting by the study design of both, which is observational, and the fact that they don’t comply with the entirety of the items in the STROBE score. This is additional to the population sample size, that even though growing in comparison to previous case series or case studies, stays low. Moreover, the HFNC technique presents a limitation when referring to ARDS. It is thought to generate low levels of PEEP, depending on the volume applied (between 35 and 60 L/min) and most importantly depending on if the patient stays mouth closed. This lack of precise knowledge about the PEEP level, confronts itself with the essence of diagnosis method of ARDS. The Berlin classification is including one crucial parameter that makes it the evolution of the AECC, the minimum PEEP level of 5 cmH2O. In this case, HFNC cannot provide such information and then block ARDS proper identification by health professionals. A post hoc analysis from two prospective studies by Coudroy et al. (2018) comes in line to defend this position and try to revise the Berlin classification on this very precise point of minimum level of PEEP. However, even though debates are forming, in this precise time, minimum level of PEEP is required to consider a diagnosis of ARDS. On the level of failure rate, it is clearly stated that the use of HFNC is related to less failure of the treatment with 35% (Frat et al., 2015) and 40% (Messika et al., 2015), in comparison to studies on the use of NIV that reports failure rate in between 37.5% and 61.5% in the case of full-face interfaces (Sehgal et al., 2015; Patel et al., 2016; Bellani et al., 2017; Menzella et al., 2021). However, one of the greatest concerns in any noninvasive ventilatory support, is not the failure rate itself, but the hospital/ICU mortality for the subjects that are part of the failure group. This is shown by Bellani et al. (2017) when taking as a study population, the largest ARDS database available nowadays. They reported with a ROX regression analysis that NIV was associated with mortality, and the use of HFNC on this matter remains unknown. If considering the Proceedings of 10 th Socratic Lectures 2024 19 of 165 results of the two articles of this review, Messika et al. (2015) are reporting 50% of deceased patients from the failure group, but this result is drawn from a sample of 18 subjects in the failure of the treatment, which is really low. Frat et al. (2015) are not reporting such data. Lack of power of both studies on such matters prevents any conclusions from being properly deduced. Messika et al. (2015) concluded that HFNC was not improving the PaO2/FiO2 ratio itself but only the FiO2, which can prove that levels of PEEP provided by HFNC is not sufficient to improve the overall gas exchange. Frat et al. (2015) with their approach of combination of both techniques with NIV, is then using the beneficial effect of oxygenation improvement from the NIV. In the combined use of HFNC and NIV, is has been found that tolerance levels were significantly better in the case of HFNC rather than NIV, and a previous study by Antonelli et al. (2007) depicted an ETI rate due to NIV intolerance of 25% (Antonelli et al., 2007) in patients with ARDS. However, when considering that patients have to keep their mouth closed to benefit from the effectiveness of HFNC, another concern can be drawn on whether the tolerance level assessment was made respecting this criterion. To continue the positive sense of combination of HFNC and NIV, Frat et al. (2015) have reported that no ETI due to interface intolerance was conducted, knowing that the interface used in this study is a full-face mask. Patel et al. (2016) have proven the efficacy of helmet interface in comparison to full-face mask on multiple level, such as failure rate and mortality. This could be a good opening for next research, evaluating the efficacy and failure rate of combined HFNC and Helmet NIV alternatively. It is advanced that HFNC have an effect on decreasing the respiratory rate (Frat et al., 2015). However, it is important to keep in mind which are the predictors of failure of NIV (Table 3) since this study is using both HFNC and NIV. As a matter of fact, respiratory rate is never going further in the predictors than a univariable analysis. By relying on multivariable analysis, then HFNC doesn’t have any substantial/statistical effect on avoiding failure of NIV. When conducting their multivariable analysis on the predictors of mortality Bellani et al. (2017) found that respiratory rate was one of them. Thus, statistically, HFNC has an effect on reducing mortality, but not on reducing failure rate. This conclusion can be turned into a positive sense, since as mentioned before, one of the greatest concerns from the analysis of the entire articles is focused on the mortality rate of the failure group. Messika et al. (2015) are observing the use of HFNC at any stage of ARDS, 33% as severe, 38% as moderate and 29% as mild. However, SAPS II scores are relatively low (median of 36 for all subjects, 29 for success and 46 for failure) and the majority of patients did not present with an additional organ failure. The use of HFNC can be done at any stage if the ETI timing is respected. The need for further research on the exact population of application is needed to establish a clear view. The questionnaire conducted in this study gives multiple information on the management of ARDS in the ICU. HFNC is designed as the major first line treatment for mild ARDS for physiotherapists in Spain (along with standard oxygen therapy), France and Chile, and the first with NIV in the case of moderate ARDS but only for physiotherapists in Spain. Limitations are multiple to this questionnaire, such as lack of power due to sample size, or lack of statistical analysis of the results. However, the conclusion of this survey is that in nowadays practice, HFNC is used broadly as the first line therapy in mild ARDS. NIV in its turn is an older technique that has been used for a long time in acute respiratory failure from COPD or cardiogenic oedema. Healthcare professionals in the ICUs are highly trained for the technique. However, in the case of ARDS, the studies of this review are creating a brand-new perspective. Results from the studies are reporting many deductions, but one crucial point here is to point out which outcome should be the main one to be analysed. Failure rate of the therapy is the first one that would seem logical, in the sense that if the patient doesn’t fail the therapy, then the outcomes are positive, which seems to be the case. (Bellani et al., 2017) is the largest study population sample of all the articles and has a failure rate of only 37.5% out of the 436 subjects enrolled. Sehgal et al. (2015) reported a failure rate of 56%, but this study presents a couple of limitations that deserves to be pointed Proceedings of 10 th Socratic Lectures 2024 20 of 165 out. The study sample is clearly different from all of the other studies, by its small sample size of 41 patients, by its aetiology of ARDS coming in most of the cases from tropical diseases (extra-pulmonary ARDS) and leaving pneumonia for less than 10% of the patients, and most importantly, including in the research patients that are staying under NIV for extremely short period of time, hence the median timing of ventilation of 3 hours for patients who failed the treatment. A limitation of this study is also the exclusion of severe ARDS patients. Failure rates in other studies are around 50% for NIV, which can be positively interpreted by saying that in case of both extrapulmonary and pulmonary ARDS, NIV is effective in half of the patients. The results of Yaroshetskiy et al. (2022) are conflicting to interpret since they report a failure rate of 73%. In this case it is important to note that they only included moderate to severe ARDS population and that is has been shown in the predictors of failure that lower PaO2/FiO2 at baseline was highly associated with NIV failure (Chawla et al., 2016; Bellani et al., 2017). This leads towards the fact that the use of NIV on more severe population leads to higher failure rates. However, Bellani et al. (2017) stated that the use of NIV was not different in practice between the three severity groups, against the current state of evidence on the subject. This raises questions on whether this “non-adequate” use of the therapy is deliberate or if lack of precision and sharpness of knowledge could be at the origin. As a result of the questionnaire, it is clear that Spanish physiotherapists are not in complete osmosis with the current knowledge since none of the participants considered ETI as a first line treatment for moderate ARDS and shows that Chilean professionals are more prone to take a scientifically based decision, with 40% of them considering ETI as first line treatment, and 60% for NIV as first line. On the contrary, most participants of the questionnaire apart from one Spanish subject (HFNC), and 2 French ones (depends on the patient) did not consider other possibilities than invasive ventilation in the case of severe ARDS, which clearly seem to be the best option when evoking failure rates and improvement of parameters. One result that points out from the studies is the comparison in the randomized control trial of Patel et al. (2016) of facemask and helmet NIV. The failure rate in the facemask group is 61.5% when in the helmet group drastically drops to 18.2%. This result is conclusive on the net effect and difference of the two interfaces when regarding the failure rate. Following a logical interpretation of the results, the focus of the question in NIV should not only be on the failure rate but also on the mortality rates (ICU, Hospital, 90-days). This concern has been raised in previous articles (Antonelli et al., 2001; Rana et al., 2006) and the results of this review are going in this sense. Bellani et al. (2017) reported 42.7% of mortality among the subjects who failed NIV which stands as the smallest and most optimistic value. Sehgal et al. (2015) reported 82.6% of mortality, Yaroshetskiy et al. (2022) 100%, Chawla et al. (2016) 69%, and Patel et al. (2016) 48.7% vs 27.3% for hospital, 56.4% vs 34.1% for 90-days, respectively in the case of facemask and helmet. Chawla et al. (2016) enhance this statistics by giving details depending on the severity of the pathology, and moderate and severe results are above 70% of mortality. Bellani et al. (2017) used multivariate COX analysis is linking NIV and ICU mortality indicating that NIV in the case of moderate and severe cases seem to lead towards high mortality rates. This link could be explained by the fact that patients under NIV are according to two studies of this review older, frailer, and with a greater number of comorbidities. Although these parameters are defending the treatment technique, it is important to be aware of the risks of delayed ETI, and the consequences it can have. By showing that NIV was used for any severity of the pathology, Bellani et al. (2017) are unrevealing some points that could include persistence of NIV against ETI criteria, thus leading to the late ETI. Lastly, the same study is showing that mortality in patients with a PaO2/FiO2 of less than 150 mmHg was 36.2% for NIV and 24.7 with IMV. This opens a new window of research in the field and calls for answers on what is the current state of practice on the ETI level. Being able to pick the right population when implementing any non-invasive technology could reduce failure rate, but mostly could allow mortality in this category to decrease. Proceedings of 10 th Socratic Lectures 2024 21 of 165 In the case of NIV, outcomes are clearly differing when using a full-face mask in comparison to the helmet interface. In the ETI reasons, Patel et al. (2016) and Sehgal et al. (2015) are pointing out respiratory failure as the first reason for ETI in the case of fullface mask NIV. As an example, this includes refractory hypoxemia or tachypnoea. As a correlation, tachypnoea can be associated to worsening of the respiratory failure, but can also be coming from psychological reasons, and most importantly stress itself (Gerritsen and Band, 2018). Frat et al. (2015) clearly showed that full-face mask comfort for the patient is not optimal, and the grades given to it on the visual analogue scale are going in this way. This would be the first conclusion drawn from the randomized control trial, that helmet is more comfortable and less aggressive for the patient, thus can have an effect on tachypnoea. Moreover, the randomized control trial shows that helmet ventilation is providing higher levels of PEEP by 3 cm H2O more than the mask. This allows the tachypnoea to be reduced as well and to correct even better the PaO2/FiO2 ratio level. Bellani et al. (2017) found that provided levels of PEEP were more important in invasive mechanical ventilation rather than NIV, but if the study had been done on helmet interfaces, the outcome would have changed, and possibly reaching the same levels. This outline one limitation of NIV through facemask, since they are not able to provide high levels of PEEP, the management of severe and even moderate ARDS patients will be far from optimal, and ETI would be recommendable. However, in the case of using helmet interfaces, the titration of PEEP is not limited by air leak and patient discomfort, which would allow successful management of this more severe population of ARDS. Continuing on the beneficial effect of helmet NIV, Patel et al. (2016) are showing that ICU length of stay (4.7 vs 7.8 days) and ventilator-free days (28 vs 12.5 days) are significantly and effectively reduced in the helmet group, and even though total hospital length of stay is not significantly different, being out of the ICU means avoidance of ICU related complications such as ICU – acquired weakness, or ventilatory related complications such as V-ILI or P-ILI or nosocomial infections. Predictors of NIV failure are then crucial. They are bringing the answer to how and when to stop the non-invasive technique to switch the patient to ETI and avoid the delay of the latter. Almost each article is reporting its set of predictors but Sehgal et al. (2015) as an example are only treating data into a univariate COX regression model and not in the multivariate model. Thus, those data are not significant enough to be considered relevant. However, 4 studies are reporting multivariate COX analysis, and their conclusions are the following. Menzella et al. (2021) only achieved to bring the SOFA score at this position, and not have the low PaO 2/FiO2 ratio. This can be interpretated that it is one of the differences between ARDS and COVID-ARDS since the general systemic state will apparently weight more than the severity of the ARDS alone. Bellani et al. (2017) are reporting higher non-pulmonary SOFA score, lower PaO2/FiO2 ratio, percentage of increase of PaCO2 over the first days as failure predictors within 28 days. SOFA score is then cited in two of the articles. Chawla et al. (2016) are then using low admission PaO2/FiO2 ratio, presence of septic shock and the severity of ARDS as predictors in their multivariate analysis. Lastly, Yaroshetskiy et al. (2022) are the most precise by stating that if after 48 hours of ventilation, 1) ROX index < 5.02, 2) PaO 2/FiO2 < 112 mmHg, 3) PETCO2 < 19.5 mmHg (using a capnometer) and 4) Patrick score  2, then failure can be predicted. Therefore, predictors can be uncovered before applying the therapy thanks to the SOFA score, lower PaO2/FiO2 ratio, presence of septic shock, severity of ARDS and can be effectively assessed after 48 hours of ventilation with the protocol established by Yaroshetskiy et al. (2022) . These findings are limited by the lack of precise information on the correlation between SOFA score, PaO2/FiO2 and severity of ARDS and failure prediction. Further investigation should be performed in order to determine the optimal limitation values when predicting failure. Figure 2 is depicting the knowledge drawn from this review. The questionnaire is limited by lack of power due to sample size. Moreover, additional statistical data interpretation would allow the aforementioned to gain power. The latter is limited knowing that it serves only as informative data. The objective of this questionnaire is to have a global idea of the actual involvement and management of Proceedings of 10 th Socratic Lectures 2024 22 of 165 the pathology of and by physiotherapists; therefore, conclusions can’t be properly drawn. They can lead to further research on the topic to provide areas of involvement of the profession. Figure 2. ARDS early ventilatory management scheme 5. Conclusions To conclude, the effective use of High-Flow Nasal Cannula (HFNC) in conjunction with Non-Invasive Ventilation (NIV) is recommended for treating mild to moderate Acute Respiratory Distress Syndrome (ARDS), but caution is advised, and HFNC should be avoided in cases of severe ARDS. NIV application in moderate and severe ARDS requires careful consideration, with a thorough assessment of predictors of failure both before and during therapy. Notably, NIV shows an association with a higher failure rate in more severe ARDS cases. Survey responses indicate widespread utilization of HFNC as a primary treatment for mild ARDS. Helmet NIV demonstrates superior outcomes in terms of mortality rates, failure rates, secondary outcomes, and patient comfort when compared to full-face masks. The use of helmet NIV is associated with enhanced comfort for patients compared to full-face masks. Comparatively, physiotherapists in Chile exhibit greater involvement in the daily care of ICU patients with ARDS than their counterparts in France and Spain. Some responses from French and Spanish professionals deviate from current guidelines on ARDS management and are identified as areas for improvement. Ongoing research is needed to investigate the potential combination of helmet NIV with HFNC in reducing mortality rates. Further exploration is required to enhance the precision of NIV predictors. Additionally, there is a need for research focusing on the use of helmet NIV in the moderate to severe ARDS population. Proceedings of 10 th Socratic Lectures 2024 23 of 165 Conflicts of Interest: The authors declare no conflict of interest. References 1. Antonelli M, Conti G, Moro M, et al. Predictors of failure of noninvasive positive pressure ventilation in patients with acute hypoxemic respiratory failure: a multi-center study. Intensive Care Med. 2001; 27:17181728. DOI:10.1007/S00134-001-1114-4 2. Antonelli M, Conti G, Esquinas A, et al. A multiple-center survey on the use in clinical practice of noninvasive ventilation as a first-line intervention for acute respiratory distress syndrome. Crit Care Med. 2007; 35:18-25. DOI:10.1097/01.CCM.0000251821.44259.F3 3. 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DOI:10.3390/DIAGNOSTICS10121053 16. Macedo LG, Elkins MR, Maher CG, Moseley AM, Herbert RD, Sherrington C. There was evidence of convergent and construct validity of Physiotherapy Evidence Database quality scale for physiotherapy trials. J Clin Epidemiol. 2010; 63:920-925. DOI:10.1016/J.JCLINEPI.2009.10.005 17. Messika J, Ahmed K Ben, Gaudry S, et al. Use of high-flow nasal cannula oxygen therapy in subjects with ARDS: A 1-year observational study. Respir Care. 2015; 60:162-169. DOI:10.4187/respcare.03423 18. Menzella F, Barbieri C, Fontana M, et al. Effectiveness of noninvasive ventilation in COVID-19 relatedacute respiratory distress syndrome. Clinical Respiratory Journal. 2021; 15:779-787. DOI:10.1111/CRJ.13361 19. Moseley AM, Sherrington C, Elkins MR, Herbert RD, Maher CG. Indexing of randomised controlled trials of physiotherapy interventions: a comparison of AMED, CENTRAL, CINAHL, EMBASE, Hooked on Evidence, PEDro, PsycINFO and PubMed. Physiotherapy. 2009; 95:151-156. DOI:10.1016/j.physio.2009.01.006 Proceedings of 10 th Socratic Lectures 2024 24 of 165 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. Munshi L, Del Sorbo L, Adhikari NKJ, et al. Prone position for acute respiratory distress syndrome: A systematic review and meta-analysis. Ann Am Thorac Soc. 2017; 14: S280-S288. DOI:10.1513/AnnalsATS.201704-343OT Patel BK, Wolfe KS, Pohlman AS, Hall JB, Kress JP. Effect of Noninvasive Ventilation Delivered by Helmet vs Face Mask on the Rate of Endotracheal Intubation in Patients With Acute Respiratory Distress Syndrome A Randomized Clinical Trial Preliminary Communication | CARING FOR THE CRITICALLY ILL PATIENT. JAMA. 2016; 315:2435-2441. DOI:10.1001/jama.2016.6338 Patel BK, Wolfe KS, Pohlman AS, Hall JB, Kress JP. Effect of noninvasive ventilation delivered by helmet vs face mask on the rate of endotracheal intubation in patients with acute respiratory distress syndrome a randomized clinical trial. JAMA - Journal of the American Medical Association. 2016; 315:2435-2441. DOI:10.1001/jama.2016.6338 Patrick W, Webster K, Ludwig L, Roberts D, Wiebe P, Younes M. Noninvasive positive-pressure ventilation in acute respiratory distress without prior chronic respiratory failure. Am J Respir Crit Care Med. 1996 ; 153:1005-1011. DOI: 10.1164/ajrccm.153.3.8630538 Peter JV, Moran JL, Phillips-Hughes J, Graham P, Bersten AD. Effect of Non-Invasive Positive Pressure Ventilation (NIPPV) on Mortality in Patients with Acute Cardiogenic Pulmonary Oedema: A MetaAnalysis. Lancet. 2006; 367:1155-1163. DOI: 10.1016/S0140-6736(06)68506-1 Rana S, Jenad H, Gay PC, Buck CF, Hubmayr RD, Gajic O. Failure of non-invasive ventilation in patients with acute lung injury: observational cohort study. Crit Care. 2006; 10: R79. DOI:10.1186/CC4923 Ranieri VM, Rubenfeld GD, Thompson BT, et al. Acute respiratory distress syndrome: The Berlin definition. JAMA. 2012; 307:2526-2533. DOI:10.1001/jama.2012.5669 Rezoagli E, Fumagalli R, Bellani G. Definition and epidemiology of acute respiratory distress syndrome. Ann Transl Med. 2017; 5:282. DOI:10.21037/atm.2017.06.62 Rochwerg B, Brochard L, Elliott MW, et al. Official ERS/ATS clinical practice guidelines: noninvasive ventilation for acute respiratory failure TASK FORCE REPORT ERS/ATS GUIDELINES. Eur Respir J. 2017; 50:1602426. DOI:10.1183/13993003.02426-2016 Saguil AMM, Fargo MV. Acute Respiratory Distress Syndrome:Diagnosis and Management. AmFam Physician. 2020; 101:730-738. Sehgal IS, Chaudhuri S, Dhooria S, Agarwal R, Chaudhry D. A study on the role of noninvasive ventilation in mild-to-moderate acute respiratory distress syndrome. Indian Journal of Critical Care Medicine. 2015; 19:593-599. DOI:10.4103/0972-5229.167037 Slutsky AS, Ranieri VM. Ventilator-Induced Lung Injury. New England Journal of Medicine. 2013; 369:2126-2136. DOI:10.1056/NEJMra1208707 Tasaka S, Ohshimo S, Takeuchi M, et al. ARDS clinical practice guideline 2021. Respir Investig. 2022; 60:446-495. DOI:10.1016/J.RESINV.2022.05.003 Thompson BT, Chambers RC, Liu KD. Acute Respiratory Distress Syndrome: 50 years. Drazen JM, ed. New England Journal of Medicine. 2017; 377:562-572. DOI:10.1056/NEJMra1608077 Vandenbroucke JP, Von Elm E, Altman DG, et al. Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): Explanation and Elaboration. Epidemiology. 2007; 18:805-35. DOI: 10.1097/EDE.0b013e3181577511. Villar J, Kacmarek RM. The American-European Consensus Conference definition of the acute respiratory distress syndrome is dead, long live positive end-expiratory pressure! Med Intensiva. 2012; 36:571-575. DOI:10.1016/j.medin.2012.08.010 Von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: Guidelines for Reporting Observational Studies. J Clin Epidemiol. 2008; 61:344-349. DOI: 10.1016/j.jclinepi.2007.11.008. Yaroshetskiy AI, Merzhoeva ZM, Tsareva NA, et al. Breathing pattern, accessory respiratory muscles work, and gas exchange evaluation for prediction of NIV failure in moderate-to-severe COVID-19-associated ARDS after deterioration of respiratory failure outside ICU: the COVID-NIV observational study. BMC Anesthesiol. 2022; 22: 307. DOI:10.1186/S12871-022-01847-7 Proceedings of 10 th Socratic Lectures 2024 25 of 165 6. Apendixes 6.1. Appendix 1 Proceedings of 10 th Socratic Lectures 2024 26 of 165 Proceedings of 10 th Socratic Lectures 2024 27 of 165 Proceedings of 10 th Socratic Lectures 2024 28 of 165 Proceedings of 10 th Socratic Lectures 2024 29 of 165 Proceedings of 10 th Socratic Lectures 2024 30 of 165 Proceedings of 10 th Socratic Lectures 2024 31 of 165 Proceedings of 10 th Socratic Lectures 2024 32 of 165 Proceedings of 10 th Socratic Lectures 2024 33 of 165 Proceedings of 10 th Socratic Lectures 2024 34 of 165 Proceedings of 10 th Socratic Lectures 2024 35 of 165 Proceedings of 10 th Socratic Lectures 2024 36 of 165 Proceedings of 10 th Socratic Lectures 2024 37 of 165 6.2. Appendix 2 Table A2.1. Scoring of articles according to STROBE statement and PEDro scale Frat et al. (2015) STROBE score (Vandenbroucke et al., 2007; Von Elm et al., 2008) PEDro scale (Moseley et al., 2009; Macedo et al., 2010) 19 out of 22 x Messika et al. (2015) 19 out of 22 x Menzella et al. (2021) 20 out of 22 x Bellani et al. (2017) 22 out of 22 x Sehgal et al. (2015) 16 out of 22 x Patel et al. (2016) x 8 out of 10 Yaroshetskiy et al. (2022) 20 out of 22 x Chawla et al. (2016) 21 out of 22 x Missing information/items - Study's funding sources - Assessment of the generalizability of the study findings. - Study's funding source - Sample size calculation - Assessment of the generalizability of the study findings. - Study's funding source - Assessment of the generalizability of the study findings. x - Study's funding - Potential sources of bias - Assessment of the generalizability of the study findings - Blinding of therapists - Blinding of assessors for mortality outcome - Treatment or “intention to treat” for mortality outcome - Study's funding source - Assessment of the generalizability of the study findings. - Assessment of the generalizability of the study findings Proceedings of 10th Socratic Lectures 2024 39 of 165 Retrospective Review of 27 Cases of Congenital Portosystemic Shunt in Dogs from 2015 to 2023 Erjavec Vladimira1, Lukanc Barbara1,* 1. University of Ljubljana, Veterinary Faculty, Small Animal Clinic, Ljubljana, Slovenia * Correspondence: Barbara Lukanc, barbara.lukanc@vf.uni-lj.si Citation: Erjavec V, Lukanc B. Retrospective Review of 27 Cases of Congenital Portosystemic Shunt in Dogs from 2015 to 2023. Proceedings of Socratic Lectures. 2024, 10, 39-46. https://doi.org/10.55295/PSL.2024.II3 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional Abstract: The study aims to conduct a retrospective review of 27 cases of a congenital portosystemic shunt in dogs from 2015 to 2023, providing insights into classification, clinical manifestations, diagnostics, anaesthesia, surgery, and perioperative care. Initiation of medical therapy at least 14 days before surgery aims to alleviate clinical signs and mitigate anaesthesia and surgery risks, emphasising a low-protein diet and lactulose to trap ammonium ions in the intestinal lumen and was strictly followed in all patients. Changes in drug uptake, metabolism, and excretion should be considered when choosing an anaesthesia protocol. All dogs, except one with absent portal vein, underwent cellophane placement, facilitating gradual shunt closure. All dogs recovered uneventfully from surgery without evidence of portal hypertension and showed clinical improvement after that. claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). Keywords: Portosystemic shunts, dogs, cellophane attenuation, perioperative preparation Proceedings of 10th Socratic Lectures 2024 40 of 165 1. Introduction A portosystemic shunt (PSS) in dogs is an abnormal connection between the portal vascular system and the systemic circulation (Buttler et al., 1990; Watson and Hertage, 1998; Mullins, 2019a). This condition diverts blood from the abdominal organs, bypassing the liver, which filters and detoxifies the blood before it returns to the heart (Sleight and Thomford, 1970; Tieman Mankin, 2015; Bertolini, 2019). PSS are classified into acquired and congenital. Congenital PSS (CPSS) result from abnormalities in the development of the portal venous system or foetal vessels and are divided into two categories: extrahepatic and intrahepatic (Bertolini, 2019; Mullins, 2019a). CPSS predominantly enter the prehepatic caudal vena cava (Mathews and Gofton, 1988) at the epiploic foramen, termed portocaval shunts (Mullins, 2019b). Portophrenic shunts entering the left hepatic vein or caudal vena cava at the diaphragm level exhibit milder symptoms than other shunts (Berent and Tobias, 2018). Portoazygos shunts, typically involving the left gastric vein, occasionally the right gastric vein, transverse the diaphragm through the oesophageal hiatus and enter the azygos vein in the thorax (Harari et al., 1990). Acquired PSS are commonly caused by portal hypertension resulting from liver cirrhosis or liver failure (Boothe et al., 1996; Tieman Mankin, 2015; Berent and Tobias, 2018). Breeds at an increased risk of extrahepatic CPSS are Cairn and Yorkshire Terriers, Maltese, Tibetan spaniels, miniature Schnauzers, Shih Tzu, Poodles (Tobias and Rohrbach; 2003), Pugs and Dachshunds (Wolschrijn et al., 2000; Tobias, 2003). The clinical manifestations of CPSS lack specificity and may fluctuate over time, impacting the central nervous system, gastrointestinal system, and urinary tract (Konstantinidis et al., 2023). General signs in dogs include poor weight gain and stunted growth (Tobias, 2012). Hepatic encephalopathy and seizures may also be seen in PSS with the signs of hepatic encephalopathy, which are ataxia, depression, changes in behaviour, circling, disorientation, head pressing, blindness, seizures, and coma. In about 30% of dogs, vomiting, diarrhoea, anorexia, and salivation occur (Howe and Boothe, 2002; Konstantinidis et al., 2023). Clinical signs of the urinary system are associated with ammonium biurat crystals and urate calculi and include dysuria, hematuria, pollakiuria, stranguria and decreased urine specific gravity (Howe and Boothe, 2002; Broome et al., 2004; Tobias, 2012; Caporali et al., 2015). In dogs, PSS can decrease the levels of various blood parameters such as total protein (TP), albumin, blood urea nitrogen (BUN), total cholesterol, and glucose. Elevated ammonia levels and serum bile acid are notable and serve as reliable indicators for diagnosing PSS in dogs (Ruland et al., 2010). Leucocytosis may be evident (Winkler et al., 2003), and mild to moderate microcytic normochromic nonregenerative anaemia is often observed alongside (Tobias, 2012; Tieman Mankin, 2015). Surgical intervention is widely acknowledged as the preferred treatment for PSS, given that medical treatment alone cannot address the underlying vascular anomaly. The primary advantage of medical therapy lies in its ability to minimise ammonia absorption from the gastrointestinal tract, aiming to prevent hepatic encephalopathy and seizures. The increased ammonia concentration in plasma could also be attributed to bacterial degradation of amino acids in the gastrointestinal tract (Self, 2016). Before undergoing anaesthesia, dogs with PSS should undergo medical stabilisation (Dugdale et al., 2020). Dogs with PSS are often young, and to avoid the risk of hypoglycemia, food should only be withheld four to six hours prior to anaesthesia (Self, 2016). Dogs with PSS require minimal premedication with opioids, induction with propofol or alfaxalone, and anaesthesia should be maintained with isoflurane or sevoflurane (Self, 2016; Dugdale et al., 2020). Low doses of alpha-2-adrenoceptor agonists can also be used for premedication, and opioids should be continued during surgery (Self, 2016). Nonsteroidal anti-inflammatory drugs should be avoided due to coagulopathies and hypoproteinaemia, and caution is advised also with paracetamol application because of hepatic insufficiency (Dugdale et al., 2020). Notably, due to their small body size and reduced metabolic heat production by the small liver, dogs with PSS are particularly prone to hypothermia (Self, 2016). Intravenous administration of Hartmann's solution is necessary during both the surgical procedure and the recovery period. Continuous monitoring of blood glucose levels and body temperature Proceedings of 10th Socratic Lectures 2024 41 of 165 is imperative during recovery, with particular attention to observing seizures. Seizures can be effectively managed by addressing metabolic disturbances such as hypoglycemia and elevated ammonia levels. Additionally, pharmaceutical options, including levetiracetam, phenobarbitone, propofol, or low doses of dexmedetomidine or medetomidine, are viable choices for seizure control (Dugdale et al., 2020). Post-surgery, ensuring adequate analgesia is essential, and opioids represent a recommended approach for managing pain (Self, 2016). 2. Materials and methods Patient data regarding CPSS were collected from medical records at the Veterinary Faculty in Ljubljana, Slovenia, covering the period from 2015 to 2023. Out of the 27 dogs included in the study, detailed medical history and laboratory results were available for 23 dogs, while CT scans were available for 25. However, due to the retrospective nature of the study, complete data retrieval was not possible for all 27 dogs (Erjavec et al.,2024). Additionally, over one-third of the dogs were referred from other clinics and were under the care of their primary veterinarian after surgery; thus, complete data for these dogs were also not available. Since this study involved the review of existing medical records and did not involve any new interventions or procedures on animals, formal ethical approval was not required. All dogs underwent surgical treatment, and among the 27 surgically treated patients, two (a Golden Retriever and a mixed breed dog) were found to have an intrahepatic PSS. The diagnosis was established based on clinical signs, laboratory findings (bile acids, blood ammonia, complete blood count (CBC) with differential, urea, creatinine, albumins, TP, potassium, sodium and chloride, glucose, ALT (alanine aminotransferase), urinalysis and confirmed with computed tomography (CT). At least 14-day preparation regimen was implemented with the following oral therapies: lactulose (Portalak 667 mg/ml, Belupo, Koprivnica, Croatia) at a dose of 0.5 ml/kg, metronidazole 10 mg/kg/12h (Metrobactin, Dechra LelyPharma BV, Lelystad, Nederland), gastroprotective drugs - esomeprazole (Nexium, Grünenthal GmbH, Aachen, Germany), hepatoprotective (Epato 1500 plus, DRN s.r.l., Italy, α IT000187AL, marketing@drnsrl.it) and ursodeoxycholic acid (Ursofalk 250 mg, Dr. Falk Pharma GmbH, Freiburg, Germany). Additionally, a low protein diet (Hill's k/d) was administered, and on the day before surgery subcutaneous vitamin K (Konakion MM, Phytomenadione 10 mg, Cheplapharm, Arzneimittel GmbH, Ziegelhof, Greifswald, Germany) at a dose of 5.0 mg/kg was given first day and continued 2 days after surgery at a dose of 2.5 mg/kg. The dogs that already had epileptic seizures received antiepileptic drugs phenobarbitone 1–4 mg/kg/12h (Epiphen 30mg, Vetoquinol, Buckingham, UK) and/or levetiracetam 20 mg/kg/8h (Keppra 100 mg/ml, UCB Pharma SA, Brussels, Belgium). Hospitalisation began a day before the scheduled surgery to facilitate preparation and blood examination (CBC with differential, urea, creatinine, albumins, TP, potassium, sodium and chloride, glucose, ALT, PT (prothrombin time), APTT (activated partial thromboplastin time). On the morning of the surgery, the dogs received their complete prescribed therapy, including lactulose, metronidazole, vitamin K, intravenously pantoprazole (Nolpaza, Krka, Novo mesto, Slovenia) and dogs with seizures antiepileptic drugs. All medicine except vitamin K was continued at least 2 weeks after surgery, i.e., to the first check-up, and then gradually discontinued according to the clinical signs and laboratory results. Induction of anaesthesia was performed with analgetic fentanyl (Fentanil Torrex 50 µg/ml, Chiesi Pharmaceuticals GmbH, Wien, Austria), 2 µg/kg or remifentanyl (Remifentanilhameln 1 mg, Siegfried Hameln GmbH, Hameln, Germany) 2 µg/kg and propofol (Propoven 10 mg/ml, Fresenius Kabi Austria GmbH, Graz, Austria) slowly to effect, 2– 3mg/kg intravenously. Anaesthesia was maintained with isoflurane in oxygen, and constant rate infusion (CRI) of fentanyl (2 µg/kg/h) or remifentanil (5 µg/kg/h) was administered during surgery and continued as CRI two to three days after surgery. During anaesthesia lactated ringer solution (Hartmannova raztopina Braun, B. Braun Melsungen AG, Melsungen, Germany) 5 ml/kg/h was given, blood glucose levels were monitored at 30-minute intervals and monitoring of arterial blood pressure was assessed using a Proceedings of 10th Socratic Lectures 2024 42 of 165 Doppler flow detector (Model 811-BL, Parks Medical Electronic), end-tidal CO2 concentration, oxygen saturation measured with a lingual SpO2 probe, body temperature with an oesophageal thermometer, and electrocardiogram (ECG) was continuous. In all animals with PSS, a ventral medial celiotomy was performed, PSS was identified, and a cellophane band was applied in all dogs except for one dog where the portal vein was absent. Before use, 1.2 cm wide strips of cellophane were prepared and sterilised. After taking them from the sterilisation pouch, cellophane strips were immersed in saline, and then one strip was folded longitudinally into 3 layers. Initially, the tissue around the shunt was carefully dissected with right-angled dissecting forceps, and subsequently, the cellophane strip was carefully passed around the shunt. Three to four titanium clips were used in alternating directions to fixate both ends of the cellophane, and the remaining parts of the cellophane were cut with scissors. Shunts were partially or not at all attenuated at the end of surgery. The abdomen was closed routinely in all dogs. Dogs were typically hospitalised for up to 3 days following surgery after which they underwent check-ups at 7-14 days post-surgery, followed by monthly appointments for 3-4 months thereafter. Further follow-up appointments were scheduled as needed based on the presence of clinical signs. 3. Results The inclusion criteria for selecting cases encompassed a thorough review of medical records pertaining to 27 dogs, operated by the same surgeon (VE), comprising 18 females and 9 males, representing various breeds such as mixed breed (6), Yorkshire Terrier (5 dogs), Pug (2), Maltese (2), Shih Tzu (2), Jack Russell Terrier (2), Miniature Schnauzer (2), and one each of Bearded Collie, Chihuahua, Cavalier King Charles Spaniel, West Highland White Terrier, Golden Retriever, and Pomeranian. The age range of the dogs on the day of the operation was 4 to 100 months, with twelve out of 27 dogs being younger than 12 months. Dogs were presented with various clinical signs, 19/23 dogs were apathic, 14/23 dogs were vomiting, neurological signs such as seizures, disorientation, ataxia, restlessness, blindness, and head pressing were found in 13/23 dogs, 7/23 had polydipsia/polyuria, 6/24 had diarrhoea, and 2/23 were salivating. On the day of surgery or a few days before blood analysis was performed, serum bile acids were elevated in all dogs (22/22), ALT was elevated in 13/25 dogs, urea was low in 17/25 dogs, PT was prolonged in 8/24, and APTT was prolonged in 15/24 dogs. Nephroliths or uroliths were found in 13/26 dogs. Ammonia was elevated in 8/8 dogs. Leucocytosis was found in 16/24 dogs. Eleven dogs from 25 (11/25) were hypoproteinaemic and hypoalbuminemic, 6/25 were anaemic with low red blood cells and low haematocrit. Before the operation, two dogs needed a transfusion. During anaesthesia, 9 of 25 dogs needed a bolus of 20% glucose (Glukoza Braun 200 mg/ml, B. Braun Melsungen AG, Melsungen, Germany) 1-2 ml/kg intravenously. Twenty out of 25 dogs required Voluvene for maintenance of blood pressure or due to hypoproteinemia (Voluven 60 mg/ml, Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany) administered either through CRI at 1–2 ml/kg/h or as a bolus (1–2 ml/kg) in case of observed hypotension. This paper does not present results from potassium, sodium, chloride, venous blood gas analysis, and urinalysis. Based on CT angiography images of 22 dogs with extrahepatic shunts, the distribution included right gastrocaval shunts in 7 dogs (32%), each of spleno- and portocaval shunts in 3 dogs (14%), splenoazygos and left gastroazygos shunts in 2 dogs each (10%), and singular occurrences of gastrophrenic, left gastric, left gastrocaval, gastroduodeno caval, and right gastrocaval with a caudal loop shunt in one dog. On the day after surgery, dogs required intensive care, and vital signs, including body temperature, pulse rate, respiration, urine production, capillary refill time, mucous membrane colour, arterial blood pressure, and blood glucose, were recorded hourly and monitored for neurological signs. Monitoring urine production and/or blood pressure was also undertaken to adjust postoperative fluid therapy. Fluid therapy using lactated Ringer's solution was given at 3 – 5 ml/kg/h. The dogs received intensive medical attention Proceedings of 10th Socratic Lectures 2024 43 of 165 until they regained the ability to move and independently consume a regular diet. Analgesia was maintained as described before, with the addition of methadone if necessary. Throughout the recovery phase, no indicators of portal hypertension, such as hypovolemic shock, progressive hypothermia, and severe abdominal pain (Tobias, 2012), were observed. We do not have uniform results after surgery because the dogs underwent check-ups at different times and with various veterinarians who did not monitor the same blood parameters. However, we found that bile acids were elevated in 12/16 dogs, ALT was elevated in 6/20 dogs, urea was low in 2/13 dogs, TP was low in 2/18 dogs, and albumins were low in 3/21 dogs (Erjavec et al., 2024). 4. Discussion The investigation of PSS in dogs provides valuable insights into the pathophysiology, clinical presentation, diagnosis and outcomes following surgical intervention. The clinical signs that raised suspicion of portosystemic shunt (PSS) in dogs in our study were primarily neurological and gastrointestinal, with a notable prevalence of apathy among the affected dogs. Notably, salivation, a more common clinical sign in cats than in dogs (Howe and Boothe, 2002), was also reflected in our study. We primarily confirmed the suspicion with results indicating elevated bile acids, ammonia levels and urinalysis, while the diagnosis was subsequently confirmed through CT imaging. Preoperative management is crucial in minimising ammonia absorption from the gastrointestinal tract, preventing hepatic encephalopathy and seizures in dogs, and enhancing postoperative recovery. When ammonia was measured, it was increased in all dogs. Although ammonia is a valuable parameter, we did not measure it in all dogs due to the complex process of sample preparation and transport to an external laboratory. While coagulation times are frequently prolonged in affected dogs, they generally do not have significant clinical implications and spontaneous bleeding is uncommon (Kummeling et al., 2006; Kelley et al., 2013) which was consistent with our study where coagulation times were prolonged, but no issues with bleeding during surgery occurred, which we attribute to the administration of vitamin K. Following PSS occlusion, gastrointestinal ulceration and gastritis may persist years after surgery in majority of dogs, prompting consideration on proton pump inhibitors, especially for those receiving NSAIDs post PSS surgery (Weisse et al., 2014; Dugdale et al., 2020). In cases of hypoalbuminemia, caution should be exercised regarding the infusion of large volumes of crystalloids. Instead, synthetic colloids or plasma infusions may be considered to maintain osmotic pressure (Self, 2016), which was carefully considered in our dogs, where the majority of dogs additionally received Voluven. The slow elimination of certain drugs may contribute to the prolonged recovery phase, potentially leading to hypothermia and hypoglycemia (Self, 2016; Dugdale et al., 2020), so maintaining body temperature is crucial, and warming measures were implemented from the premedication phase through full recovery. Cellophane banding emerges as a straightforward surgical procedure for the progressive attenuation of PSS in dogs (Youmans and Hunt, 1998). This method involves applying a cellophane band around the shunt to reduce blood flow. While the benefits of surgically attenuating shunts are widely acknowledged, rapid closure or ligation of the shunt may not yield optimal results for many dogs, as it can lead to the development of portal hypertension and cardiovascular compromise. Because the occlusion of the PSS with cellophane progresses slowly through inflammatory reaction, the shunt closes gradually, while the portal vein can uptake larger volumes of blood (Holt, 1994; Youmans and Hunt, 1998; Frankel et al., 2006). Since this process occurs concurrently, no portal hypertension occurred. Using inexpensive and readily available cellophane allows progressive attenuation in the weeks following cellophane placement. Slow attenuation of CPS may also allow more time for the cardiovascular and central nervous systems to adapt to changing hepatic metabolism (Youmans and Hunt, 1998). The incorporation of preoperative imaging can reduce both surgical duration and the extent of dissection required for evaluating the shunt (Or et al., 2016). Attenuation location for different shunt types vary; portocaval shunts are attenuated at the level of epiploic Proceedings of 10th Socratic Lectures 2024 44 of 165 foramen, portophrenic at the abdominal surface of the diaphragm and newer studies suggest attenuation of portoazygos shunts within the thorax (Mullins, 2019b), while older literature suggested attenuation at the diaphragm level (Or et al., 2016), where azygos shunts were also attenuated in our study. The following considerations guided us during the surgeries: A healthy canine circulatory system should exhibit no large vessels entering the caudal vena cava between the phrenicoabdominal vein and the porta hepatis (Berent and Tobias, 2018). Moreover, it is common to observe turbulent blood flow in the caudal vena cava where the shunting vessel enters (Mullins, 2019a). After the surgery, laboratory parameters such as ALT, urea, TP, albumines, and bile acids improved in most dogs, alongside clinical signs. Subsequently, medical therapy was discontinued. Based on clinical signs, lactulose treatment should be sustained for a minimum of 4 weeks, coupled with a protein-restricted diet until liver function normalises, indicated by normal albumin levels. Serum bile acids may remain abnormal for over a year following shunt ligation (Tobias, 2012). However, if abnormalities persist nine months post-surgery, the likelihood of normalisation decreases. In such cases, additional laboratory values, including albumin, urea, and liver enzymes, should be utilised to monitor the patient's condition. If blood values fail to normalise six months post-surgery, a reevaluation is advised to explore potential issues such as incomplete shunt occlusion, multiple acquired shunts, or other liver diseases (Tobias, 2012). However, the primary goal of surgery is for the animal to be free of clinical signs and not require therapy. It is important to note that while laboratory findings often improve after surgery, they may still frequently remain abnormal, as observed in our study. 5. Conclusion The absence of significant complications observed during the surgical procedure can be attributed to administering appropriate therapy to the dogs for a sufficient duration before the operation. This therapeutic approach was consistently maintained throughout the recovery period. Intensive patient monitoring was crucial in the initial 12-24 hours post-surgery. Furthermore, a thorough observation was kept for any neurological signs, ensuring prompt initiation of assistance if such signs manifested. No signs of portal hypertension were detected in any of the cases post-surgery. Acknowledgements: We thank the veterinarians who referred these dogs for treatment and the veterinarians and technical staff at the Veterinary Faculty in Ljubljana, who assisted with their treatment and follow-up. Funding: This research was supported by Slovenian Research Agency (research program No. P4-0053). Institutional Review Board Statement: All procedures complied with the relevant Slovenian governmental regulations (Animal Protection Act, the Official Gazette of the Republic of Slovenia, 43/2007). Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. Berent AC, Tobias KM. Hepatic Vascular Anomalies. In: Johnston SA, Tobias KM, editors. Veterinary Surgery: Small Animal 2nd edition. St. Louis, Elsevier Saunders. 2018; pp. 1852-1886. Bertolini G. Anomalies of the Portal Venous System in Dogs and Cats as Seen on Multidetector-Row Computed Tomography: An Overview and Systematization Proposal. Vet Sci. 2019; 6:10. DOI: 10.3390/vetsci6010010 Boothe HW, Howe LM, Edwards JF, Slater MR. Multiple extrahepatic shunts in dogs: 30 cases (1981– 1993). J Am Vet Med Assoc. 1996; 208: 1849–1854. 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DOI: 10.1080/01652176.2000.9695032 Proceedings of 10th Socratic Lectures 2024 48 of 165 Data of 27 Cases of Congenital Portosystemic Shunt in Dogs from 2015 to 2023 Erjavec Vladimira1, Celinšek Barbara1, Jelovčan Petra1, Krofič Žel Martina1, Lisjak Anja1, Milevoj Nina1,2, Nemec Svete Alenka1, Pavlica Matic1, Pogorevc Estera1,3, Praper Ksenija1, Ravnik Verbič Urška1, Suhadolc Scholten Sara1, Šilc Ema1, Turk Živa1, Verk Barbara1, Lukanc Barbara1,* 1. 2. 3. * University of Ljubljana, Veterinary Faculty, Small Animal Clinic, Ljubljana, Slovenia The Royal (Dick) School of Veterinary Studies, University of Edinburgh, UK ESV, Estera Pogorevc s.p. Correspondence: Barbara Lukanc, barbara.lukanc@vf.uni-lj.si Citation: Erjavec V, Celinšek B, Jelovčan P, Krofič Žel M, Lisjak A, Milevoj N, Nemec Svete A, Pavlica M, Pogorevc E, Praper K, Ravnik Verbič U, Suhadolc Schol- Abstract: Congenital portosystemic shunts (PSS) present a significant challenge in veterinary medicine, requiring comprehensive analysis for effective diagnosis and management. This repository offers an extensive overview of 27 cases of congenital PSS in dogs, spanning the period from 2015 to 2023. The data are shown in Table 1. ten S, Šilc E, Turk Ž, Verk B, Lukanc B. Data of 27 cases of congenital portosystemic shunt in dogs from 2015 to 2023. Keywords: Portosystemic shunts, dogs, clinical signs, types of PSS, laboratory results Proceedings of Socratic Lectures. 2024, Funding: This research was supported by Slovenian Research Agency (research program No. P4-0053). 10, 48-49. https://doi.org/10.55295/PSL.2024.II4 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted publication for possible under the open terms access and conditions of the Creative Commons Attribution (CC0) license Proceedings of 10th Socratic Lectures 2024 49 of 165 Table 1. Data on dogs with Congenital Portosystemic Shunt from 2015 to 2023 X: mixed breed, CKCS: Cavalier King Charles Spaniel, CHD: Chihuahua, WHWT: West highland white terrier, BC: Bearded collie, MS: Miniature Schnautzer, GR: Golden Retriever, L: left, R: right Proceedings of 10th Socratic Lectures 2024 51 of 165 Exotic Animals’ Vascular System Characteristics and its Application in Clinical Practice Vejzović Anel1*, Tandir Faruk1, Hadžiomerović Nedžad1, Avdić Rizah1, Dučić Nejra1 University of Sarajevo, Veterinary Faculty, Department of Basic Sciences of Veterinary *Corresponding author: anel.vejzovic@vfs.unsa.ba 1 Citation: Vejzović A, Tandir F, Hadžiomerović N, Avdić R, Dučić N. Exotic Animals’ Vascular System Characteristics and its Application in Clinical Practice. Proceedings of Socratic Lectures. 2024, 10, 51-57. https://doi.org/10.55295/PSL.2024.II5 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Abstract: Veterinary practice commonly involves caring for domestic and companion animal. Since the trend of owning exotic pets is rapidly increasing, veterinarians often treat exotic patients as well. As a result, veterinarians are facing increased demands for specialized skills and knowledge. This article aims to provide a concise review of the anatomy of the vascular system in exotic animals and its relevance in clinical practice. Snakes, lizards and chelonians have 3-chambered heart with 2 atria and one partially divided ventricle whereas crocodiles have 4-chambered heart. It is important to consider these characteristics during the anesthesia in reptiles since blood shunting affects changes in blood pressure, oxygen level and other anesthetic parameters. In birds, the cardiovascular system is crucial in enduring the exhausting physical activities such as swimming, flying or diving and running. Birds have renal portal system which consists of cranial and caudal renal portal vein. The recommendation of avoiding drug application in the hindlimbs of birds is present for a long time. This is valid not only for birds, but for reptiles, amphibians and most fish. The ventricle of amphibians is trabeculated, which minimizes blood shunting through various anatomical and physiological features. During biphasic systole, the left and right side of the ventricle contract separately, directing blood in different parts of aortic arches. Due to the complexity of the exotic animals, thorough education added in veterinary curricula is needed. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licens es/by/4.0/). Keywords: amphibians, birds, cardiovascular, clinical, reptiles Proceedings of 10th Socratic Lectures 2024 52 of 165 Introduction Common veterinary practice deeply relies on working with domestic and companion animals. Even though they are perceived as the most frequent patients, trend of owning exotic pets is rapidly increasing. Statistics show that the number of pet birds in Europe is around 52 million with 29 million small mammals, 10 million aquatic animals and around 9 million reptiles (Ostović et al., 2022). In the USA, the exotic animal trade market has seen significant growth in recent years. Between 2000 and 2006, United States imported around 1.5 billion wild-life animals from which 92 % was acquainted as pets (Smith et al., 2009). These numbers increase steadily every year. Thus, skills and knowledge required from veterinarians increase and become challenging (Espinosa García-San Román et al., 2023). Contrary to this, education about exotic pets and practical skills are not included in most of the veterinary schools or they are included insufficiently (Rosenthal, 2006). Institutions are often reluctant towards introducing the courses about exotic animals, not realizing that exotic pets as patients are more common than before and students should have some basic knowledge after the graduation. Recommendation of American College of Zoological Medicine regarding veterinary curricula implies that all students should be able to take history and perform physical examination of exotic patients. They also suggest that students acquire specific knowledge of physiology, methods of restrain, sample collection, and understanding of clinical data (Stoskopf et al., 2001). Many specificities characterize exotic animals in comparison with usual companion animals. However, the studies regarding cardiovascular system of exotic animals are scarce. Existing studies primarily focus on laboratory animals due to their widespread use in biomedical researche. Although American mink (Mustela vison) is not a typical laboratory animal, its cardiovascular system has been extensively studied. Several studies (Hadžiomerović et al., 2016; Mrvić et al., 2017; Mrvić et al., 2021) investigated mink’s vascular system including kidneys, liver and lungs. They described branching of hepatic, renal and pulmonary veins and arteries comparing it to the dog as a similar carnivore model. In other studies, Ninomiya et al. (2008) and Mazensky et al. (2012) explored vascular features of rabbit eyes using vascular corrosion casting and branching of renal veins and arteries. This article aims to condense specific anatomy of exotic animal’s vascular system and to refer on its use in clinical practice. It further intends to help veterinary practitioners in process of clinical examination of these animals and to highlight what to be aware of regarding their anatomy and physiology. Reptiles Reptiles belong to the large class of Reptilia consisting of several orders. Snakes and lizards (order - Squamata), chelonians and crocodiles make the majority of this class where squamates are the most species-rich order with approximately 7000 species (Jensen et al., 2014). Their cardiovascular anatomy greatly varies. Snakes, lizards and chelonians have 3chambered heart with 2 atria and one partially divided ventricle whereas crocodiles have 4-chambered heart. All species have 2 aortic arches, right and left with 4 systemic veins including 2 cranial caval veins and caudal caval vein (Sladky and Mans, 2012; Jensen et al., 2014). These anatomical features cause mixing of arterial and venous blood. Even though considered pathophysiological in mammals and birds, blood shunting is proven to have several roles in reptile organism unlike previous hypothesis that treats shunting as evolutive remnant without physiological significance or even detrimental. It enables stabilization of oxygen level during breaks in respiration. Furthermore, right to left shunt can partially help heating process by increasing the amount of blood in systemic circulation. Left to right shunt, where the blood level in lungs is reduced, happens during the diving apnea of aquatic reptiles (Hicks, 2002; Mosley, 2005). It is important to consider these characteristics during the anesthesia in reptiles since blood shunting affects changes in blood pressure, oxygen level and other anesthetic parameters. The shunting can also delay or hasten the excretion of the inhalation anesthetics. Another vascular system characteristic specific to reptiles is renal portal system. Cranial and caudal portal veins collect blood from the tail, pelvis, limb and caudal part of the spine and intestines forming the plexus around the kidneys. Blood flow is controlled by autonomic nervous system and valves which direct blood through or around the kidneys (Sladky and Mans, 2012). Renal portal system could possibly affect the pharmacodynamics and pharmacokinetics of drugs Proceedings of 10th Socratic Lectures 2024 53 of 165 administered in the caudal part of the reptile’s body. Some authors suggest that drugs administered in the caudal part could be nephrotoxic or that the excretion time of the drug could be decrease, consequently affecting the effective dose and treatment of the animal. Contrary to this, some studies oppose these statements due to the lack of scientific evidence. However, current recommendations advise against the administration of nephrotoxic drugs and those excreted primarily by kidneys in the caudal part of reptiles (Mosley, 2005; O’Malley, 2017). During the clinical examination of patient, it is significant to know the sites for blood sample collection as well as drug administration sites. In lizards, ventral tail vein, ventral abdominal vein, cranial vena cava, brachial vein and jugular vein are commonly used while in chelonians, for intravenous route and blood sample collection, jugular, dorsal tail vein and brachial vein are used (Figure 1). Figure 1. A: Venipuncture sites in turtle – jugular vein (Courtesy of the University of California, Davis). B: Anesthetic administration in brachial plexus of turtle (Courtesy of Kurt K. Sladky). Intravenous route for drug administration in snake is jugular, palatine, and ventral tail vein. Important note for the examination of exotic animals is to restrain animal properly but in most cases, sedation or general anesthesia is necessary (Coutant et al., 2018). Birds Avian heart is located in the median plane of cranioventral part of coelomic cavity surrounded by the lobes of liver. It consists of two atria and two ventricles. Mass of the avian heart in regard to body mass surpasses the mammal heart (Strunk and Wilson, 2003). The avian heart is well-adapted due to the high demands for oxygen. The cardiovascular system is crucial in enduring the exhausting physical activities in different birds such as swimming, flying or diving and running (Dzialowski and Crossley, 2022). Even though heart of mammals and birds share substantial amount of similarities, during the evolution, birds developed many physiological characteristics which reflect on the gross anatomy. For example, heart size of goose increases before the period of migration (Bishop et al., 1995). Peripheral resistance is lower in pulmonary blood vessels, therefore the right ventricle wall is thinner compared to left ventricle (Dzialowski and Crossley, 2022). Generally lower peripheral resistance in birds compared to mammals demands higher arterial pressure for the high cardiac output. Heart rate in birds is variable and depends on the size of the bird, physical activity and different physiological needs (King and Lelland, 1984). With powerful heart muscle birds achieve bigger heart stroke volume, large cardiac output and are able to increase the heart rate over 1000 beats per minute (Dzialowski and Crossley, 2022). Birds major systemic blood vessels consist of aorta which originates from right aortic arch and three caval veins including right and left cranial vena cava and caudal vena cava. Aorta gives branches to the two brachiocephalic trunks for the vascularization of cranial part of the body and continues as descending aorta. Right cranial vena cava and caudal vena cava terminate in right atrium separately from left cranial vena cava which enters to sinus venosus (Strunk and Wilson, 2003). Amongst other Proceedings of 10th Socratic Lectures 2024 54 of 165 characteristics in birds, vascular system of kidney is to be highlighted. Birds have renal portal system which consists of cranial and caudal renal portal vein. These veins drain blood from common iliac, external iliac, internal iliac, caudal mesenteric and ischiatic veins along with the internal vertebral venous sinus (Figure 2). Arterial supply is done by renal arteries which enter each lobe separately (BurgosRodriguez, 2010). Kidneys of the bird itself have specific structure, embedded in renal fossa of the sacrum and divided into three parts, cranial, middle and caudal lobe. Birds have two types of nephrons, mammalian-like and reptile-like. Mammalian type is of more complex structure with longer proximal and distal tubules while reptilian type of nephron is missing Henley loop and the tubules are numerous but shorter (Burgos-Rodriguez, 2010; Hadžiomerović et al., 2021). Detailed description of kidneys and renal portal system encompasses the objectives of the study but it is assessed briefly to emphasize a complexity of these features in birds. This has clinical significance, since the recommendation of avoiding drug application in the hindlimbs of birds is present for a long time. Due to the lack of extensive studies that would distinguish which drugs are safe for application, most of the practitioners still avoid it. Many existing studies have opposite results and claims, therefore it is most appropriate to administer drugs in the cranial part of animal. This is valid not only for birds, but for reptiles, amphibians and most fish (Coutant et al., 2018). For the intravenous route in birds and blood sample collection in birds, mostly there are three blood vessels available including ulnar superficial vein, also known as basilic or wing vein, medial metatarsal vein and jugular vein. Important to note is that right jugular vein is often bigger then the left, hence it is more accessible (Coutant et al., 2018). Figure 2. Venous vascularization of fowl’s kidney and blood vessels of renal portal system (corrosion technique). 1. V. cava caudalis; 2. V. iliaca communis; 3. V. iliaca externa; 4. V. portalis renalis cranialis; 5. V. portalis renalis caudalis; 6. V. renalis cranialis; 7. V. renalis caudalis; 8. V. ischiadica; 9. V. mesenterica caudalis; 10. V. iliaca interna Amphibians Among three types of animals reviewed in this article, species from the group of amphibians represent the rarest patients for veterinarians. Earlier studied mostly by Proceedings of 10th Socratic Lectures 2024 55 of 165 biologists, in the last few decades veterinarians are more involved in working with amphibians. Amphibians are divided into three groups: Anura, Caudata and Caecilians. Frogs and salamanders are the representatives of first two groups as the commonly studied species (Forzán et al., 2017). The three-chambered heart of amphibians is generally similar among species, but some differences can be noted. In anurans and salamanders, the right atrium is usually larger than the left, except in a few species of anurans, such as African Clawed frog (Xenopus laevis). The interatrial septum is fenestrated in salamanders, allowing for the mixing of blood, while it is complete in anurans. Blood from both atria enters the undivided ventricle (Sharma, 1961; Heinz-Taheny, 2009). Trabeculated ventricle in amphibians and different anatomical and physiological features minimize the blood shunting. During biphasic systole, the left and right side of ventricle contract separately forcing the blood in different parts of aortic arches. Separation of the blood is enabled by spiral valve in the conus arteriosus. Three separate trunks exit the ventricle, systemic arch, carotid and pulmocutaneous arch (Figure 3). In the first phase of heart contraction, deoxygenated blood from the right side of the ventricle enters the pulmocutaneous arch which gives pulmonary and cutaneous arterial vessels carrying the blood for gas exchange in the lungs and skin of amphibians. In the second phase, blood is sent to the systemic and carotid arches for oxygenation of the cranial and caudal parts of the animal. Therefore, very small portion of blood from the right side of ventricle goes to the left side. The blood from the left side is directed mainly to the systemic circulation avoiding right side (Heinz-Taheny, 2009). Along with systemic and pulmocutaneous circulation, amphibians also have renal portal system. According to O’Malley (2005), since the blood from the hindlimbs does not bypass the kidneys, drug administration in the caudal part of the body may cause changes in drug distribution. Additionally, ventral abdominal vein, often used in clinics passes through the liver as a part of the hepatic portal system. This could affect pharmacokinetics of drug administered in this vein and its branches. Hence, it is suggested to avoid the application of drugs primarily metabolized in the liver in the caudal part of the body (Heinz-Taheny, 2009). Furthermore, taking in considerations amphibians are poikilothermic animals, clinicians should keep the patients in their optimal temperature zones due to the fact physiology, behavior and therapy are influenced by temperature (Whitaker et al., 1999; Wright, 2001). Venipuncture sites differ between the species. In the frogs, cardiac puncture, ventral abdominal vein, femoral vein and lingual vein are used. Ventral tail vein is most commonly used in salamanders. Amount of blood sample should not exceed 1% of body weight in healthy animal nor 0.5 % in diseased animal (Heinz-Taheny, 2009). Figure 3. Anatomy of the anuran heart. Large block arrows indicate blood flow into the heart. Thin black arrows indicate flow of oxygenated blood (Courtesy of Frank Taheny). Proceedings of 10th Socratic Lectures 2024 56 of 165 Conclusion The number of exotic pets is increasing over the past few years. Aside from usual work with companion animals, veterinarians often treat exotic patients as well. This demands certain level of knowledge about specific anatomy and physiology of these animals. The article provides a brief insight in vascular system of reptiles, birds and amphibians with an emphasis on important clinical considerations regarding these characteristics. However, due to the complexity of the exotic animals, thorough education is needed. Funding: This article received no grant from any funding agency. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Bishop CM, Butler PJ, Egginton S, el Haj AJ, Gabrielsen GW. 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Future Directions in Training of Veterinarians for Small Exotic Mammal Medicine: Expectations, Potential, Opportunities, and Mandates. Journal of Veterinary Medical Education. 2006; 33:382–385. DOI: https://doi.org/10.3138/jvme.33.3.382 22. Sharma HL. The circulatory mechanism and anatomy of the heart of the frog Rana pipiens. Journal of Morphology. 1961; 109:323–349. DOI:10.1002/jmor.1051090307 23. Sladky KK, Mans C. Clinical Anesthesia in Reptiles. Journal of Exotic Pet Medicine. 2012; 21:17-31. DOI: https://doi.org/10.1053/j.jepm.2011.11.013 24. Smith KF, Behrens M, Schloegel LM, et al. Ecology. Reducing the risks of the wildlife trade. Science. 2009; 324:594595. DOI: 10.1126/science.1174460 25. Stoskopf MK, Paul-Murphy D, Kennedy-Stoskopf S, Kaufman G. American College of Zoological Medicine recommendations on veterinary curricula. J Am Vet Med Assoc. 2001; 219:1532-1535. DOI: https://doi.org/10.2460/javma.2001.219.1532 26. Strunk A, Wilson GH. Avian cardiology. Vet Clin North Am Exot Anim Pract. 2003; 1:1-28. DOI: https://doi.org/10.1016/S1094-9194(02)00031-2 27. Whitaker BR, Wright KM, Barnett SL. Basic husbandry and clinical assessment of the amphibian patient. Vet Clin North Am Exot Anim Pract. 1999; 2:265-290. DOI: 10.1016/s1094-9194(17)30125-1. 28. Wright KM, Applied Physiology. In Wright KM, Whitaker BR, editors. Amphibian medicine and captive husbandry. Florida, USA, Krieger Publishing. 2001; pp. 31–34. Proceedings of 10 th Socratic Lectures 2024 59 of 165 Milestones in Research of Small Cellular Particles. Membrane Modeling and Theoretical Description in Connection with In vitro and Ex vivo processes Romolo Anna 1,2, Kralj-Iglič Veronika 1,2,* University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics , Ljubljana, Slovenia Extracellular Vesicles and Mass Spectrometry Group, Institute of Biosciences and Bioresources, National Research Council of Italy, Napoli, Italy * Correspondence: Veronika Kralj-Iglič; veronika.kralj-iglic@zf.uni-lj.si 1. 2. Citation: Romolo A, Kralj-Iglic V. Milestones in Research of Small Cellular Particles. Membrane Modeling and Theoretical Description in Connection with In vitro and Ex vivo processes. Proceedings of Socratic Lectures. 2024, 10, 59-68. https://doi.org/10.55295/PSL.2024.II6 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). Abstract: Sub-micron sized small cellular particles (SCPs) (microparticles, microvesicles, exosomes, extracellular vesicles, extracellular particles) are recently gaining attention due to their potential role in all fields involving living organisms (e.g. medicine, food science, agriculture, ecology). Due to their small size and dynamic nature, their properties and biological roles are not yet understood up to a level to be widely used and remain potentially important. Employing the knowledge from different fields is highly warranted to progress towards better understanding of physiological and patophysiological processes based on SCPs. This contribution outlines four milestones in SCP research: M1: Model of the biological membrane, M2: Membrane budding and vesiculation, M3: Theoretical description of the membrane shape and M4: Transferrin release from the reticulocyte membrane in the form of membrane – enclosed SCPs. Also we outline the increasing interest of the scientific society for SCPs reflected in highly cited papers and present a synthesis of the theoretical and experimental view on membrane-enclosed SCPs within the fluid crystal mosaic model of the membrane. Keywords: Extracellular vesicles; Extracellular particles; Extracellular vesicles; Small cellular particles; Microvesicles; Exosomes Proceedings of 10 th Socratic Lectures 2024 60 of 165 1. Milestone 1: Model of biological membrane In 1972, a paper was published by Singer and Nicolson (1972) following thorough study of biological membrane thermodynamics and proposing the Fluid mosaic model. According to this model, the membrane is composed of a lipid bilayer with embedded proteins that are integral to the membrane (Figure 1A). The proteins are arranged with the ionic and highly polar groups protruding from the membrane into the aqueous phase, and the nonpolar groups largely buried in the hydrophobic interior of the membrane. The proteins are more or less free to move laterally over the membrane analogously to a twodimensional solution of integral proteins (or lipoproteins) in bilayer solvent. In the next 50 years the agreement of this model with experimental results established it as a base for the description of membrane features. However, already in the paper (Singer and Nicolson, 1972), it was suggested that direct interactions between the embedded entities may take place and may have also consequences for biological features involving the membrane. Lateral segregation of the protein molecules was observed in connection with the pinocytosis of the membrane – endovesiculation - which refers to the uptake of substances by a cell. This concept was later further developed and reported in Simons and Ikonen (1997) and Brown and London (1998). Simons and Ikonen (1997) presented a model based on the dynamic clustering of sphingolipids and cholesterol to form rafts (Figure 1B) that move laterally within the fluid bilayer and function as platforms for the attachment of proteins. Furthermore, lateral segregation into rafts was connected with membrane curvature within the entities called calveolae. It was suggested that spingolipid– cholesterol rafts may be an essential feature of all organellar membranes involved in biosynthetic and endocytic traffic. Brown and London (1998) further up-graded the model by considering order within the particular regions in the lipid bilayer. Figure 1. Illustration of the fluid mosaic model of the membrane in its original form (A) and by considering formation of membrane rafts (B). Adapted from Kralj-Iglič (2012). 2. Milestone 2: Membrane budding and vesiculation Erythrocytes are convenient for the study of the properties of the cell membrane. Mature cells do not have nucleus nor cytoskeleton. Their shape is largely determined by the properties of the membrane (lipid bilayer with underlying membrane skeleton). The process of microvesiculation (Figure 2A) and the pinched-off vesicles (Figure 2B) were imaged and described (Figure 3) by Weed and Reed (1966). Figure 2. A: Budding of the erythrocyte. B: Electron micrograph of “fragments”. Panel B has magnification 14000 X. Adapted from Weed and Reed (1966). Proceedings of 10 th Socratic Lectures 2024 61 of 165 Figure 3. Scheme of erythrocyte membrane fragmentation as presented by Weed and Reed (1966). In 1967, SCPs were identified as fragments shed from platelets (Wolf, 1967). Already then, their potential biological role was indicated (Wolf, 1967). Later it was observed that blood contains a mixture of young and old erythrocytes which differ in the size, density and surface charge (Lutz et al., 1977). It was suggested that microvesiculation is responsible for the loss of the erythrocyte membrane and therefore increase of the volume to area ratio is reflected in senescent rounding of erythrocytes (Lutz et al., 1977). In the following decades, erythrocyte shape change, hemolysis and vesiculation were thoroughly studied experimentally (Nelson et al., 1983, Isomaa, 1979, Isomaa et al., 1987, Hagerstrand et al., 1992). It was found that the erythrocyte membrane may bud inward or outward and if this process continues, it terminates in vesiculation (Isomaa et al., 1987, Hagerstrand et al., 1992). Procedures were developed to isolate and image microvesicles (Isomaa st al., 1987, Hagerstrand et al., 1992). It was found that the shape of the buds and of SCPs may vary depending on the added compounds that intercalate into the membrane (Figure 4). Figure 4. Budding of erythrocytes (panels A, C and D) and isolated microvesicles (panels B, E and F). The process was induced by adding amphiphilic molecules into the erythrocyte suspension (A,B: dodecyl maltoside; D,E,F: dodecyl zwittergent). A, C, D and F: scanning electron micrographs; B and E: transmission electron micrographs. Adapted from (Schara et al., 2009). Budding and vesiculation of nano to micro – sized particles were detected and observed in different biological systems. Figure 5 shows budding and vesiculation in a microalgae Ochromonas danica. Assessment and visualization of SCPs in different samples led to the conclusion that cells of all types are prone to shed their fragments in the surrounding solution (Yanez-Mo et al., 2015). Being freed from the mother cell, they can travel with surrounding liquid, reach nearby or distant cells and interact with them. Proceedings of 10 th Socratic Lectures 2024 62 of 165 Figure 5. SCPs emerging from the surface of the cell membrane of a 9-day-old Ochromonas danica cell; see arrows. Adapted from (Aaronson et al., 1971). 3. Milestone 3: Theoretical description of the bilayer membrane shape Erythrocyte shape was described theoretically (Canham, 1970) by considering the membrane as a thin, locally slightly curved elastic shell. The contour of the rotationally symmetric shape was approximated by Cassini ovals and the bending energy of the shell was minimized at chosen geometrical constraints by determining the parameters of the ansatz (Canham, 1970). Good agreement was obtained between calculated shapes and shapes observed under the optical microscope (Canham, 1970). Theoretically advanced expression for the membrane free energy including the assumption that the membrane has spontaneous curvature was presented by Helfrich (1973). The description was improved by stating the variational problem for minimization of the free energy by a set of Euler-Lagrange differential equations (Deuling and Helfrich, 1976). This enabled a rigorous solution of the set of differential equations for the axisymmetric shapes. The calculated shapes shown in Figure 6 (Kralj-Iglič et al., 2022) were obtained by using this method. Figure 6. Observed and calculated membrane shapes corresponding to a sequence leading to formation of internal vesicle (endovesiculation) in SCPs found in isolates from blood and to a sequence leading to formation of external vesicle (exovesiculation) in a giant phospholipid vesicle. The theoretical shapes were taken as axisymmetric with respect to the vertical axis and were calculated by solving the variational problem presented as a system of the Euler-Lagrange differential equations. Note the relevance of the model over 100 fold size difference. From Kralj-Iglič et al., (2022). Proceedings of 10 th Socratic Lectures 2024 63 of 165 Bilayer couple principle considering that the two membrane layers are in close contact but can slide over each other (Sheetz and Singer, 1974) was implemented to explain intercalation of compounds into a particular lipid layer (Helfrich, 1974, Evans, 1974) thereby changing average mean curvature of the membrane. The models were theoretically elaborated to yield the equilibrium membrane shape by minimization of the membrane free energy (reviewed by Seifert, 1997). Membrane budding and vesiculation were described by an increase (in the case of exovesiculation) or a decrease (in the case of endovesiculation) of the average mean curvature of the membrane. The pinching off of the bud was preceeded by the thinning of the neck connecting the bud and the mother membrane. When the bud is pinching off, the opening in the membrane is very small and can be sealed with little energy change by rearranging the adjacent molecules. In considering the models of the membrane it was taken into account that besides lateral redistribution and segregation the anisotropic membrane constituents may undergo orientational ordering with respect to the axis perpendicular to the membrane. This introduced curvature deviator as an important parameter (Fischer, 1992; Kralj-Iglič et al., 1999). Changing of the constraints and/or parameters (e.g. relative volume (expressing volume to area ratio v, average mean curvature <h>, average curvature deviator <d>) yields different shapes of membrane-enclosed entities without internal structure. A (v, <h>, <d>) phase diagram featuring some shapes corresponding to the minimal free energy is presented in Figure 7. Regions are defined within limiting shapes composed of spheres, cylinders, tori and flat regions. Shapes composed of spheres lie in the <d>=0 region. Red, blue and green lines indicate transformations of shapes due to changes in v, <h> and <d>. Endo and exo vesiculation (Figure 6) are presented by the blue and red – marked sequences, respectively. Figure 7. A (v, <h>, <d>) phase diagram of shapes calculated by minimization of the membrane free energy. Two aspects (side view (left) and top view (right)) are shown. From (Kralj-Iglič et al., 2020). Taking into consideration orientational ordering of anisotropic membrane constituents indicated stability of thin (below cca 100 nm) tubular vesicles (Kralj-Iglič et al., 2002) and required revision of the model of the membrane (Kralj-Iglič, 2012). As these effects were similar to the ones observed in liquid crystals, the up-graded model which becomes relevant in nano-sized anisotropic membrane regions was called the Fluid crystal mosaic model (Figure 8). Figure 8. Illustration of the fluid crystal mosaic model of the membrane. Adapted from Kralj-Iglič (2012). Proceedings of 10 th Socratic Lectures 2024 64 of 165 Milestone 4: Transferrin release from the reticulocyte membrane in the form of membrane – enclosed SCPs. While internal structures filled with SCPs were observed already in 1971 (Coons and Axtell, 1971) (Figure 8A), the importance of this feature was revealed in the study of transferrin release from maturing reticulocytes (Harding et al., 1983; Pan and Johnstone, 1983). It was suggested and supported by measurements and observations that transferrin was released in the form of small vesicles that are formed in invaginated compartments of reticulocytes (Figure 8B). These results were considered as a base for the general mechanism of formation of »exosomes« - vesicles that form within internal compartments of the cell and are eventually released into the surrounding solution. Figure 8. A: Cross-section of an excretory tube cell of the mesostigmatid mite Macrocheles muscaedomesticae showing a group of lysosomes (arrows). Each is bounded by a single membrane and contains many round bodies of varying degrees of size and electr on density. X 20,000. Adapted from (Coons and Axtell, 1971). B: Release of SCPs labelled with gold (black dots) from the invagination of an unfixed reticulocyte. The cell was incubated for 30 min with AuTf, subjected to a 20-min chase with unconjugated transferrin, and then quick-frozen without prior fixation and freeze-substituted. Bar, 200 rim. X 61,000. Adapted from (Harding, 1983). Notorious SCPs Although the existence of SCPs has been acknowledged, their roles and underlying mechanisms are not yet completely understood and are a subject of extensive study. As SCPs were discovered in different samples by scientists from different fields, they were called different names. International Society for Extracellular Vesicles (ISEV) is coordinating effort to nominate SCPs and impose quality requirements on experimental procedures. For his purpose ISEV has issued several »position papers«. Some of these works have become highly cited. We have browsed Google Scholar database for papers using keywords »extracellular vesicles«, »microparticles«, »microvesicles« and »exosomes« and outlined papers cited more than 2000 times in Table 1. It can be seen that the majority of highly cited papers are reviews, however the one listed with the highest number of citations in Table 1 is the ISEV position paper (Thery et al., 2018). Recently, the requirements were updated in a new publication MISEV2023 (Welsh et al., 2024) with contribution of over 1000 ISEV members. The goal of this paper is to provide »snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues«. As SCP-based mechanisms are very basic and relevant in all living systems, new SCP research areas are emerging. As the biological roles of SCPs are acknowledged, their better understanding is highly waranted. However, further efforts are needed to accomplish this goal and achieve breakthrough of SCP-based methods in everyday practice. Model approach and theoretical work is therefore an important element in SCP research. For comparison, Table 2 shows a list of highly cited papers on membrane models and theoretical descriptions and also some milestone papers referred to in this work. Proceedings of 10 th Socratic Lectures 2024 65 of 165 Table 1. Highly cited papers on cellular fragments found in first 20 pages of Google Scholar database by keywords »extracellular vesicles«, »microparticles«, »microvesicles« and »exosomes« subject to more than 2000 citations. Paper N Expression browsed Journal Type of the paper Thery et al., 2018 8043 Extracellular vesicles J Extracellular Vesicles ISEV Position paper Raposo and Stoorvogel, 2013 8027 Exosomes/microvesicles J Cell Biol Review Van Niel et al., 2018 5864 Extracellular vesicles Nature Rev Mol Cel Biol Review Thery et al., 2006 5821 Exosomes Curr Prot Cell Biol Research Colombo et al., 2014 5639 Exosomes/Extracellular Vesicles Ann Rev Cell Develop Biol Review Thery et al., 2002 5624 Exosomes Nature Reviews Immunology Review Kalluri and LeBleu, 2020 5503 Exosomes Science Review Skog et al., 2008 5481 Microvesicles Nature Cell Biology Review Yanez Mo et al., 2015 4887 Extracellular vesicles J Extracellular Vesicles Review Johnstone et al., 1987 3217 Vesicles J Biol Chem Research Tkach and Thery, 2016 3001 Extracellular vesicles Cell Review El Andalousi et al., 2013 2985 Extracellular vesicles Nature Reviews Drug Discovery Research Mathivanan et al., 2010 2728 Exosomes J Proteomics Review Lotvall et al., 2014 2633 Extracellular vesicles J Extracellular Vesicles ISEV Position paper Costa Silva et al., 2015 2548 Exosomes Nature Cell Biology Research Mulcahy et al., 2014 2468 Extracellular vesicles J Extracell Vesicles Review Pisitkun et al., 2004 2438 Exosomes Proc Nat Acad Sci Research Gyorgy et al., 2011 2305 Extracellular vesicles Cell Mol Life Sci Review Witwer et al., 2013 2251 Extracellular vesicles J Extracellular Vesicles ISEV position paper Al Nedawi et al., 2008 2275 Microvesicles Nature Cell Biology Doyle and Wang, 2019 2212 Extracellular vesicles MDPI Cells Review Vlassov et al., 2012 2192 Exosomes Biochim Biophys Acta Review Pan and Johnstone, 1983 2166 Vesicles Cell Research Cocucci et al., 2009 2141 Exosomes Robbins and Morelli, 2014 2121 Extracellular vesicles Nature Reviews Immunology Review N: number of citations on March 19, 2024. Table 2. Selected milestone papers. Paper N The scope Journal Type of the paper Singer and Nicolson, 1972 13186 Membrane model Science Research Simons and Ikonen, 1997 12015 Membrane model Nature Research Helfrich, 1973 7186 Membrane shape Z Naturforschung Theory Brown and London, 1998 3908 Membrane model Ann Rev Cell Develop Biol Research Seifert U,1997 2015 Membrane shape Advances in Physics Review Canham, 1970 2005 Red blood cell shape J Theor Biol Theory Wolf, 1967 1999 Platelet dust Br J Haematol Research Harding et al., 1983 1988 Vesicles and tubules J Cell Biol Research Sheetz and Singer, 1974 1940 Membrane model Proc Nat Acad Sci Research Weed and Reed, 1966 268 Cell fragments Am J Med Research N: number of citations on March 19, 2024. Proceedings of 10 th Socratic Lectures 2024 66 of 165 Conclusions Biological roles of SCPs inspire great effort of the scientific community to fulfill the potential effects and uses of SCPs in health improvement of living organisms. As SCPs are tiny and dynamic entities, their harvesting, assessment and manipulation presents a challenge. Modeling and theoretical description are powerful tools which should be included in consideration of SCPs. Acknowledgement Due to increasing interest in SCPs and large number of published works we are aware that some important works may not have been included in the survey. Funding: This research was supported by European Commission: HORIZON-MSCA2022-SE-01-01 project FARM EVs (Grant agreement number 101131175) and Slovenian Research Agency (Research cores P3-0388, J3-3066, J2-4447 and J2-4427). Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Aaronson S, Behrens U, Orner R, et al. Ultrastructure of intracellular and extracellular vesicles, membranes, and myelin figures produced by Ochromonas danica. J Ultrastruct Res. 1971; 35: 418 –430. 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DOI: 10.3402/jev.v4.27066 Proceedings of 10 th Socratic Lectures 2024 70 of 165 Effect of Time, pH, Alcohol and Sugar Content on Nicotine Re-lease from Pouches Available on Slovene Market Nenadović Milena1, Kržišnik Katja2, Trebše Polonca2*, Bavcon Kralj Mojca2 University of Belgrade, Faculty of Biology, Belgrade, Serbia University of Ljubljana, Health faculty, Ljubljana, Slovenia * Correspondence: Polonca Trebše; trebsep@zf.uni-lj.si 1. 2. Citation: Nenadović M, Kržišnik K, Trebše P, Bavcon Kralj M. Effect of Time, pH, Alcohol and Sugar Content on Nicotine Release from Pouches Available on Slovene Market. Proceedings of Socratic Lectures. 2024, 10, 70-75. https://doi.org/10.55295/PSL.2024.II7 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Abstract: Tobacco-free nicotine pouches, first introduced in Sweden, are made as a less hazardous product for cigarette consumers. Instead of tobacco leaves, they consist of nicotine-containing powder and other ingredients such as water, salts, natural aromatic oils and others, that will boost the flavor and effect and make it last longer. This research aimed to investigate the extraction efficiency of nicotine from poaches of different brands. The effects of alcohol and sugars present in saliva at different pHs and consumption times were investigated as well. We optimized a High performance liquid chromatography with diode-array detection method (HPLC-DAD) for nicotine determination and its quantification. We used reversed-phase chromatography (RP-HPLC), and a mixture of sodium hydrogen carbonate and acetonitrile as a mobile phase (85:15, v/v). Different brands of nicotine pouches were used. Nicotine extraction rate showed positive linear dependence on time. The percentage of extraction was measured at different intervals, up to two hours, without getting a plateau. Nicotine extraction lowers with the increasing volume of saliva. The obtained results have shown that the increasing ethanol concentration in saliva (from 5-40 %) leads to a higher extraction rate, coming up to 85 %. In the case of sugar addition, the results were variable. Experiments with White fox pouches have shown that extraction of nicotine decreases with a higher concentration. On contrary, Siberia pouches do not exhibit that correlation. When it comes to pH, we used pH range that usually varies in saliva and the results have shown no significant differences. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/ by/4.0/). Keywords: Nicotine, Pouches, HPLC-DAD, Extraction ______________________________________________________________________________ Proceedings of 10 th Socratic Lectures 2024 71 of 165 1. Introduction As people’s awareness regarding the hazard of cigarettes grows, many companies have come up with alternative/potentially less harmful products to replace their use. One of these products, nicotine pouches, also known as tobacco-free snus, hailing from Sweden. These pouches are weighing up to 1g, do not contain tobacco, but have nicotine powder, sweeteners, flavorings, salts, and plant-based fibers. They are placed between the lip (usually the upper lip) and the gums, and the holding time can be up to an hour, although it is usually 10-15 minutes. The long-term health impact of nicotine pouches is still unknown. Nicotine pouches contain nicotine which is harmful to young people in any form. Due to the absence of tobacco leaf, the Food and Drug Administration does not classify oral nicotine pouches as a smokeless tobacco product (Robichaud et al., 2020). Side effects of use can include irritation of the gums, sore mouth, hiccups, nausea, nicotine addiction and more. Only 10-20 % of nicotine reaches the systemic circulation. This means that only 1-2 mg of nicotine is absorbed into the blood from a one-gram pouch containing around 16 mg of nicotine (Miller-Holt et al., 2022). Nicotine is absorbed quite rapidly from Swedish snus. Studies on Swedish snus users have shown that the plasma steady-state levels of nicotine and its main metabolite, cotinine, are like Swedish snus users and cigarette smokers (Scherer et al., 2022, Miller-Holt et al., 2022). Various brands of tobacco-free snus are available on the Slovenian market, and for the purposes of this research, we chose only some of them and compared the results obtained. We investigated how much nicotine is released from the bag into the saliva, trying to simulate people’s daily habits. As you can eat and drink prior to using snus, in addition to varying the pH and the time of use, we investigated the effect of alcohol and sweeteners on nicotine release, wanting to simulate people’s everyday life this way (Bishop et al., 2022). Photo 1. Nicotine pouches; White fox and Siberia. 2. Materials and Methods 2.1. Materials Different brands of nicotine pouches were used: White fox, Siberia (GN Tobacco, Sweden) (Photo 1), Velo (R.J. Reynolds Vapor Company, USA), and Thunder (V2 Tobacco, Denmark), but the first two expressed better extraction rate, so we continued our research on them. We used three types of White fox pouches: The original (blue) and Double-mint (green), both with 12 mg of nicotine per poach (0,75 grams), and Full-charge (red) with 16 mg of nicotine per poach (1 gram). Siberia pouches were Regular (big) with 16.4 mg of nicotine per pouch and Slim, (small) with 10.66 mg of nicotine per pouch. To stay in accordance with scientific ethics, the results will be presented by the alternative names previously stated in brackets (blue, green, red, big and small). In Slovenia, nicotine pouches are available in every tobacco shop and gas stations, so we obtained them from one of these. Proceedings of 10 th Socratic Lectures 2024 72 of 165 The artificial saliva solution was prepared by dissolving 0.69 g of Sodium phosphate monobasic monohydrate (NaH2PO4), 0.4 g of sodium chloride (NaCl), 0.795 g of calcium chloride (CaCl2), 0.4 g potassium chloride (KCl), 0.005 g sodium sulfide (Na 2S) and 1 g of urea, in distilled water in final volume of 1 liter. 2.2. Methods For examining dependence of: • volume of saliva, we used vials filled with 2, 4, 6, 8 and 10 milliliters of saliva • pH of saliva, we used 1 M HCl and 1 M of NaCl for establishing wanted pH (5, 6, 7 and 8) • alcohol concentration, we used different percentage (5, 10, 15, 20, 30 and 40 %) of ethanol mixed with saliva • sugar (saccharose) concentration, we used 5, 10 and 15 % of sugar dissolved in saliva; for each of these experiments, we put 2 ml of prepared solution in the vials (except for investigating the impact of volume of saliva, where we put 2, 4, 6, 8 and 10 ml), placed one pouch inside, and left it in water bath on 37 degrees. After 15 minutes, 1 ml from each vial was taken and placed in the vials for further detection on HPLC-DAD. For measuring time dependence, we used big vial filled with 10 ml of saliva. Then we placed pouch in vial and put it in the water bath on 37 degrees. We had 2 repeats per each type of nicotine pouches. 1 ml of saliva was taken after 5, 10, 15, 30, 45, 60, 90 and 120 minutes and placed into the vials for HPLC-DAD. Experiments are done in duplicate (for volume, time, and alcohol), or in triplicate (for pH and sugar dependence). Calibration curve was constructed for nicotine measurement from six standard solutions specifically: 50, 100, 150, 200, 250 and 500 mg/ml. The standard solutions were prepared by serial dilution of proper amount from stock standard solutions with saliva. Chromatography Conditions: we used HPLC equipped with a diode array detector (DAD) for nicotine detection. C18 Agilent HP-5MS (19091S-433) Capillary Column (30M x 0.25mm x 0.25µm) was used. Mobile phase was consisting of acetonitrile (ACN) and sodium hydrogen carbonate buffer (pH 9.2, 2 M) (15:85, v/v), at a flow rate of 1,2 ml/min, running time 15 min and a UV detection achieved at 259 nm. For buffer preparation, we used 20 ml of sodium carbonate solution (2,2 g/100 ml, 0.2M), and 230 ml of sodium bicarbonate (1.68 g/100 ml, 0.2M). Filled to 1 liter with distilled water (Bansal et al., 2018). 3. Results The results are shown in the Tables 1-6. We measured areas under the signals in chromatograms and calculated the percentage of nicotine extraction based on the known concentration of nicotine in pouches and in solutions diluted from the stock. Table 1. Nicotine extraction rate (%) depending on the volume of saliva (White fox - green). volume [ml] green Area mg of nicotine % 2 23935.35 5.08 42.36 4 16611.5 3.53 29.40 6 9343.6 1.98 16.54 8 6892.6 1.46 12.20 10 6027.65 1.28 10.67 Proceedings of 10 th Socratic Lectures 2024 73 of 165 Table 2. Nicotine extraction rate (%) depending on the alcohol percentage found in saliva (White fox - blue, red) blue red Alcohol % average mg of nicotine % average mg of nicotine % 5 1037.2 0.22 18.35 20419.45 4.34 27.10 10 1347.2 0.29 23.84 19915.6 4.23 26.43 15 1373.4 1.29 24.30 21842.25 4.64 28.99 20 1641.45 0.35 29.04 53908.7 11.45 71.55 30 1853.05 0.39 32.79 52031.2 11.05 69.06 40 2104.7 0.45 37.25 56161.45 11.93 74.54 Table 3. Nicotine extraction rate (%) depending on the sugar percentage found in saliva White fox - (blue, red) blue red Sugar % average mg of nicotine % average mg of nicotine % 0 4073.4 0.87 72.09 2891.55 0.61 51.17 5 2928.93 0.62 51.83 2800.6 0.59 49.56 10 2675.8 0.57 47.35 2554.63 0.54 45.21 15 2194.33 0.47 38.83 2311.37 0.49 40.90 Table 4. Nicotine extraction rate (%) depending on the sugar percentage found in saliva (Siberia - small, big) small big Sugar % average mg of nicotine % average mg of nicotine % 0 1658.40 0.35 33.04 2088.4 0.44 27.04 5 1462.73 0.31 29.14 1163.43 0.25 15.06 10 1070.83 0.23 21.33 1362.83 0.29 17.65 15 1443.93 0.31 28.76 1717.8 0.36 22.24 Table 5. Nicotine extraction rate (%) depending on the pH of the saliva (White fox green small pH average mg of nicotine % average 5 20703.47 4.40 36.64 19902.80 6 23041.60 4.89 40.78 19617.23 7 21986.43 4.67 38.91 20003.13 8 19621.23 4.17 34.72 15342.93 - green, Siberia - small) mg of nicotine 4.23 4.17 4.25 3.26 % 39.65 39.08 39.85 30.56 Table 6. Nicotine extraction rate (%) depending on the time pouches spent in saliva (White fox - green, Siberia - small, big) green small big time average mg of % average mg of % average mg of % [min] nicotine nicotine nicotine 5 2797 0.59 4.95 943.75 0.20 1.88 2521.95 0.54 3.27 10 3597.5 0.76 6.37 1886.85 0.40 3.76 4650.25 0.99 6.02 15 4308.15 0.91 7.62 2241 0.48 4.64 4565.45 0.97 5.91 30 6565.05 1.39 11.62 2747.55 0.58 5.47 4925.1 1.05 6.38 45 8649.35 1.84 15.31 4326.95 0.92 8.62 6223.2 1.32 8.06 90 10683.05 2.27 18.91 7564 1.61 15.07 12780.35 2.71 16.55 120 14238.75 3.02 25.20 7791.35 1.65 15.52 14246.85 3.03 18.45 Proceedings of 10 th Socratic Lectures 2024 74 of 165 30 45 25 35 30 % of extraction % of nicotine 40 25 20 15 10 20 15 10 5 5 0 0 0 2 4 6 8 10 0 12 50 100 150 time(min) volume (ml) 80 70 60 50 40 30 20 10 0 nicotine extraction (%) nicotine extraction (%) Figure 1. Nicotine extraction rate (%) depending on the volume of saliva (left) and time pouches spent in the saliva (right); White fox - green pouches. 0 10 20 30 EtOH percentage (%) blue red 40 50 80 70 60 50 40 30 20 10 0 0 5 10 15 20 sugar concentration (%) red blue Figure 2. Nicotine extraction rate (%) depending on the percentage of alcohol (left) or sugar (right) found in saliva (White fox – blue, red pouches). 4. Discussion Time dependence: With all pouches, nicotine extraction rate showed positive linear dependence with time. The percentage of extraction was measured at different intervals, up to two hours, without getting a plateau. At the beginning, we have a linear increa se of the extraction % with the time, but after 45 minutes, the increase is slower, but it continues to rise. Maximum of extraction was achieved after 120 minutes, and it was 25 % with the green pouches. Nicotine extraction lowered with the increasing volume of saliva. For 2 ml of saliva (which was the minimal volume enough to cover pouches completely), we found an extraction rate 42 % in some brands of pouches. Ethanol dependence: The obtained results have shown that the increasing ethanol concentration in saliva (from 5-40% EtOH) leads to a higher extraction rate, coming up to 85% with some pouches. We had a second peak following the first one, possibly as a product of nicotine degradation, or the difference occurred because of the protonated groups. Sugar dependence: In the case of sugar addition, the results were variable. Experiments with red and blue pouches have shown that extraction of nicotine decreases with a higher concentration (5, 10 and 15 %) of sugar. On Proceedings of 10 th Socratic Lectures 2024 75 of 165 the contrary, big and small pouches do not exhibit that correlation, potentially because of the difference in pouch material or ingredients in them. We observed that the sacks are less tight and nicotine could be distributed unevenly. When it comes to pH, we used a pH range that usually varies in saliva (from 5 to 8), and the results have shown no significant differences. In some cases, we had very big areas measured under the peaks, so we had to dilute them 10 times to make it calculable. 5. Conclusion We standardized the existing HPLC-DAD method for nicotine extraction and used it for nicotine quantification in saliva under specific conditions of extraction. As shown in the results, some of the substances, such as alcohol or sugar, can modulate nicotine extraction rate from pouches. Consumers could potentially adjust nicotine consumption by using products that contain these substances. Funding: This research was financially supported by Slovene Research Agency (Research Programme Health Protection Mechanisms P3-0388) Conflicts of Interest: The authors declare no conflict of interest. References 1. Bansal M, Sharma M, Bullen C, Svirskis D. A Stability Indicating HPLC Method to Determine Actual Content and Stability of Nicotine within Electronic Cigarette Liquids. Int J Environ Res Public Health. 2018; 15:1737. DOI: 10.3390/ijerph15081737 2. Bishop E, East N, Bozhilova S, et al. An approach for the extract generation and toxicological assessment of tobaccofree 'modern' oral nicotine pouches. Food Chem Toxicol. 2020; 145:111713. DOI: 10.1016/j.fct.2020.111713 3. Miller-Holt J, Danielson T, Pithawalla YB, Brown AP, et al. Method development and validation of dissolution testing for nicotine release from smokeless tobacco products using flow-through cell apparatus and UPLC-PDA. J Chromatogr B Analyt Technol Biomed Life Sci. 2020; 1141:122012. DOI: 10.1016/j.jchromb.2020.122012 4. Miller-Holt J, Baskerville-Abraham I, Sakimura M, et al. In vitro evaluation of mutagenic, cytotoxic, genotoxic and oral irritation potential of nicotine pouch products. Toxicology Reports. 2022; 9:1316-1324. https://doi.org/10.1016/j.toxrep.2022.06.008 5. Robichaud MO, Seidenberg AB, Byron MJ. Tobacco companies introduce 'tobacco-free' nicotine pouches. Tob Control. 2020; 29:e145-e146. DOI: 10.1136/tobaccocontrol-2019-055321 6. Scherer G, Mütze J, Pluym N, Scherer M. Assessment of nicotine delivery and uptake in users of various tobacco/nicotine products. Curr Res Toxicol. 2022 ; 3:100067. DOI: 10.1016/j.crtox.2022.100067 Proceedings of 10th Socratic Lectures 2024 77 of 165 New Approaches for Testing the (Geno)Toxic Activity of Nano-particles In Vitro Rozman Iza1*, Štern Alja1, Žegura Bojana1 1. National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia * Correspondence: Iza Rozman; iza.rozman@nib.si Citation: Rozman I, Štern A, Žegura B. New Approaches for Testing the (Geno)Toxic Activity of Na-noparticles In Vitro. Proceedings of Socratic Lectures. 2024, 10, 77-85. https://doi.org/10.55295/PSL.2024.II8 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Abstract: The safety of nanomaterials, whether they are made of natural or artificial substances, represents a significant challenge because nanotechnology, as a young and up-and-coming field, is developing very quickly, while nanotoxicology and nanoecotoxicology are falling behind. Since the production, use, and consequently, the exposure of people to nanomaterials is increasing significantly, the acquisition of data on potential acute and chronic toxicity plays a crucial role. It is known that nanomaterials due to their high surface-to-volume ratio, high reactivity, and unique physical, chemical, and biological properties exhibit a greater risk of toxicity than the corresponding bulk material and that is why a comprehensive assessment of the toxicity of nanoparticles should always be done prior to their use. We develop 3D cell models as a new in vitro methodological approach for nanoparticle (geno)toxicity assessment to better understand the impact nanomaterials have on environmental and human health. Currently, as a part of our ongoing study, core-shell iron nanoparticles are being examined, where the core consists of FeO, and the shell is made of Fe3O4. So far, in vitro cyto- and genotoxicity were assessed in the human hepatocellular carcinoma cell line HepG2, using the ATP assay and the comet assay, respectively, but due to ongoing genotoxicity testing and reservations about data publishing, the results will not be presented in this scientific contribution. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and Keywords: 3D cell models; 2D cell models; Cytotoxicity; DNA damage; Genotoxicity; Iron-based nanoparticles conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). __________________________________________________________________________ Proceedings of 10th Socratic Lectures 2024 78 of 165 1. Introduction Nanotechnology is a technological intersection with the nanoscale which straightforwardly links the macroscopic world of our perceptions with the nanoscopic world of individual biomolecules (Contera, 2019). It represents one of the most promising technologies of the 21st century (Bayda et al., 2019) strongly intertwined with our everyday life and society (He et al., 2019). The prefix “nano” is of Greek origin meaning “dwarf” or something very small and depicts one thousand millionth of a meter (10−9 m) (Bayda et al., 2019) hence by the word nanomaterials we describe materials with one or more components that have at least one dimension in the range of 1 to 100 nm (Borm et al., 2006; T. Singh et al., 2017). In the last decade, the production and use of nanomaterials have grown tremendously, and as a result, so has human exposure to these materials (Borm et al., 2006; Zhu et al., 2019). Since human exposure to nanoparticles is inevitable (Yang W, et al., 2021) much attention has been drawn to nanoparticle toxicology (Yang Y, et al., 2017) – especially to the potential acute and chronic adverse effects that nanoparticles may cause on humans (W. Yang et al., 2021) – mostly because nanomaterials due to their high surface-to-volume ratio, high reactivity, and unique physical, chemical, and biological properties (Awashra and Młynarz, 2023; Yang W,et al., 2021) exhibit a greater risk of toxicity than the corresponding bulk material (Hoet et al., 2004). The study of nanoparticle adverse effects and toxicity is referred to as nanotoxicology (Elsaesser and Howard, 2012) and even though exposure to nanoparticles is increasing, information on their toxicological properties remains inadequate (Gornati et al., 2009). For a comprehensive assessment of the toxicity of nanoparticles, structure, and corresponding physicochemical properties need to be fully characterized because only then can the observed toxic effects be attributed to specific properties of nanoparticles in order to establish specific nanoparticle structure-activity/ toxicity functional relationships ( Yang W, et al., 2021). Furthermore, to better understand the mechanisms of nanoparticle toxicity studies at the cellular and sub-cellular levels need to be done (Awashra and Młynarz, 2023). 2. 2.1. In vitro Cell Models Two-dimensional (2D) cell models Genetic toxicity testing is an essential part of drug and material safety assessment since DNA damage can lead to genetic changes, including mutations, chromosome damage, and genomic instability that can lead to cancer development (David, 2020; Maynard et al., 2011). Current EU legislation for chemical and material safety assessment demands testing of chemical/material on two types of in vitro tests: (i) an Ames test (a bacterial test) and (ii) one of two mammalian cell tests – micronucleus test or chromosomal aberration test – followed by an in vivo animal model study (Cimino, 2006; Corvi et al., 2013). For an adequate evaluation of genotoxicity, the evaluation of three parameters is required: gene mutations, structural changes, and numerical changes in chromosomes (R et al., 2013). In in vitro toxicology, the golden standard for studying absorption, distribution, metabolism, excretion, and toxicity of compounds is considered to be human primary hepatocytes, which, compared to permanent liver cell lines, better reflect the properties and phenotype of hepatocytes in vivo, but in practice, due to the limited availability of human liver samples, they are replaced by cell lines such as HepG2, HepaRG, Huh7, SKHep-1, and others. In addition, primary liver cells are difficult to maintain under in vitro conditions. Not only can they be grown for a short period, but they also quickly lose their cuboidal morphology and liver-specific functions during cultivation. The high price and differences between donors due to polymorphisms are also a problem (Arzumanian et al., 2021; Klingmüller et al., 2006; Sefried et al., 2018; Shulman and Nahmias, 2012; Štampar et al., 2020; Zeilinger et al., 2016). Both human primary hepatocytes and human hepatic cell lines are normally cultured as a monolayer of cells (2D cell model) for genotoxicity testing (Laohathai and Bhamarapravati, 1985; Shulman and Nahmias, 2012) with one main difference: primary human hepatocytes cannot be cultured indefinitely as they stop dividing under in vitro conditions (Shulman and Nahmias, 2012). In this specific respect using human hepatic cell lines represents a good alternative for genotoxicity studies. However, one must consider that hepatic cell Proceedings of 10th Socratic Lectures 2024 79 of 165 lines are immortalized, cancer cells, which means that despite retaining a certain degree of the properties of primary hepatocytes, they also show similarities to tumor cells (Arzumanian et al., 2021). While both primary hepatocytes and human hepatic cell lines as model systems have their advantages and disadvantages, both are usually used in 2D culture. 2D cell models have certain limitations for genotoxicity testing. They are indeed associated with simple and low-cost maintenance and performance of functional tests, yet they do not mimic the natural structures of tissues (Kapałczyńska et al., 2016) – 2D models lack the cell-cell and cell-extracellular matrix (ECM) signaling (Breslin and O’Driscoll, 2013; Kapałczyńska et al., 2016), which in turn leads to reduced cell differentiation and modified signaling of metabolic pathways (Aucamp et al., 2017). Furthermore, they lose the diversity of phenotype (Richter et al., 2021; von der MARK et al., 1977), and have unlimited access to the ingredients of the medium such as oxygen, nutrients, metabolites, and signal molecules (Kapałczyńska et al., 2016), forcing them into a polarization that does not reflect physiological conditions (Fontoura et al., 2020). Furthermore, the shortcomings of 2D cell models include the non-robustness of the models (Xiao et al., 2022) and misleading results (Saji et al., 2019) due to which according to ECHA (European Chemical Agency) additional in vivo studies need to be done (“ECHA - European Chemical Agency,” n.d.; R et al., 2013). For these purposes, the European REACH regulation promotes the 3R strategy (Replacement, Reduction, Refinement), to replace and reduce the use of animals in in vivo studies, while at the same time refining test systems, obtaining more relevant results for humans (Törnqvist et al., 2014). As a result of this and the many disadvantages of 2D models, a lot of attention is being paid to the development of new alternative models that will reflect the in vivo conditions more accurately (Ipek et al., 2023) – and one of these are 3D cell models. 2.2. Three dimensional (3D) cell models In toxicology, 3D cell models also known as spheroids are a powerful tool for studying the genotoxic effects of chemicals/materials because they better mimic in vivo conditions (İpek et al., 2023; Wang et al., 2021). Cells inside the spheroid comprise different cell layers (Figure 1) (Edmondson et al., 2014). The external layer is composed of cells displaying high proliferation rates, towards the middle resting cells can be observed, and non-dividing (necrotic) cells can be found in the core of the spheroid (Alvarez-Pérez et al., 2005; Edmondson et al., 2014; Nath and Devi, 2016). The high proliferation rate of cells in the spheroid's external layer can be explained by their easier access to oxygen and nutrients (Tredan et al., 2007). In contrast, cells within spheroids remain in a necrotic state due to the absence of oxygen (hypoxia) and nutrients (Minchinton andTannock, 2006; Tredan et al., 2007). Figure 1. Structure of a spheroid (Edmondson et al., 2014; Nath and Devi, 2016). Created with BioRender.com Proceedings of 10th Socratic Lectures 2024 80 of 165 Apart from the 2D cell model, cells in the 3D model form better cell-to-cell connections and produce a matrix that promotes tissue-specific cell binding, direct cell-to-cell interactions, and cell-to-extracellular matrix interactions (Langhans, 2018). They also preserve their natural morphology (Costa et al., 2016) and maintain high viability for several weeks (Štampar et al., 2021). Gene and protein expression levels in spheroids better resemble levels found in cells in vivo (Costa et al., 2016; Langhans, 2018; Ravi et al., 2015). Therefore, by using 3D cell models, we can reduce the differences between in vitro and in vivo studies, decreasing the likelihood of needing to use animal models (Costa et al., 2016; Langhans, 2018; Ravi et al., 2015). Despite all the advantages of 3D models, they still have certain drawbacks, including more demanding and expensive cell maintenance in culture (Costa et al., 2016; Langhans, 2018; Ravi et al., 2015), difficult replication of experiments, and more demanding interpretation of data (Kapałczyńska et al., 2016). 2.3. Spheroid formation We are developing human hepatocellular carcinoma (HepG2) 3D cell models as a new methodological approach for nanoparticle (geno)toxicity assessment. In general, several approaches and materials can be used for culturing cells in 3D for instance, different hydrogel substrates, e.g., beads, injectable gels, moldable gels, and macroporous structures (Białkowska et al., 2020), or/and different methods such as the forced floating method (Štampar et al., 2019), pellet culture method, liquid overlay method, hanging drop method, etc. (Ryu et al., 2019). Within our department, HepG2 spheroids are routinely formed using the forced floating method (Figure 2), which will be described further on. Figure 2. A simplified scheme of spheroid preparation with the forced floating method (Štampar et al., 2019). Created with BioRender.com When preparing spheroids from HepG2 cells using the forced floating method, 96-well Ubottom low attachment microtiter plates are needed. The cell suspension is mixed with an appropriate volume of cold HepG2 medium (4 °C) – HepG2 medium is composed of MEME medium (MEME-10370-047) containing NEAA supplemented with 10% FBS, all from Gibco (Praisley®, Scotland, UK) and 2.2 g/L NaHCO3, 2 mM L-glutamine, 100 IU/mL penicillin/streptomycin and 1 mM sodium pyruvate from Sigma-Aldrich (St. Louis, MO, USA) – and 4 % methylcellulose in a way that cell density equals 15.000 cells/mL. Then 200 µL of the mixture is pipetted into each well and plates are centrifugated for 90 minutes at 28 °C and 900 g. Due to the centrifugal force, the cells formed clusters (aggregates), which Proceedings of 10th Socratic Lectures 2024 81 of 165 after 72 hours of incubation (37 °C, 5 % CO2) matured into spheroids suitable for further research (Štampar et al., 2019). 3. Methods for assessment of In vitro cell models 3.1. ATP Luminescent Cell Viability Assay The ATP Luminescent Cell Viability Assay is a method for determining the number of viable cells in culture based on the quantification of the ATP present. The measurement of ATP using firefly luciferase is the most frequently applied method for estimating the number of viable cells (Riss et al., 2016) – ATP serves as an indicator of metabolically active cells because when cells lose membrane integrity, they lose the ability to synthesize ATP and endogenous ATPases rapidly deplete any remaining ATP from the cytoplasm (Riss et al., 2016). The ATP assay proved sensitive and user-friendly cell viability assay. It was reported that ATP assay is less prone to artifacts than other viability assay methods (Riss et al., 2016). Another advantage of this assay is that an incubation step with a population of viable cells is not prerequisite to convert a substrate into a colored compound (as in MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, or MTS – 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays), which also eliminates a plate handling step because cells do not need to be returned to the incubator to generate a signal (Riss et al., 2016). 3.1.1. Luminescent cell viability assay protocol For the performance of the cell viability assay, we prepared the spheroids as described in the section 2.3 and exposed them to graded nanoparticle concentrations prepared as described in the section 3.3. After 24-hour or 96-hour exposure, we transferred 5 spheroids for each concentration from the U-bottom microtiter plate to the white opaque walled microtiter plate and added 50 µL of the reagent (CellTiter-Glo®, Promega, Madison, Wisconsin, USA) which contains a detergent to lyse the cells, ATPase inhibitors to stabilize the ATP that is released from the lysed cells, luciferin as a substrate, and the stable form of luciferase to catalyze the reaction that generates photons of light (Riss et al., 2016). Then we resuspended the mixture of reagent and cells and incubated it for 10 minutes at room temperature before measuring the luminescence signal. 3.2. Comet Assay The comet assay, or Single-Cell Gel Electrophoresis (SCGE), is an extremely sensitive and fast quantitative in vitro method that detects DNA damage at the level of a single cell (Nickson and Parsons, 2014). The method enables the detection of single- and doublestrand DNA breaks, alkali-labile sites, DNA-DNA, and DNA-protein cross-linking (Tice et al., 2000). DNA damage can be either endogenous or exogenous. Most of the endogenous DNA damage arises from the chemically active DNA engaging in hydrolytic and oxidative reactions with water and reactive oxygen species (ROS), that are naturally present within cells while exogenous DNA damage occurs when environmental, chemical, and physical agents such as UV and ionizing radiation, alkylating agents, and crosslinking agents damage the DNA (Chatterjee and Walker, 2017). Nowadays the method represents one of the standard methods for DNA damage evaluation with applications in genotoxicity testing, human biomonitoring, molecular epidemiology, eco/genotoxicology, and basic research on DNA damage and repair (Collins, 2004; Cordelli et al., 2021). In the SCGE method, the cells are embedded in an agarose gel, and lysed so that only the nuclei (DNA) remain in the gel. The gel is then exposed to an electric field in the electrophoresis and because the damaged DNA migrates at a different rate than nondamaged DNA using specific dyes we can observe structures, resembling a comet. The undamaged DNA in the comet structure is referred to as the “head” while the trailing damaged DNA band is referred to as the “tail”. The percentage of DNA in the tail is directly proportional to the percentage of DNA damage that has occurred in a particular cell (Nickson and Parsons, 2014; Vandghanooni and Eskandani, 2011). A simplified procedure scheme is shown in Figure 3. Proceedings of 10th Socratic Lectures 2024 82 of 165 Figure 3. A simplified procedure scheme of the comet assay (Nickson and Parsons, 2014; Vandghanooni and Eskandani, 2011). Created with BioRender.com 3.2.1. Comet Assay Protocol After the treatment (section 3.3), a suspension of viable single cells was obtained by the combination of enzymatic digestion and mechanical degradation. For each tested concentration we harvested 5 spheroids and transferred them to a 1,5 mL Eppendorf tube. Then we centrifuged them for 4 minutes at 1000 Rotations Per Minute (RPM) and discarded the media. We washed them with 1 mL 1x Phosphate Buffered Saline (PBS) and repeated the centrifugation step. After that, we discarded the PBS and added 50-100 µL of an enzymatic mixture (10x diluted collagenase (50 mg/ml) solution with serum-free medium (MEME-10370-047, Gibco, Praisley®, Scotland, UK) and TrypLE (Gibco; 12604013, Waltham, Massachusetts, USA) in the ratio of 1:2) into the Eppendorf Safe-Lock Microtube (Eppendorf, Hamburg, Germany) with spheroids. We incubated the spheroids for 8-10 minutes at 37 °C and 5 % CO2. After the incubation, we resuspended the spheroids and added 500 µL of HepG2 growth media with supplements to deactivate the collagenase type I and centrifuged them for 4 minutes at 1000 rpm. We discarded the media and then the comet assay was conducted according to Singh et al. (1988) with minor modifications by Štampar et al. (2019). 3.3. Nanoparticle sample preparation So far, our work has focused on (geno)toxicity assessment of core-shell iron nanoparticles – the core consists of FeO while the shell is made of Fe3O4. First, we evaluated the cytotoxicity of graded concentrations of nanoparticles using the ATP Luminescent Cell Viability Assay. Nanoparticles were dispersed into cell media with the highest concentration of 80 µg/cm2 and the lowest concentration of 0,2 µg/cm2 for 24-hour exposure while the highest concentration for 96-hour exposure was 40 µg/cm2 and the lowest concentration was 0,02 µg/cm2. The concentrations were determined based on the OECD (Organisation for Economic Co-operation and Development) TG 487 guidelines (OECD, 2022) for testing manufactured nanomaterials in a way that the concentration of 40 µg/cm2 equaled the concentration of 100 µg/mL. Usually, it is recommended that the top dose is restricted to 100 µg/mL or 100 µg/cm2, whichever is higher, because doses higher than this are not physiologically relevant, and can result in interference with scoring due to high deposition on cells (OECD, 2022). As a positive control, 15 % DMSO Proceedings of 10th Socratic Lectures 2024 83 of 165 was used, since currently, there are no suitable nanoparticles that can be used as positive controls for the in vitro assays (OECD, 2022). 4. Conclusion In general, the toxicity of nanoparticles can be assessed with several different approaches, among which, the most beneficial ones in terms of cost and time saving are the in vitro studies. In vitro studies are essential to identify biochemical and molecular mechanisms of nanoparticles' cyto- and genotoxicity and are also the first step in identifying potentially harmful effects for humans and the environment. When it comes to in vitro studies, 3D cell models also known as spheroids are a powerful tool for studying the genotoxic effects of chemicals/materials. Not only do they mimic in vivo conditions better, but their usage allows us to minimize testing on animals, thus following the 3R (Replacement, Reduction, and Refinement) principle. Furthermore, spheroids represent a promising model for nanoparticle (geno)toxicity assessment and regarding that, significant progress has already been made. However, a lot of inadequate information is available and that is why more research needs to be done to identify the most appropriate approach for assessing nanoparticle toxicity. 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Zeilinger K, Freyer N, Damm G, Seehofer D, Knöspel F. Cell sources for in vitro human liver cell culture models. Exp Biol Med (Maywood). 2016; 241:1684-198. DOI: 10.1177/1535370216657448 Zhu S, Gong L, Li Y, Xu H, Gu Z, Zhao Y. Safety Assessment of Nanomaterials to Eyes: An Important but Neglected Issue. Adv Sci (Weinh). 2019; 6:1802289. DOI: 10.1002/advs.201802289 Proceedings of 10 th Socratic Lectures 2024 87 of 165 The Effect of Size and Surface Treatment of Nucleating Agents on Polyamide 6 Morphology Studied by Flash Differential Scanning Calorimetry Nardin Blaž1, Pešl Teja1, Rozman Tamara1, Bolka Silvester 1,* 1. Faculty of Polymer Technology, Slovenj Gradec, Slovenia * Correspondence: Silvester Bolka; silvester.bolka@ftpo.eu Citation: Nardin B, Pešl T, Rozman T, Bolka S. The Effect of Size and Surface Treatment of Nucleating Agents on PA6 Morphology Studied by Flash DSC. Proceedings of Socratic Lectures. 2024, 10, 87-97. https://doi.org/10.55295/PSL.2024.II9 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Abstract: The use of recycled polymer materials with low carbon footprint, which enables circular economy, is becoming increasingly interesting for commercial use in the world. The main reasons for this are the accumulation of waste after the use of polymer products and the warming of the atmosphere due to the overloading of the environment with greenhouse gases produced during the extraction of these materials. We were interested in the differences in properties between differently modified samples of thermoset waste in a thermoplastic matrix. Our main focus was on the influence of the modification of the interface on the stiffness and strength of the prepared material. The results of tensile and bending tests showed that with the right combination of compatibilizers, we were able to successfully increase the stiffness and strength of the composites. The simultaneous increase in stiffness and strength is a very good indicator that with the right combination of compatibilizers (polypropylene grafted maleic anhydride (PP-g-MA) and modified thermoplastic polyurethane (TPU) co-polymer) we were able to ensure good surface interaction between the fibres and the polymer matrix in these samples. Waste paper was added to the thermoplastic matrix as a reference. The difference in the performance of the composites can be explained by the better wettability of the waste paper with the SEBS-g-MA compatibilizer and the better interfacial interactions of the modified TPU copolymer with the thermoset and the PP-g-MA with the glass fibres in the case of the waste thermoset composite. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). Keywords: waste thermoset, compatibilizer, thermoplastics, different fraction of waste thermosets, composites Proceedings of 10 th Socratic Lectures 2024 88 of 165 1. Introduction The recycling of plastic waste, especially post-consumer recycling (PCR), has begun and is still developing very slowly. Compared to other materials such as paper, glass and metals, recovery and recycling rates of plastics are generally low. Even in countries with advanced waste management systems and years of experience in recycling, the recycling rates of plastics are generally much lower than those of the other materials mentioned above. This is partly due to the wide variety of uses of plastics, types of additives and types of polymer composites. This diversity of uses is one of the main advantages of plastics, but it also poses a problem for recycling (Shen and Worrell, 2014). The high melting point of polyamides is the result of strong hydrogen bonding in the crystals. This also enables stiffness above the glass transition temperature and almost up to the melting point. The effect is further enhanced when reinforcing materials such as glass fibres are added. The melting point is determined by the number of hydrogen bonds between the chains, which depends on the density of the amide groups. The melting temperature decreases with increasing length of the aliphatic chain between the amide groups. Polyamides crystallize quickly, which can be advantageous for small particles to further accelerate this process; this can shorten the cycle time and increase productivity. Nucleating agents are substances in the form of fine particles that accelerate the crystallization of polyamides. Nucleation increases tensile strength and stiffness, but makes the material more brittle (Wypych, 2016) (Palmer, 2000). The glass transition temperature depends on the cold crystallization of polyamide 6 (PA6) and is influenced by the cooling conditions. The ratio between the rigid and the mobile amorphous fraction in PA6 is determined by the thermal history (Parodi et al., 2017). Nucleating agents are additives used to increase the crystallization rate during processing by controlling the morphology of the polymers. For this reason, nucleating agents are often specific to a particular polymer or family of polymers and should be carefully selected depending on the desired change in physical properties. Nucleating agents at low cooling rates (10 K/min) cause a shift of crystallisation temperature (Tc) to higher temperatures. The crystallization rate of PA6 is not affected by nucleating agents up to 90 °C, as the density of nuclei is very high at temperatures close to the glass transition temperature. In order to measure the effectiveness of nucleating agents, Flash differential scanning calorimetry (DSC) measurements must be carried out by rapidly cooling the melt to a crystallization temperature between the glass transition temperature (Tg) and melting temperature (Tm) (Mileva et al., 2012). The reported crystallization rate for PA6 is lower compared to PA66, which is due to the odd (PA6)/even (PA66) number of CH2 groups (Poel, Istrate and Mathot, 2016). With Flash DSC the fast-scan chip-calorimeter measurements in a broad temperature range were conducted, also in low temperature region. In the case of nylon, the crystallization rate showed a parabolic shape depending on the crystallisation temperature with a maximum between Tg and Tm (Li et al., 2020). 2. Methods & materials Commercially available PA6 (Akulon K222-D) was purchased from DSM, Netherlands (PA6 in Table 1). Commercially available antioxidant (AT 10 – AO in Table 1) was purchased from AMIK ITALIA, Italy. Commercially available CaCO 3 was provided by Calcit, Slovenia. Trade names of CaCO3 are Calplex Extra (NA01 in Table 1), Calplex 5 (NA02 in Table 1) Calplex 40 (NA03 in Table 1), Polyplex Extra (NA11 in Table 1), Polyplex 5 (NA12 in Table 1) and Polyplex 40 (NA13 in Table 1). Commercially available talc (Plustalc H15 – NA T in Table 1) was purchased from Elementis Minerals, Netherlands. Commercially available nucleating agent (Prisma AD PE Nucleant 91225 – NA AD in Table 1) was purchased from Frilvam, Italy and the only material in the pellet form. The composition of the samples is listed in Table 1. For all tests, the materials were mixed separately and extruded on the Labtech LTE 20-44 twin screw extruder. The screws had a diameter of 20 mm, an L/D ratio of 44:1, a screw speed of 600 rpm and an increasing temperature profile from the hopper (245 °C) to the die (260 °C) (Pracella et al., 2010). Proceedings of 10 th Socratic Lectures 2024 89 of 165 The first sample (Sample 0) was pure PA6, which we extruded and injection moulded. Then we produced a PA6 sample with 0.5 % antioxidant (Sample 1, with AO). Composites with 0.1 % calcium carbonate were produced in 6 different versions; in three the calcium carbonate was uncoated (Samples 2-4, with NA01, NA02 and NA03, respectively) and in three it was coated with stearic acid (Samples 5-7, with NA11, NA12 and NA13, respectively). We prepared two more versions: in one we added 0.1 % talc (Samples 8, with NA T) and in the last one we added 1 % nucleating agent based on polyethylene with 50 % talc (Samples 9, with NA AD). All composites contained 0.5 % antioxidant. Table 1. Sample composition. Sample AO (%) NA0 1 (%) NA0 2 (%) NA0 3 (%) NA1 1 (%) NA1 2 (%) 0 0 0 0 0 0 0 1 0.5 0 0 0 0 2 0.5 0.1 0 0 3 0.5 0 0.1 4 0.5 0 5 0.5 6 NA1 3 (%) NA T (%) NA AD (%) PA6 (%) 0 0 0 100 0 0 0 0 99.5 0 0 0 0 0 99.4 0 0 0 0 0 0 99.4 0 0.1 0 0 0 0 0 99.4 0 0 0 0.1 0 0 0 0 99.4 0.5 0 0 0 0 0.1 0 0 0 99.4 7 0.5 0 0 0 0 0 0.1 0 0 99.4 8 0.5 0 0 0 0 0 0 0.1 0 99.4 9 0.5 0 0 0 0 0 0 0 1 98.5 The injection moulding was carried out on a Krauss Maffei 50-180 CX with a screw diameter of 30 mm. The temperature profile increased from the hopper (220 °C) to the nozzle (230 °C). The mould temperature was set to 80 °C and the cooling time to 20 s. Injection moulded pieces were shaped according to ISO 527 (for tensile tests and DMA) and ISO 179 (for impact test) (Jazani et al., 2011). The tensile tests were carried out with the Shimadzu AG-X plus in accordance with ISO 527-1 (Huang et al., 2013). Tensile modulus (Et), tensile strength (σm), elongation at tensile strength (ɛm) and elongation at break (ɛtb) were evaluated in the tensile tests. Five measurements were carried out for each sample. The thermomechanical properties were analysed using a Perkin Elmer DMA 8000 (Wunderlich, 2005). The samples were heated at 2 °C/min from 25 °C to 210 °C in an air atmosphere. A frequency of 1 Hz and an amplitude of 20 μm were used in dual cantilever mode (Jazani et al., 2011). Thermogravimetric analyses (TGA) were performed using a Mettler Toledo TGA/DSC 3+ thermal analyser. Analyses were carried out in a nitrogen atmosphere (20 mL/min) from 40 to 550 °C with a heating rate of 10 °C/min, followed by an isothermal segment in an oxygen atmosphere (20 mL/min) at 550 °C for 10 minutes using an Al crucible. Changes in the weight are evaluated as degradation in % and peak of the first derivative of the measured curve as degradation temperature. Thermal measurements were performed with a Differential Scanning Calorimeter (DSC 2, Mettler Toledo) in a nitrogen atmosphere (20 mL/min). The temperature of the samples was raised from 0 to 260 °C at a heating rate of 10 °C/min and held in the molten state for 5 minutes to clear the thermal history. After cooling at 10 °C/min, the samples were reheated at 10 °C/min to 260 °C. Tc, the crystallisation enthalpy (ΔHc), Tm and the melting enthalpy (ΔHm), Tg and the change in specific heat capacity (Δcp) were determined from the cooling and the second heating scan. The crystallinity (Wc,h) was calculated from melting enthalpy, divided by the melting enthalpy of 100 % crystalline PA6 (Wunderlich, 2005). Flash DSC 1 with Huber TC45 intercooler and nitrogen purge gas (50 mL/min) was used to preformed crystallization kinetics measurements. The test samples were heated (230 °C) and cooled (20 °C) three times at a high heating and cooling rate (2.000 °C/s) so that Proceedings of 10 th Socratic Lectures 2024 90 of 165 most of the nuclei that formed during cooling from previous processing have melted. During the first heating, the sample melts and spreads over the entire sensor. During the second heating the thermal history is erased and during the third heating we obtain realistic results of the material. The sample is cooled to different temperatures and held there for 5 seconds. The temperatures at which the measurement stopped were between 55 °C and 180 °C in 5 °C increments. After the measurements were completed, we evaluated the rate of crystallization as an exothermic peak during this isothermal segment. The impact tests were performed using the Dongguan Liyi Test Equipment pendulum, type LY-XJJD5 (Lin et al., 2011), according to ISO 179. The distance between the supports was 60 mm and a 5 J pendulum was used. Impact strength was determined as the quotient between the energy required to fracture the sample divided by the cross-section of the sample. 3. Results & Discussions 3.1. Tensile tests The results of the tensile tests are shown in Table 2. Sample 0 is the reference material, pure PA6, with a tensile modulus of 2.29 GPa, tensile strength of 70.0 MPa, elongation at tensile strength of 4.52 % and elongation at break of 114 %. In Sample 1, only the antioxidant was mixed into the matrix. The addition of the antioxidant reduced the tensile modulus Et by 15.7 %, the tensile strength σm by 0.1 %, the elongation at break ɛtb by 24.6 % and increased the elongation at tensile strength ɛm by 3.5 % (Table 2). Samples 2, 3 and 4 consisted of matrix, antioxidant and uncoated calcium carbonate, which differ in particle size. Sample 2 and 3 showed a decrease in tensile modulus Et at tensile strength σm by 1.3 % and 6.1 %, respectively; Sample 4 showed an increase in tensile modulus Et at tensile strength σm by 3.9 %. All three samples showed a decrease in tensile strength σm by 1.2 % for Sample 2, 2.7 % for sample 3 and 2.7 % for Sample 4. The elongation at tensile strength ɛm did not change drastically. The elongation at break ɛm decreased by 15.2 % for sample 2, 52.5 % for sample 3 and 42.1 % for sample 4 (Table 2). Samples 5, 6 and 7 consist of a matrix, an antioxidant and coated calcium carbonate with different particle sizes. For all three samples, we observe a decrease in tensile modulus Et, tensile strength σm and elongation at tensile strength ɛm and an increase in elongation at break ɛtb. Of these three samples, Sample 5, which contained Polyplex Extra with the smallest particles, achieved the highest stiffness, strength and toughness. It was as we expected: the smaller the particles, the better the effect of adding calcium carbonate (Table 2). To improve the tensile modulus Et, we need to use Calplex 40, which is uncoated and has larger particles than Calplex Extra and Calplex 5. Other calcium carbonates reduced the tensile modulus Et (Table 2). All samples showed a reduction in tensile strength σm compared to neat PA6, but this was lowest for Calplex Extra, which is uncoated and contains the smallest particles. The Calplex additives are optimal for slight changes in tensile strength σm, which hardly change it at all; the Polyplex additives reduce it slightly (Table 2). Elongation at break ɛtb increased the most with Polyplex 40, which contains larger particles and is coated. Talc is added to Sample 8. Talc reduced the tensile modulus Et by 3.5 % compared to pure PA6, while the tensile strength σm increased slightly (by 0.3 %). The elongation at tensile strength ɛm decreased by 6.2 %; the elongation at break ɛtb decreased by 71.6 % compared to the reference (Table 2). A nucleating agent consisting of 50 % talc and 50 % LDPE reduced the tensile modulus Et by 2.2 %, the tensile strength σm by 2.6 %, the elongation at tensile strength ɛm by 1.6 % and the elongation at break ɛtb by 33.1 % (Table 2). Proceedings of 10 th Socratic Lectures 2024 91 of 165 Table 2. Tensile tests results with recycled PP matrix. Et (GPa) σm (MPa) εm (%) εtb (%) 0 2.29 ± 0.12 70.0 ± 1.1 4.52 ± 1.14 144 ± 36 1 1.93 ± 0.34 70.0 ± 0.5 4.68 ± 0.07 86 ± 57 2 2.26 ± 0.15 68.1 ± 0.2 4.52 ± 0.04 173 ± 46 3 2.15 ± 0.03 68.1 ± 0.6 4.52 ± 0.09 173 ± 41 4 2.38 ± 0.14 68.1 ± 0.6 4.51 ± 0.14 66 ± 42 5 2.25 ± 0.06 65.7 ± 0.4 4.39 ± 0.08 174 ± 52 6 2.01 ± 0.01 65.3 ± 0.5 4.47 ± 0.11 186 ± 0 7 2.02 ± 0.01 64.1 ± 1.2 4.46 ± 0.07 208 ± 37 8 2.21 ± 0.01 70.2 ± 0.4 4.24 ± 0.08 32 ± 9 9 2.24 ± 0.02 68.2 ± 0.5 4.45 ± 0.13 76 ± 22 Sample 3.2. Dynamic mechanical analysis tests The storage modulus (Figure 1) of the samples agrees with the results of the tensile test, with the exception of Sample 4. Sample 0 has the highest storage modulus, while Sample 6 has the lowest storage modulus, measurement of stored energy representing the elastic protion. Uncoated calcium carbonate lowers the storage modulus, namely Calplex Extra by 7.7 %, Calplex 5 by 10.1 % and Calplex 40 by 16.6 %. Coated calcium carbonate reduces the storage modulus more, namely by 18.5 % for Polyplex Extra, 24.9 % for Polyplex 5 and 22.4 % for Polyplex 40. Talc lowers the storage modulus by 19.0 %, while the combination of talc and LDPE lowers it by 24.4 %. The curve is constantly decreasing, which means that we did not observe any cold crystallization during heating. Figure 1. Storage modulus vs. temperature for the samples. Proceedings of 10 th Socratic Lectures 2024 92 of 165 The peak of the loss factor curve (Figure 2, Table 3) indicates the glass transition. If the peak is lower, it means that the material has a higher elastic response, i.e. a higher degree of crystallinity. This can be observed in Sample 8, to which talc was added, and is consistent with the results of the tensile tests. The loss factor, quotient between viscous and elastic portion, is highest for Sample 0 and lowest for sample 9. Uncoated calcium carbonate lowers the loss factor. The smallest particle size (Calplex Extra) reduces the loss factor by 2.2 %, the medium size (Calplex 5) by 3.3 % and the largest size (Calplex 40) by 12.2 %. Coated calcium carbonate or Polyplex Extra, Polyplex 5 and Polyplex 40 reduce the loss factor by 10.5 %, 8.3 % and 12.2 % respectively. Talc reduces the loss factor by 11.6 % and the mixture of talc and LDPE by 16.6 %. From this we can conclude that the loss factor decreases with the addition of talc and calcium carbonate. Figure 2. Loss factor (Tan δ (-)) vs. temperature (°C) for the samples. Table 3. Loss factor results at DMA measurements for the samples. Tg (°C) 0 tan δ (-) 0.181 62.1 1 0.177 62.1 2 0.177 62.0 3 0.175 62.4 4 0.159 61.2 5 0.162 57.6 6 0.166 60.1 7 0.159 58.0 8 0.160 56.8 9 0.151 61.6 Sample Proceedings of 10 th Socratic Lectures 2024 93 of 165 3.3. Thermogravimetric analysis tests The degradation temperature of the samples varied within less than 0.3 % (Figure 3), which means that the additives we used did not drastically affect the degradation temperature. The inorganic filler content changed (Figure 3), which is completely logical as we have added inorganic fillers. Pure PA6 has 0.04 % inorganic fillers. The addition of an antioxidant increases the proportion of inorganic fillers to 0.08 %. In Samples 2 to 6, the inorganic fillers were 0.1 % - as much as we have added calcium carbonate. Sample 7 had only 0.04 % inorganic fillers. Sample 8 had 0.07 % inorganic fillers. Sample 9 had 0.34 % inorganic fillers, which means that some of the nucleating agent, which is low density polyethylene (LDPE), has degraded. Since the scattering of the results are smaller than the amount of inorganic residues in neat PA6 we can conclude that the distribution of the fillers is homogeneous and constant. Figure 3. Degradation temperature and degradation amount at TGA measurements for the samples. 3.4. DSC tests The results of the DSC analysis are shown in Table 4. The melting temperature Tm of sample 0 is at 220.1 °C. Most of the additives had no drastic influence on the melting temperature. The glass transition temperature Tg did not change drastically due to the additives (less than 2 % with the exception of Calplex 5, which lowered it by 3.7 %). In theory, it was stated that nucleating agents have no effect on the melting temperature (Poel, Istrate and Mathot, 2016), which is consistent with our results. The melting enthalpy (ΔHm) was most strongly affected by talc, which increased it by 5.2 % (Table 4, Sample 8). This was followed by Sample 9, where ΔHm was 4.4 % higher than that of pure PA6. ΔHm was reduced only by antioxidants (by 2.3 %) and Calplex 40 (by 2.6 %); all other additives increased it by between 0.8 % (Calplex 5) and 5.2 % (talc) (Table 4, Samples 3 and 8, respectively). The degree of crystallinity of pure PA6 is 31.5 %. The addition of antioxidants reduced it by 1.8 %. Calplex Extra increased it by 4.2 %, Calplex 5 by 1.4 % and Calplex 40 reduced it by 2.1 % (Table 4). This indicates that smaller particles are more effective because they have a larger surface area in the case of uncoated calcium carbonate. The addition of Polyplex Extra increased the degree of crystallinity by 2.0 %, Polyplex 5 by 3.5 % and Polyplex 40 by 4.0 %. With coated calcium carbonate it is exactly the opposite as with uncoated calcium carbonate. Larger particles achieve a higher degree of crystallinity. The most optimal inorganic filler for increasing the degree of crystallinity is talc. It increases it by 5.9 %, while the combination of talc and LDPE increases it by 4.9 %. Proceedings of 10 th Socratic Lectures 2024 94 of 165 The crystallization temperature is an indicator of heterogeneous crystallization, which is modelled by the addition of nucleating agents. Sample 8 has the highest crystallization temperature, followed by sample 2, which is consistent with the tensile tests and the DMA results. Table 4. DSC tests results for the samples. Sample Tg (°C) Tm (°C) ΔHm (J/g) Wc,h (%) Tc (°C) 0 55.5 220.1 72.5 31.5 191.7 1 54.9 220.8 70.8 31.0 191.4 2 54.5 220.0 75.1 32.9 192.1 3 53.5 220.7 73.1 32.0 191.5 4 54.3 220.4 70.6 30.9 191.8 5 54.6 220.3 73.5 32.1 191.8 6 55.3 220.7 74.6 32.6 191.5 7 55.1 220.6 75.0 32.8 191.5 8 54.4 220.5 76.3 33.4 193.0 9 54.6 220.0 75.7 33.1 191.9 3.5. Flash DSC tests The Flash DSC analysis (Figure 4) shows the peak temperature of the crystallization and the crystallization rate. The highest is peak temperature, the fastest is the crystallization. The height of the graph represents the rate of crystallization. If the graph is higher, it means that the material crystallizes faster. All materials were tested under the same conditions. If the peak of the crystallization curve is shifted towards higher temperatures, this means that smaller crystals are formed (Poel, Istrate and Mathot, 2016). This gives the material greater transparency and toughness (Poel, Istrate and Mathot, 2016). Sample 8, where talc was added into PA6 matrix, crystallized the fastest. From this we can conclude that Sample 8 had the highest degree of crystallinity, which was confirmed by our DSC analysis results (Table 4). Sample 2 crystallized fastest at a higher temperature, which means that smaller crystals were formed and it was more transparent after injection moulding. From this we can conclude that Calplex Extra provides nuclei for crystallization because it increases the crystallization temperature. For calcium carbonate, Calplex Extra proved the best product because it is uncoated and contains the smallest particles. It increased the degree of crystallinity, the rate of crystallization when cooling PA6 and the temperature at which the material crystallizes fastest. Stiffness increased and strength was reduced. Coated calcium carbonate also increased the degree of crystallinity, but the other properties were not improved as much. With sufficiently small nucleating agents, no steric hindrances occured, which is why the degree of crystallinity also increased. Proceedings of 10 th Socratic Lectures 2024 95 of 165 Figure 4. Flash DSC crystallization rate evaluation for the samples. 3.6. Impact tests Figure 5 presents the impact strength of the samples. The impact strength of Sample 0 was 114 kJ/m2; the sample did not crack. The impact strength decreased with all additives except talc. Antioxidant decreased the impact strength by 5.5 %; Calplex Extra decreased it by 7.0 %; Calplex 5 decreased it by 3.3 %; Calplex 40 decreased it by 9.0 %. Polyplex Extra had the greatest effect on impact strength, reducing it by 17.1 %. Polyplex 5 reduced it by 5.4 % and Polyplex 40 by 2.0 %. Talc was the only agent that increased impact strength by 0.6 %. The mixture of talc and LDPE reduced the impact strength by 1.6 %. All additives that act as nucleating agents (except talc) reduced the impact strength. Figure 5. Impact strength for the samples. Proceedings of 10 th Socratic Lectures 2024 96 of 165 4. Conclusions Generally, it was observed that the type, size, and surface treatment of the nucleating agents influenced the morphology behaviour of PA6. Among them, NA01 was the best, as it increased the degree of crystallinity the most. The degree of crystallinity increased with all variants of added calcium carbonate, except for NA03. Uncoated particles larger than 90 μm already hinder crystallization. The highest crystallization temperature was achieved with Calplex Extra, which increased productivity. The difference between coated and uncoated calcium carbonate was large. Our results showed that it is better to use smaller particles as they have a larger specific surface area. The addition of calcium carbonate lowered the tensile modulus and tensile strength of PA6. The addition of coated calcium carbonate increased the elongation at break, while the addition of uncoated calcium carbonate decreased it. The impact toughness was reduced the most by Polyplex Extra and the least by Polyplex 40. The inorganic filler that increases the degree of crystallinity of PA6 the most was talc (Sample 8). It increased it by almost 6 % without changing the melting point of PA6. PA6 with added talc is also characterised by its crystallization rate, but crystallizes at a low temperature (Sample 8), compared to other tested samples. Talc lowered the tensile modulus and the elongation at break and slightly increased the tensile strength of PA6. Of the versions of calcium carbonate tested, Calplex Extra proved to be the most effective nucleating agent. Although talc increased the degree of crystallinity most successfully, Calplex Extra achieved the highest crystallization temperature. For further investigations, we would recommend determining the optimum concentration of calcium carbonate. The effect of the particle size and concentration of the nucleating agent should be further investigated. Based on the results of our study, we would suggest an investigation in the direction of even smaller particle size of nucleating agents. From the present study it is evident, that too high concentration has no positive effect on the degree of crystallinity. The initial concentration would be 0.1 %; it would be increased in steps of 0.1 %; the final concentration would be 1.0 %. Conflicts of Interest: The authors declare no conflict of interest. References 1. Huang R, Xu X, Lee S, et al. High Density Polyethylene Composites Reinforced with Hybrid Inorganic Fillers: Morphology, Mechanical and Thermal Expansion Performance. Materials. 2013; 6: 4122–4138. DOI: 10.3390/ma6094122 2. 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Elsevier Inc. 2014; DOI: 10.1016/B978-0-12-396459-5.00013-1. 11. Wunderlich B. Thermal Analysis of Polymeric Materials - Bernhard Wunderlich. Springer. 2005. 12. Wypych G. Handbook of nucleating agents. G Wypych (ed.). Toronto: ChemTec Publishing. 2016 Proceedings of 10th Socratic Lectures 2024 99 of 165 The Use of Different Fractions of Waste Thermosets for Ther-moplastic Composites Nardin Blaž 1, Pešl Teja1, Rozman Tamara1, Bolka Silvester 1,* 1. Faculty of Polymer Technology, Slovenj Gradec, Slovenia * Correspondence: Silvester Bolka; silvester.bolka@ftpo.eu Citation: Nardin B, Pešl T, Rozman T, Bolka S. The Use of Different Fractions of Waste Thermosets for Ther-moplastic Composites. Proceedings of Socratic Lectures. 2024, 10, 99-111. https://doi.org/10.55295/PSL.2024.II10 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the Abstract: The use of recycled polymer materials with a low carbon footprint, which enable a circular economy, is becoming increasingly interesting for commercial use in the world. The main reasons for this are the accumulation of waste after the use of polymer products and the warming of the atmosphere as a result of the overloading of the environment with greenhouse gases produced during the extraction of these materials. We were interested in the differences in properties between differently modified samples of thermoset waste in a thermoplastic matrix. We paid the most attention to the influence of the modification of the interface on the stiffness and strength of the prepared material. The results of tensile and bending tests showed that with the right combination of compatibilizers, we could successfully increase the stiffness and strength of the composites. The simultaneous increase in stiffness and strength is a very good indicator that with the right combination of compatibilizers (PP-g-MA and modified TPU co-polymer) we were able to ensure a good surface interaction between the fibres and the polymer matrix in these samples with the right combination of compatibilizers (PP-g-MA and modified TPU copolymer). Waste paper was added to the thermoplastic matrix as a reference. The difference in the performance of the composites can be explained by the better wettability of the waste paper with the SEBS-g-MA compatibilizer and the better interfacial interactions of the modified TPU copolymer with the thermoset and the PP-g-MA with the glass fibres in the case of the waste thermoset composite. terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/ by/4.0/). Keywords: waste thermoset, compatibilizer, thermoplastics, different fraction of waste thermosets, composites Proceedings of 10th Socratic Lectures 2024 100 of 165 1. Introduction The recycling of plastic waste, especially post-consumer recycling (PCR), has started and is still developing very slowly. Compared to other materials such as paper, glass and metals, recovery and recycling rates are generally low. Even in countries with advanced waste management systems and years of experience in recycling, the recycling rates of plastics are generally much lower than those of the other materials mentioned above. This is partly due to the wide variety of uses of plastics, types of additives and types of polymer composites. This diversity of uses is one of the main advantages of plastics, but it also poses a problem for recycling (Shen and Worrell, 2014). Industrial waste is generally better separated than municipal waste, which is why municipal waste is sorted much more frequently than industrial waste. The same applies to washing, as municipal waste is usually much more contaminated. It is therefore easier to recycle industrial waste. As a result, we can produce recyclable materials of significantly higher quality and with better or, above all, more homogeneous properties (mechanical, chemical, optical) from this waste (Ragaert et al.,2017). Polyolefins are a type of polymer that can be perfectly mechanically recycled, modified and reused in a variety of applications with suitable separation processes from industrial and municipal waste. The most common polyolefins include polypropylene (PP) and polyethylene (PE). Precisely because of their mechanical recycling properties and the possibility of modification, polyolefins are very often modified by adding various fillers in order to reduce the price and increase rigidity and strength. Due to their low cost, low density and high stiffness, natural fibres such as wood, cellulose, jute, bamboo, conifers and, last but not least, waste paper have attracted the attention of researchers. Thermoset waste and waste paper make up a large proportion of municipal waste which, if properly separated and processed, can be an excellent addition to the polymer matrix as a reinforcing agent, improving above all the mechanical properties of the biocomposite. The incorporation (dispersion) of fibres and the interfacial adhesion of thermoset waste and waste paper with the polyolefin matrix can be problematic. Therefore, improving the interfacial interactions between hydrophilic glass and natural fibres and hydrophobic polyolefins is an important research task, as the interfacial adhesion between glass and natural fibres and polyolefins plays an important role in determining the properties of composite materials. In this way, we can reduce the impact on the environment and realise the idea of transforming waste materials into valuable, commercially viable biocomposites (Bolka et al., 2020). Thermoset composites are cured by a chemical reaction or by heat or radiation (Boquillon and Fringant, 2000; Hay and O’Gara, 2006; Walczyk and Kuppers, 2012). Their main advantage is excellent properties at the same time low production cost when combining the thermoset matrix with reinforcing fibers (Gore and Kandasubramanian, 2018; Blanco et al., 2021; Caydamli et al., 2021). At the end of the life of the composites, the waste stream can be divided into three main streams. Waste stream of smaller products that are not systematically collected at national or European level and end up as mixed waste. The second waste stream consists of large composite parts (parts of wind turbines, aeroplanes, ships). The third waste stream consists of industrial waste from the production of composite materials. For this waste stream, the composition and quantity of the waste are known (Colledani, 2022). There are mechanical, thermal and chemical recycling technologies for composites. Regardless of the technology, the reinforcement fibres obtained cannot completely replace the virgin fibres, but can only be added to the virgin fibres to a certain percentage in order to maintain the properties of the composites. Mechanical recycling always takes place in several steps. Firstly, large composite parts are cut into smaller pieces that can be shredded. This is followed by shredding and, if necessary, screening, during which fractions of different sizes are obtained. The smallest fraction in the form of dust can be used as filler or reinforcement (Bernardeau et al., 2018), but is usually utilised for energy by incineration. Larger fractions are usually used in the manufacture of new composite products (Pickering, 2006; Oliveux et al., 2015). Proceedings of 10th Socratic Lectures 2024 101 of 165 This paper presents a two-pronged review of thermoset composite waste. In the first part, the use of the smallest fraction of waste thermoset composites in combination with a thermoplastic polyamide matrix (Bernardeau et al., 2018) is presented. In the following, the use of a larger fraction as reinforcement for a thermoplastic recycled polypropylene matrix (Bream and Hornsby, 2000; 2001) is presented, using composites with waste paper for comparison (Bourmaud and Baley, 2007; Zhidan et al., 2011; Xiaolin, Xiangfeng and Rumin, 2013; Akbulut et al., 2016; Scholten and Meiners, 2019). 2. Methods & materials Commercially available PA 6 (Badamid B70) was purchased from Bada, Germany (PA6 in Table 1). Commercially available recycled polypropylene was donated by TAB-IPM , Slovenia in ground form (rPP in Table 2). Commercially available antioxidant (AT 10 – AO in Tables 1 and 2) was purchased from AMIK ITALIA, Italy. Commercially available lubricant (Crodamide ER – SA in Table 1) was purchased from Croda, Italy. Commercially available ethylene elastomer modified with compatibiliser (Fusabond N416) was purchased from DuPont, Switzerland (C1 in Table 1). The commercially available compatibilizer PP-g-MA (Exxelor PO 1020) was purchased from Exxon Mobil, Netherlands (C2 in Table 2). The commercially available compatibilizer SEBS-g-MA (Taipol 7126) was purchased from TSRC Corporation, Taiwan (C3 in Table 2). Commercially available compatibilizer modified TPU copolymer (Kuramiron U TU-S5265) was purchased from Kuraray Europe, Germany (C4 in Table 2). The waste paper was donated by Papirnica Vevče, Slovenia in ground form (WP in Table 2). The thermoset composite waste in dust form was donated by the company Iskra ISD plast, Slovenia (rTC1 in Table 1). Table 1. Sample composition with PA6 matrix. Sample PA6 (wt.%) AO (wt.%) SA (wt.%) C1 (wt.%) rTC1 (wt.%) PA6 95.5 0.5 0 0 0.0 PA6 AO rTC10.1 99.4 0.5 0 0 0.1 PA6 AO rTC10.5 99.0 0.5 0 0 0.5 PA6 AO rTC11.0 98.5 0.5 0 0 1.0 PA6 AO SA1 C15 rTC130 63.5 0.5 1 5 30.0 Thermoset composite waste as a fraction sieved through a 4 mm sieve was donated by Technol, Slovenia (rTC2 in Table 2). The composition of the samples is listed in Tables 1 and 2. For all tests, the materials were mixed separately and extruded on the Labtech LTE 20-44 twin-screw extruder. The screws had a diameter of 20 mm, an L/D ratio of 44:1, a screw speed of 600 rpm and an increasing temperature profile for PA6 and rPP from the hopper (165 °C and 220 °C, respectively) to the die (190 °C and 250 °C, respectively) (Pracella et al., 2010). The injection moulding was carried out on a Krauss Maffei 50-180 CX with a screw diameter of 30 mm. The temperature profile for PA6 and rPP increased from the hopper (175 °C and 220 °C) to the nozzle (190 °C and 250 °C, respectively). The mould temperature for PA6 and rPP was set to 80 °C and 45 °C, respectively and the cooling time to 10 s (Jazani et al., 2011). The tensile tests were carried out with the Shimadzu AG-X plus in accordance with ISO 527-1 (Huang et al., 2013). Tensile stiffness (Et), tensile strength (σm), elongation at yield (ɛm) and elongation at break (ɛtb) were evaluated in the tensile tests. Five measurements were carried out for each sample. The thermomechanical properties were analysed using a Perkin Elmer DMA 8000 (Wunderlich, 2005). Proceedings of 10th Socratic Lectures 2024 102 of 165 Table 2. Sample composition with recycled PP matrix. Sample rPP (wt.%) AO (wt.%) C2 (wt.%) C3 (wt.%) C4 (wt.%) WP (wt.%) rTC2 (wt.%) A 100.00 0.00 0.0 0.0 0.0 0 0 B 69.62 0.38 0.0 0.0 0.0 30 0 C 64.62 0.38 0.0 0.0 5.0 30 0 D 64.62 0.38 2.5 0.0 2.5 30 0 E 64.62 0.38 0.0 2.5 2.5 30 0 F 69.62 0.38 0.0 0.0 0.0 0 30 G 64.62 0.38 0.0 0.0 5.0 0 30 H 64.62 0.38 2.5 0.0 2.5 0 30 I 64.62 0.38 0.0 2.5 2.5 0 30 The samples were heated at 2 °C/min from 25 °C to 210 °C and 170 °C under air atmosphere for PA6 and rPP matrix based composites, respectively. A frequency of 1 Hz and an amplitude of 20 μm were used in dual cantilever mode (Jazani et al., 2011). Thermogravimetric analyses (TGA) were performed on a Mettler Toledo TGA/DSC 3+ thermal analysis instrument. The analyses were carried out in a nitrogen atmosphere (20 mL/min) from 40 to 550 °C with a heating rate of 10 °C/min, followed by an isothermal segment in an oxygen atmosphere (20 mL/min) at 550 °C for 30 min. using an Al2O3 crucible. Thermal measurements were performed using a differential scanning calorimeter (DSC 2, Mettler Toledo) under a nitrogen atmosphere (20 mL/min). The temperature of the samples was raised from 0 to 260 °C for PA6 and 0 to 180 °C for rPP samples at a heating rate of 10 °C/min and kept in a molten state for 5 minutes to extinguish the thermal history. After cooling at 10 °C/min, the samples were reheated to 260 °C for PA6 and to 180 °C for rPP at 10 °C/min. The crystallisation temperature (Tc), the crystallisation enthalpy (ΔHc), the melting temperature (Tm) and the melting enthalpy (ΔHm), the glass transition temperature (Tg) and change in the specific heat capacity (Δcp) were determined from the cooling and the second heating scan. (Wunderlich, 2005). The impact tests were performed with the Dongguan Liyi Test Equipment pendulum, type LY-XJJD5 (Zhidan et al., 2011), in accordance with ISO 179. The distance between the supports was 60 mm and a 1 J pendulum was used. 3. Results and Discussion 3.1. Tensile tests A comparison of Et in Table 3 shows that the addition of waste thermoset composite powder as nucleating agent greatly increases Et and that 0.5 wt.% of added waste thermoset composite powder is the optimum amount. When 30 wt.% of waste thermoset composite is added and in combination with a compatibilizer, E t even decreases slightly. The tensile strength is also increased when waste thermoset powder is added as a nucleating agent, and the highest increase in σm is also achieved with 0.5 wt.% waste thermoset powder added. At an addition of 30 wt.% and in combination with a compatibilizer, the tensile strength decreases drastically, as this is a consequence of the shape of the particles, which only act as a filler and not as a reinforcing agent. In terms of elongation, both εm and εtb, the trend is that elongation decreases with increasing addition of thermoset composite powder. However, they decrease drastically when 30 wt.% of waste thermoset composite powder is added and in combination with a compatibilizer. Proceedings of 10th Socratic Lectures 2024 103 of 165 Table 3. Tensile tests results with PA6 matrix. Et (GPa) σm (MPa) εm (%) εtb (%) PA6 2.31 ± 0.20 66.3 ± 0.4 4.3 ± 0.1 158.6 ± 64.8 PA6 AO rTC10.1 2.78 ± 0.25 68.1 ± 0.2 4.4 ± 0.1 15.3 ± 7.2 PA6 AO rTC10.5 2.97 ± 0.20 69.9 ± 1.5 4.2 ± 0.3 6.2 ± 3.0 PA6 AO rTC11.0 2.90 ± 0.17 65.9 ± 6.5 3.9 ± 0.7 5.1 ± 2.2 PA6 AO SA1 C15 rTC130 2.25 ± 0.27 47.0 ± 0.2 3.5 ± 0.2 3.8 ± 0.4 Sample We can conclude that the waste thermoset composite powder acts as a nucleation agent and that the optimum amount to add is 0.5 wt.%. The waste powder of the thermoset composite could also be used as a filler, but the σm and strains are drastically reduced. The Et comparison shown in Table 4 clearly shows that the rPP samples with added fibres achieved significantly higher Et values than the pure rPP polymer matrix samples. This is of course the expected result, as E t increases when fibres are added to the polymer, regardless of whether they are synthetic or natural fibres. The E t value of the pure rPP matrix was 0.9 GPa. Among the rPP samples with added waste paper fibres, the highest E t value was achieved by the D sample and the lowest by the C sample. The Et values of the rPP and waste paper samples with various additives (modifiers) were between 1.26 GPa and 1.69 GPa. Among the samples containing waste thermoset composite with glass fibres and various additives, the highest Et value was achieved by the F sample and the lowest by the G sample. The values of the tensile modulus of the samples containing the thermoset composite with glass fibres were between 1.52 GPa and 1.98 GPa. We can conclude that the addition of fibres to the polymer matrix improves the E t of composite. Even better properties can be achieved by modifying the interface between matrix and fibres, namely by adding antioxidants, modifiers and compatibilizers. Improving the interface between fibres and matrix means better interfacial interactions, as the modifiers act as binders both on the side of the polymer matrix and on the side of the fibres. A comparison of σm and εm is shown in Table 4. Table 4 for σm shows that σm of the pure rPP sample is 25.7 MPa. In the following, we can observe a slightly higher σm of the rPP materials to which paper fibres and additives were added, while at the same time a decrease in εm can be observed for all samples, which is due to the added fibres, as their elongation is much more limited than that of the rPP matrix. It can be observed that the B sample, which contained rPP and paper fibres but no additives, reached the same σm value (27.7 MPa) as the samples made of pure rPP and that at the same time its εm value decreased significantly, namely to an elongation of 5.4 %. For all other rPP samples with added paper and additives, we can observe a concrete increase in the σ m value and at the same time a decrease in the εm value compared to the samples made of pure rPP. The highest σm value was achieved by the sample D, which reached 33.3 MPa at an elongation of 5.7 %. The worst tensile properties of the rPP samples with added paper and additives were exhibited by the sample C, which achieved a value of σm of 25.6 MPa at an elongation of 5.3 %. It can be concluded that all combinations of additives in the samples proved to be effective, as all combinations achieved better results than the rPP sample with added paper without additives. The exception was the addition of modified TPU copolymer to the C sample, which achieved comparable results to the B sample. The combination of the compatibilizers PP-g-MA and modified TPU copolymer had the greatest influence on the improvement in tensile strength. The σm values of the rPP samples with the addition of a waste thermoset composite with glass fibres and additives decreased and were between 19.8 MPa and 21.7 MPa. The only exception was the sample H, which reached σm of 28.0 MPa. In addition, εm decreased for all samples. The elongations of the samples varied between 3.1 % and 4.7 %. The lower tensile strengths can be attributed to poorer interfacial interactions between the matrix and the waste thermoset composite with glass fibres, the lower elongations are the result of significantly lower elongations of the glass fibres and the proportion of waste thermoset composites compared to cellulose fibres. Similar to the rPP samples with added waste paper, the combination of the compatibilizers PP-g-MA and modified TPU copolymer proved to be the best additive. Proceedings of 10th Socratic Lectures 2024 104 of 165 The comparison of εtb shown in Table 4 is extremely revealing as we can see how the addition of fibres to the matrix of pure rPP significantly affects the ability of the composite to elongate before it collapses. The elongation at break decreased from 289.5 % for the pure rPP sample to a minimum of 6.4 % to a maximum of 7.4 % for the rPP samples with added waste paper and additives and to a minimum of 3.8 % to a maximum of 11.2 % for the samples with added waste thermoset composites with glass fibres and additives. The decrease in elongation at break can be attributed to the limited tensile properties of the added waste paper fibres and glass fibres, which allowed less plastic deformation (more brittle fracture) due to their presence in the matrix. Table 4. Tensile tests results with recycled PP matrix. Et (GPa) σm (MPa) εm (%) εtb (%) A 0.90 ± 0.12 25.7 ± 0.7 7.8 ± 0.4 289.5 ± 128.4 B 1.64 ± 0.24 24.7 ± 0.5 5.4 ± 0.3 7.3 ± 0.4 C 1.26 ± 0.17 25.6 ± 0.3 5.3 ± 0.2 6.5 ± 0.7 D 1.69 ± 0.37 33.3 ± 0.3 5.7 ± 0.1 6.4 ± 0.3 E 1.64 ± 0.20 29.5 ± 0.3 5.9 ± 0.2 7.0 ± 0.5 F 1.98 ± 0.18 21.6 ± 0.2 3.2 ± 0.4 7.6 ± 2.4 G 1.52 ± 0.14 19.8 ± 0.2 4.7 ± 0.2 11.2 ± 1.8 H 1.83 ± 0.19 28.0 ± 0.2 3.1 ± 0.2 3.8 ± 0.3 I 1.54 ± 0.21 21.7 ± 0.1 3.2 ± 0.1 8.9 ± 1.4 Sample With the right combination of compatibilizers, we were able to successfully increase the stiffness and strength of the composites, and in all cases the elongation at break was drastically reduced. We were able to increase the stiffness of the composites with waste paper by 82 % and the strength by 33 %, and the stiffness of the waste thermoset composites by 120 % and the strength by 9 %. The simultaneous increase in stiffness and strength is a very good indicator that we have succeeded in ensuring good surface interactions between the surface of the fibres and the thermoplastic matrix in these composites with the right combination of compatibilizers. By using different combinations of compatibilizers, we can produce a composite material with precisely defined properties. The lower strength increase in composites with a waste thermoset composite is to be expected because the waste thermoset composite contains approx. 60 % cured thermoset, which is very brittle, and only 40 % glass fibres. For composites with added waste paper, the partial volumes containing the waste paper particles are much smaller than in the waste thermoset composites, so the partial strains at the interface between the waste paper and the rPP matrix are also much smaller compared to the waste thermoset composite. The size of the partial volumes is reflected in the differences in the tensile strength of these composites. 3.2. DMA tests The storage modulus (Figure 1) in the range from 40 to 90 °C for samples with PA6 matrix is consistent with the results of the tensile test. The more waste thermoset composite powder is added, the higher the storage modulus. The highest storage modulus is found in the entire temperature range for the composite PA6 AO SA1 C15 rTC130, to which 30 wt.% of waste thermoset composite powder was added. All samples show drastic drop in the storage modulus (around 50 °C) in the area of the glass transition of PA6, although this depends on the amount of waste thermoset powder added. Proceedings of 10th Socratic Lectures 2024 105 of 165 Figure 1. Storage modulus vs. temperature for the composites with PA6 matrix. The glass transition increases with the amount of waste thermoset powder added. This can also be seen in Figure 2, which shows the loss factor. The level of the loss factor illustrates the elastic response of the material and is lowest at 0.5 wt.% for the versions to which the waste thermoset composite powder is added. The result is consistent with the tensile tests, in which the PA6 AO rTC10.5 sample showed the highest stiffness and strength. The PA6 AO SA1 C15 rTC130 sample has the peak of loss factor at the highest temperature and the lowest peak height. This means that the sample has the highest glass transition and reacts most elastically of all the samples measured. This is due to the waste thermoset composite powder added at 30 wt.% which acts as a filler but still influences the stiffness and dynamic behaviour of the composite. Figure 2. Loss factor vs. temperature for the composites with PA6 matrix. The storage modulus for the samples with rPP matrix (Figures 3 and 4) is significantly higher for composites with waste paper and waste thermoset composites in the entire temperature range than for the pure rPP matrix. The highest storage modulus is exhibited Proceedings of 10th Socratic Lectures 2024 106 of 165 by the composite samples to which a combination of PP-g-MA compatibilizers and modified TPU copolymer was added. The storage modulus is lower for composites with waste paper than for composites with waste thermoset composites. This result was expected, as glass fibres have a much higher stiffness than cellulose fibres in waste paper, despite their low proportion in the waste thermoset composite. Regardless of the composition, the temperature resistance of the composites is also drastically improved compared to the pure rPP matrix. For composites with waste thermoset composite, the F sample, to which no compatibilizer has been added, is comparable to the H sample. This means that the waste thermoset composite is homogeneously mixed with the rPP matrix. At a temperature of 70 °C, the storage modulus of the F sample starts to decrease compared to the H sample, as there are no good interfacial interactions between rTC 2 and the rPP matrix. Figure 3. Storage modulus vs. temperature for the composites with rPP matrix and waste paper. Figure 4. Storage modulus vs. temperature for the composites with rPP matrix and recycled thermoset composites. The loss factor (Figure 5) in the rPP sample does not show a pronounced peak. The samples with added rTC2 show pronounced peaks, which can be attributed to the glass transition of the thermoset resin in the source of the waste thermoset composite. The peak of the glass transition for the samples is between 100 °C and 110 °C, because this means that the compatibilizers in the samples are differently compatibilized for both the rPP matrix and the thermoset resin. The combination of the compatibilizers PP-g-MA and modified Proceedings of 10th Socratic Lectures 2024 107 of 165 TPU copolymer shows the best compatibility, as the glass transition for the thermoset resin has moved to lower values (closest to the glass transition for the rPP matrix). Figure 5. Loss factor vs. temperature for the composites with rPP matrix and recycled thermoset composites. From the results, we can conclude that the compatibilizer PP-g-MA has good interactions between the glass fibers and the rPP matrix and has a great influence on the mechanical properties of the composite, while the modified TPU copolymer compatibilizer has good interactions between the thermoset resin and the rPP matrix and has great influence on the thermal properties of the composite, but has a weaker influence on the mechanical properties of the composite. 3.3. TGA tests The characterization of the inorganic residues for the samples with PA6 matrix (Figure 6) after TGA annealing shows that there are quite a lot of inorganic additives in the pure PA6 matrix, which are probably processing additives. In the versions to which 0.1 to 1.0 wt.% rTC1 was added, there were no differences in the inorganic residues after TGA annealing. In the PA6 AO SA1 C15 rTC130 sample, the inorganic residue was just under 4 wt.%, which means that only about 13 wt.% glass fibres are contained in the rTC1. Proceedings of 10th Socratic Lectures 2024 108 of 165 Figure 6. Inorganic residues after TGA measurements for the samples with PA6 matrix. The characterization of the inorganic residues for the samples with rPP matrix (Figure 7) after tempering on the TGA shows that 0.3 wt.% of inorganic impurities are contained in the rPP matrix. In the samples of composites with waste paper, the inorganic residues are between 10.9 and 11.8 wt.%. In samples of composites with rTC2, the inorganic residues are between 11.7 and 12.3 wt.%. In composites with recovered paper, there is an inorganic residue of CaCO3, which is added to the paper as a filler and is present in the recovered paper between 36 and 39 wt.%. In rTC2, the inorganic residue consists of glass fibres, which account from 39 to 41 wt.% in rTC2. Figure 7. Inorganic residues after TGA measurements for the samples with rPP matrix. 3.4. DSC tests For samples with a PA6 matrix (Table 5), the addition of small percentages of rTC1 has practically no effect on the glass transition or crystallization on cooling. From this we can conclude that we cannot detect any change in the morphology of the composites with DSC measurements. In the PA6 AO SA1 C15 rTC130 sample, however, the glass transition is significantly higher, the crystallization temperature, the melting enthalpy and also the melting point decrease slightly. Table 5. DSC tests results for samples with PA6 matrix. Tg (°C) Cp (J/gK) Tc (°C) ΔHc (J/g) Tm (°C) ΔHm (J/g) PA6 55.2 0.05 192.2 31.1 220.9 38.5 PA6 AO rTC10.1 55.6 0.06 191.9 32.4 221.1 36.8 PA6 AO rTC10.5 53.6 0.03 191.8 30.0 221.1 37.4 PA6 AO rTC11.0 55.0 0.05 192.1 32.6 220.7 38.5 PA6 AO SA1 C15 rTC130 61.4 0.02 190.7 21.0 220.0 24.5 Sample For the samples with rPP matrix (Table 6), the first melting point is the melting of the low PE content in the rPP matrix and thus the crystallization temperature for the PE content. The melting point and crystallization temperature for the rPP matrix are in the temperature range of 165.4 °C to 166.4 °C and 121.5 °C to 124.4 °C respectively. Composites with a combination of PP-g-MA compatibilizers and modified TPU copolymer exhibit the high- Proceedings of 10th Socratic Lectures 2024 109 of 165 est crystallization temperatures, which means that this combination triggers heterogeneous crystallization of the rPP matrix during cooling. These two composites also achieve the highest degree of crystallinity in the versions with added compatibilizers. Table 6. DSC tests results for samples with recycled PP matrix. 2nd Heating Cooling Tm1 ΔHm1 Tm2 ΔHm2 Tc1 ΔHc1 Tc2 ΔHc2 Sample (°C) (J/g) (°C) (J/g) (°C) (J/g) (°C) (J/g) A 126.4 11.4 165.6 79.0 112.5 9.5 124.0 76.5 B 126.1 8.1 165.8 52.3 112.6 6.9 123.6 51.0 C 126.0 6.9 165.9 49.9 112.3 6.0 122.7 48.9 D 125.9 8.9 166.2 55.3 111.1 6.3 124.4 52.7 E 125.6 6.3 165.4 47.7 112.3 5.5 121.9 46.7 F 126.5 6.8 166.4 54.9 112.4 6.2 123.1 54.2 G 126.3 7.5 166.1 52.5 112.3 6.0 122.6 51.5 H 125.9 8.8 165.8 53.7 111.2 6.6 123.3 50.8 I 125.8 6.0 165.6 50.4 112.0 5.5 121.5 50.2 3.5. Notched impact tests With regard to the notched impact strength (Figure 8), a drastic decrease in all composites samples can be seen, both with waste paper and with added rTC 2. The notched impact strength is slightly higher for composites with waste paper than for composites with rTC2, which is due to the low glass fibre content of rTC 2. Compatibilizers increase the notched impact strength in all variants. This increase in notched impact strength is more pronounced in composites with rTC2, as the polyester resin without compatibilizer does not have good interfacial interactions with the rPP matrix. The sample G has the highest notched impact strength, which means that the modified TPU copolymer with the compatibilizer improves the interactions between the rPP matrix and the polyester resin the most. The results are in very good agreement with the results of the tensile and DMA tests. Figure 8. Notched impact strength for the composites with rPP matrix and recycled thermoset composites. Proceedings of 10th Socratic Lectures 2024 110 of 165 4. Conclusions The tests carried out have shown that with the right choice of thermoplastic matrix, both fine powder from thermoset waste and larger fractions can be used to improve the properties of the newly created composite. The fine powder from thermoset waste works perfectly as a core in the PA6 matrix and the composite material improves stiffness, strength, temperature resistance and glass transition, but drastically reduces elongation at break and toughness. Larger proportions of thermoset waste, which in our case contained only about 40 wt.% of glass fibres, improve the stiffness, strength and temperature resistance of the composite in combination with the right compatibilizers and the rPP matrix, but drastically reduce the elongation at break and toughness. For the rPP matrix, PP-g-MA and modified TPU copolymer proved to be the best combination of compatibilizers, with PP-g-MA providing good interfacial interactions between glass fibers and rPP matrix and modified TPU copolymer between thermoset matrix particles and rPP matrix. The results provide a good basis for further research into the mechanical recycling of thermoset waste as nucleating agent or reinforcement for thermoplastic matrices. In this way, mechanical recycling could be used to utilise the entire amount of waste thermoset composite, as fine dust is a by-product or waste from grinding for larger fractions. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Akbulut T, Akkus M, Ayrilmi N. Mechanical Properties of Wood Polypropylene Composites Produced by Using Resin Impregnated Paper Waste. Machines Technologies Materials. 2016; 2: 70–72. 10:38-40 Available from: https://stumejournals.com/journals/mtm/2016/5/38 Bernardeau F, Caro AS, Perrin D et al. Valorization of waste thermoset material as a filler in thermoplastic: Mechanical properties of phenolic molding compound waste-filled PP composites. Journal of Applied Polymer Science. 2018; 135: 1–11. DOI: 10.1002/app.45849 Blanco D, Rubio EM, Lorente-Pedreille RM, Sáenz-Nuño MA. Lightweight Structural Materials in Open Access: Latest Trends. Materials. 2021; 14:6577. https://doi.org/10.3390/ma1421657 Bolka S, Pešl T, Lorber R. From Recycled Polypropylene To Engineering Plastic Composite Via Addition of Waste Paper. Contemporary Materials. 2020; 1–7. Boquillon N, Fringant C. Polymer networks derived from curing of epoxidised linseed oil: Influence of different catalysts and anhydride hardeners. Polymer. 2000; 41:8603–8613. DOI: 10.1016/S0032-3861(00)00256-1 Bourmaud A, Baley C. Investigations on the recycling of hemp and sisal fibre reinforced polypropylene composites. 2007; 92:1034–1045. DOI: 10.1016/j.polymdegradstab.2007.02.018 Bream CE , Hornsby PR. Structure development in thermoset recyclate-filled polypropylene composites. Polymer Composites. 2000; 21: 417–435. DOI: 10.1002/pc.10199 Bream CE, Hornsby PR. Comminuted thermoset recyclate as a reinforcing filler for thermoplastics - Part II Structure-property effects in polypropylene compositions. Journal of Materials Science. 2001; 36: 2977–2990. DOI:10.1023/A:1017914806565 Caydamli Y, Heudorfer K, Take J, et al. Transparent fiber-reinforced composites based on a thermoset resin using liquid composite molding (Lcm) techniques. Materials. 2021; 14:6087. DOI: 10.3390/ma14206087 Colledani M. Systemic Circular Economy Solutions for Fiber Reinforced Compositesnull. 2022; DOI: 10.1007/9783-031-22352-5 Gore P, Kandasubramanian B. Functionalized Aramid Fibers and Composites for Protective Applications: A Review. Industrial and Engineering Chemistry Research. 2018; 57: 16537–16563. DOI: 10.1021/acs.iecr.8b04903 Hay JN, O’Gara P. Recent developments in thermoset curing methods. Proceedings of the Institution of Mechanical Engineers, Part G: Journal of Aerospace Engineering. 2006; 220:187–195. DOI: 10.1243/09544100JAERO35 Huang R, Xu X, Lee S, et al. High Density Polyethylene Composites Reinforced with Hybrid Inorganic Fillers: Morphology, Mechanical and Thermal Expansion Performance. Materials. 2013; 6:4122–4138. DOI: 10.3390/MA6094122 Jazani OM, Arefazar A, Beheshty MH Study on the effect of processing conditions on the impact strength of PP/SEBS/PC ternary blends using Taguchi experimental analysis. Journal of Polymer Engineering. 2011; 31: 237–241. Proceedings of 10th Socratic Lectures 2024 111 of 165 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. DOI: 10.1515/POLYENG.2011.048 Oliveux G, Dandy LO, Leeke GA. Current status of recycling of fibre reinforced polymers: Review of technologies, reuse and resulting properties. Progress in Materials Science. 2015; 72: 61–99. DOI: 10.1016/j.pmatsci.2015.01.004 Pickering SJ Recycling technologies for thermoset composite materials-current status. Composites Part A: Applied Science and Manufacturing. 2006; 37: 1206–1215. DOI: 10.1016/j.compositesa.2005.05.030 Pracella M, Haque MMU, Alvarez V. Functionalization, compatibilization and properties of polyolefin composites with natural fibers. Polymers. 2010; 2: 554–574. DOI: 10.3390/polym2040554 Ragaert K, Delva L, Van Geem K. Mechanical and chemical recycling of solid plastic waste. Waste Management. 2017; 69: 24–58. DOI: 10.1016/j.wasman.2017.07.044 Scholten A, Meiners D. Use of recycled waste paper as fiber reinforcement for polypropylene-relationship of fiber extraction process and mechanical properties of the composites. AIP Conference Proceedings. 2019; 2055. DOI: 10.1063/1.5084825 Shen L, Worrell E. Plastic Recycling, Handbook of Recycling: State-of-the-art for Practitioners, Analysts, and Scientists. Elsevier Inc. 2014; pp. 179-190. DOI: 10.1016/B978-0-12-396459-5.00013-1 Walczyk D, Kuppers J. Thermal press curing of advanced thermoset composite laminate parts. Composites Part A: Applied Science and Manufacturing. 2012; 43: 635–646. DOI: 10.1016/j.compositesa.2011.12.008 Wunderlich B. Thermal Analysis of Polymeric Materials - Bernhard Wunderlich. 2005. Edited by B. Wunderlich. Springer. Xiaolin Z, Xiangfeng B, Rumin W Study on mechanical properties and water absorption behaviour of wastepaper fibre/recycled polypropylene composites. Polymers and Polymer Composites. 2013; 21: 395–402. DOI: 10.1177/096739111302100608 Zhidan L, Chao C, Zixian G, et al. A Compatibilized Composite of Recycled Polypropylene Filled with Cellulosic Fiber from Recycled Corrugated Paper Board: Mechanical Properties, Morphology, and Thermal Behavior. Journal of Applied Polymer Science. 2011; 122: 2789–2797. DOI: 10.1002/app.34321 Proceedings of 10 th Socratic Lectures 2024 113 of 165 Dark Energy and Dark Matter Žulj Tim1,* Faculty of Electrical Engineering, University of Ljubljana, 1. * Correspondence: Tim Žulj, tz3432@student.uni-lj.si Citation: Žulj T. Dark Energy and Dark Abstract: Study of space is never ending quest of humanity. An emerging question regards dark energy and dark matter which are the subjects of this contribution. We will consider the difference between the dark energy and dark matter, history of matter and contemporary efforts to find experimental evidence on dark energy and dark matter. We will briefly consider galaxy rotation curves, gravitational lensing and cosmic microwave background. Matter. Proceedings of Socratic Lectures. 2024, 10, 113-118. https://doi.org/10.55295/PSL.2024.II11 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/b y/4.0/). Keywords: Dark matter, Dark energy, Universe Proceedings of 10 th Socratic Lectures 2024 114 of 165 1. Introduction Dark matter theories focus on questions such as how did stellar systems and galaxies evolve throughout time, and how is universe changing with time. Despite its significance, knowledge on cosmology is yet rudimentary and better understanding of dark energy and dark matter presents a challenge. Dark matter is defined as a substance that pulls galaxies together, while dark energy pushes them apart (causes universe expansion). It was estimated that roughly 27% of the matter in the universe is dark matter and 68% of the energy in the universe is dark energy (NASA, 2024). Normal matter was estimated to form about 5% of the matter in the universe (NASA, 2024). Normal matter, also known as baryonic matter is composed of particles call baryons, which include protons and neutrons. These baryons make up atoms, which interact with electromagnetic field and can emit, absorb and reflect light. Therefore, normal matter is observable. Dark matter on the other hand is a form of matter that does not interact with electromagnetic field and does not emit, absorb or reflect light. Despite its invisibility dark matter has observable effects on the motion of galaxies (Bertone et al., 2005). 2. History of dark matter Throughout history, philosophers have been speculating about what are we, what is everything around us made of, and what is the nature of the matter. The ancient Greeks were thinking about these things and they indicated that there might be forms of matter that are imperceptible or not noticeable, simply because it was too far away or invisible and undetectable by our senses. The oldest records on the matter were found from 5th century Before the Christian Era (BCE) (Bertone and Hooper, 2018). Leucippus and Democritus were convinced that all matter was made of the same fundamental and invisible building blocks called atoms and that these atoms were infinite in numbers (Bertone and Hooper, 2018). Sir Isaac Newton contributed significantly to the history of cosmology; in 1687 he published »Philosophia Naturalis Principia Mathematica«. He developed scientific tools leading to discoveries about universe. John Michell and Simon Laplace found that there could exist objects so massive that even light would not be able to escape the gravitational pull (Montgomery et al., 2009). This was considered the first mention of black holes (Bertone and Hooper, 2018). In particular after development of astronomical photography, it was acknowledged that stars were not evenly distributed in the sky. In dense stellar fields, dark regions were observed (Bertone and Hooper, 2018). Two explanations were suggested: either stars were lacking or – an absorbing matter were present along the line of sight (Bertone and Hooper, 2018 ).The second explanation was considered the more probable one. Lord Kelvin estimated the amount of dark matter in the Milky Way galaxy (Bertone and Hooper, 2018). He claimed that »if stars in Milky Way can be described as a gas of particles, acting under the influence of gravity, then one can establish a relationship between the size of the system and the velocity dispersion of the stars« (Bertone and Hooper, 2018). The Coma Cluster of galaxies was observed with the telescope (Morrison and Zwicky, 2015, Primack, 2024) and reported on thousands of galaxies. A question was posed what would happen if all these galaxies were stationary? (Morrison and Zwicky, 2015). The answer led to inevitable collapse due to gravity (Morrison and Zwicky, 2015). On the other hand, if the galaxies would be moving above some threshold velocity, they would fly apart (Morrison and Zwicky, 2015). It was concluded that since they are still there after 8000 million years after they were formed, there must exist a fine balance between energy of motion (kinetic energy) and the gravitation attraction (potential energy). If one could measure the kinetic energy, mass of the respective volume of space could be calculated. The result of the calculation indicated that there Proceedings of 10 th Socratic Lectures 2024 115 of 165 is about 6 or 7 times more mass in The Coma Cluster than it was observed (Bertone and Hooper, 2018; Morrison and Zwicky, 2015; Primack, 2024). 3. Evidence of dark matter 3.1. Galaxy rotation curves Galaxy rotation curves are rotational velocities of the stars and gas in the galaxy as a function of their distance from the galactic centre. Under some reasonable simplifying assumptions, it is possible to infer the mass distribution of galaxies from their rotation curves. In 1960s and 1970s, image tube spectrograph was developed ( Bertone and Hooper, 2018) to preform spectroscopic observations of the Andromeda Galaxy. The results on the mass in outer parts of some galaxies disagreed with rotation curves predicted from photometry (Bertone and Hooper, 2018 ). The stars at the edge of galaxies were moving so fast that they should be (according to the theoretical estimations) flung off into space. Instead, the stars stayed in their orbits. This was explained by the gravitational pull exerted by something undetectable (NOVA, 2018). Newton's law of gravity describes the gravitational force Fg acting on an object with mass m in rotational orbit: Fg = G Menc m/r2 , (1) where G is gravitational constant, Menc is the mass enclosed within the orbit and r is the distance from the centre of mass. The gravitational force acts as the centripetal force Fc needed to keep the object in rotational motion, Fc = mv 2/r , (2) where v is the orbital velocity. When we set these two forces equal to each other and express v, we get v = (G Menc/r)1/2 . (3) The calculated velocity was however considerably smaller than the measured velocity (Figure 1). Agreement however be obtained by assuming the presence of the additional mass, or dark matter, within galaxies. The equations are fundamental to understanding galaxy rotation curves and indicate the existence of dark matter in galaxies. Figure 1: Rotation curves of the Andromeda Galaxy. Measured rotational velocities of the outer stars are the blue line, while velocities that would be expected from the estimated mass of the visible matter in the galaxy are the green line. Adapted from (Nerich, 2011). Proceedings of 10 th Socratic Lectures 2024 116 of 165 3.2 Gravitational lensing Gravitational lensing is a phenomenon, where the gravitational field of a massive object, such as a galaxy or a cluster of galaxies, bends and distorts the light from distant objects, like background galaxies. Already Newton considered the possibility that light could be deflected by gravity (Bertone and Hooper, 2018). But within the general relativity theory published by Einstein, this phenomenon was quantitatively predicted (Bertone and Hooper, 2018 ). The empirical test of gravitational lensing and of the general relativity theory was conducted in 1919 by Arthur Eddington who organized expedition to observe solar eclipse (Bertone and Hooper, 2018). Experiment consisted of measurement of the deflection of light by the gravitational field of the massive object (sun). Bending of light lead to the distortion or magnification of distant objects like background galaxies or quasars. The results of measurement were in favour of Einstein’s predictions (Bertone and Hooper, 2018). After the confirmation, the gravitational lensing became a powerful tool for cosmologists and astrophysicists to study the distribution of matter in universe including dark matter and dark energy. It was used for discovery and study of distant galaxies and for estimation of the expansion rate of the universe (Bertone and Hooper, 2018). Following Bovy (2023), we describe the deflection of light due to gravity as illustrated in Figure 2 where a two dimensional representation of the three-dimensional configuration is shown. In the figure, the source is on the left, the lens is in the middle, and the observer is on the right. The extent of the lens are considered much smaller than the distances between the source, lens, and observer so that the lens is taken as very thin in the direction perpendicular to the light ray. The perturbed light path is taken to be a straight line until it reaches the lens at ξ where it changes its direction by the deflection angle α̂ and continues its path in a straight line until being observed. The angle β represents the direction of light in the absence of the lens while the angle θ represents the observed direction. All the angles in Figure 2 are taken to be small and the relation between the angles is (Bovy, 2023)  = θ - α̂(ξ) DLS/DS (4) where β is the observed angular position of the source, θ is the observed angular position of the image of the source, α̂ is the deflection angle, DLS is the distance between the lens and the source and DS is the distance to the source (Figure 2). It follows from the general theory of relativity that in case that point mass is acting as a gravitational lens, α̂(x) = 4GM / (DL θ c2) , (5) where G is the gravitational constant, M is the mass of the lensing object and c is the speed of light (Einstein, 1916). Eqs.(4) and (5) relate the apparent position of a distant source θ to its observed position by taking into account the gravitational influence of the mass (M) causing the lensing. Proceedings of 10 th Socratic Lectures 2024 117 of 165 Figure 2: Geometry of lensing system. From (Bovy, 2023). There are two types of gravitational lensing. Strong lensing: This occurs when the gravitational field is strong enough to produce clearly visible distortions or multiple images of the background object. The lens equation is crucial for understanding and predicting the positions of these multiple images. Weak lensing: In this case, the gravitational distortion is subtle and doesn't produce multiple images but instead causes a slight stretching or shearing of the background object. 3.3 Cosmic microwave background Cosmic Microwave Background (CMB) is a faint glow of radiation that dates from the very beginning of everything. It's what's left of the early stages of the universe, from the era known as »recombination era«, which occurred 380 000 years after Big Bang (Bertone and Hooper, 2018). At that point the universe cooled down enough for electrons and protons to combine and form different atoms. The first atoms were hydrogen atoms, which made universe transparent for radiation. The light particles (called photons) that were present at that time have been traveling freely through space ever since and we observe them today as cosmic microwave background (Peebles, 1993). CMB supports the Big Bang theory and has provided information about the scale, composition and evolution of the universe, in particular early universe's structure and dynamics. The radiation spectrum of CMB is given by the Planck's law which law describes the spectral density of electromagnetic radiation emitted by a black body in thermal equilibrium at a given temperature T, when there is no net flow of matter or energy between the body and its environment, I()= (2h3)/(c2(eh/kT-1)) , (6) where I() is the intensity of radiation,  is the frequency of the electromagnetic radiation, h is the Planck constant, c is speed of light, k is the Boltzmann constant (Peebles, 1993). The temperature T of CMB is approximately 2.73 K. To describe CMB, we also need to consider Wien's displacement law that determines at what wavelength  the intensity of radiation emitted from a blackbody reaches its maximum,  = b/T (7) where b is the Wien’s displacement constant. 3.4 Other evidences Large scale formation refers to the distribution of matter in the universe on a scale much larger than galaxies such as galaxy clusters and superclusters. By studying these Proceedings of 10 th Socratic Lectures 2024 118 of 165 structures scientists can test the prediction of different cosmological scenarios. The consistency between theoretical models or advanced computer simulations incorporating dark energy and the observed large scale structure may provide evidence for the existence of the dark energy (Blumenthal et al., 1984). Baryon Acoustic Oscillations (BAO) are unique features imprinted on the distribution of matter in the early universe (Bertone and Hooper, 2018). They originate from acoustical waves in the hot, dense plasma of the early universe, before the formation of atoms. These waves created regions of over-dense and under-dense matter and can be observed in the universe today. 4 Conclusions The possible realms of dark matter and dark energy continue to captivate and challenge our understanding of the universe. There are evidences and theories that provide explanation of the observations that indicate existence of dark matter and dark energy. This is reflected in accelerated expansion of the universe, gravitational lensing and cosmic microwave background. The pursuit of knowledge regarding dark matter and dark energy, not only expands our scientific comprehension but also pushes us to re-evaluate or re-assess the fundaments of our description by space and time. Technological advances will likely play an important role; we expect a step forward in the field of exploring the universe as in January 2025 Large Synoptic Survey Telescope (LSST) in Chile is expected to start operating (Primack, 2024). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Bertone G, Hooper D, Silk J. Particle Dark Matter: Evidence, Candidates, and Constraints. Physics Reports. 2005; 405:279-390. DOI: 10.1016/j.physrep.2004.08.031.3 Bertone G, Hooper D. Review of modern physics, volume 90: History of dark matter. 2018; 90:2-6. DOI: 10.1103/RevModPhys.90.045002. Blumenthal GR, Faber SM, Primack JR, Rees MJ. Formation of galaxies and large scale structure with cold dark matter. Nature. 1984; 299:37. DOI: 10.1038/311517a0.5 Bovy J. Dynamics and Astrophysics of Galaxies. Toronto University; 2023. Chapter 16: Gravitational lensing; Ebook Available from: Gravitational lensing (Dynamics and Astrophysics of Galaxies) (galaxiesbook.org) Einstein A. Die Grundlage der allgemeinen Relativitätstheorie. Annalen der Physik. 1916; 354: 769 –822. Morisson I, Zwicky F. The Father of Dark Matter. Gresham College. 2015. Available from: https://www.youtube.com/watch?v=TV0c1EFIKy4. Accessed January 16, 2024. Montgomery C, Orchiston W, Whittingham I. Michell, Laplace and the origin of the black hole concept. Journal of Astronomical History and Heritage. 2009; 12: 90 - 96. Available form: https://adsabs.harvard.edu/full/2009JAHH...12...90M NASA. Dark energy, Dark Matter. Available from: https://science.nasa.gov/astrophysics/focus-areas/what-isdark-energy/. Accessed: January 16, 2024. Nerich S. Could dark matter not matter? Available from: https://phys.org/news/2011 -12-dark.html. Accessed March 8, 2024. NOVA PBS OFFICIAL. How scientists discovered dark matter. 2018. Available from: https://www.youtube.com/watch?v=lNmRHccI4Z8. Accessed January 16, 2024. Peebles PJE. Principles of Physical Cosmology. Princeton University; 1993. Chapter: The thermal cosmic background radiation; p. 131-135. Primack J. A brief history of dark matter. University of California, Santa Cruz. Available from: https://kiss.caltech.edu/workshops/dark_matter/presentations/primack.pdf. Accessed January 16, 2024. Vera Rubin observatory, Monthly updates. Available from: https://www.lsst.org/about/project-status. Accessed March 8, 2024. Proceedings of 10th Socratic Lectures 2024 120 of 165 Artificial Intelligence in Law Enforcement: Current State and Development Prospects Lunhol Olha1,*, Torhalo Pavlo1 1. 2. Donetsk State University of Internal Affairs * Correspondence: Lunhol Olha; olyalungol@gmail.com Citation: Lunhol O, Torhalo P. Artificial Intelligence in Law Enforcement: current state and development prospects . Proceedings of Socratic Lectures. 2024, 10, 120-124. https://doi.org/10.55295/PSL.2024.II12 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the Abstract: This article provides an analysis of the current state and future prospects of Artificial Intelligence (AI) implementation in law enforcement. As advancements in technology continue to reshape various sectors, the integration of AI in policing has become a focal point, revolutionizing traditional methods and offering new opportunities. The article begins by outlining the contemporary landscape of AI applications in law enforcement, encompassing predictive policing, facial recognition, data analysis, and crime pattern identification. The discussion delves into the benefits and challenges associated with these technologies, addressing concerns related to privacy, bias, and ethical considerations. Furthermore, the article explores the evolution of AI in law enforcement, examining how machine learning algorithms enhance predictive capabilities, streamline investigative processes, and contribute to proactive crime prevention. It also highlights successful case studies and realworld implementations, showcasing the positive impact AI has had on solving complex criminal cases and optimizing resource allocation. In exploring development prospects, the article considers emerging trends such as explainable AI, human-AI collaboration, and continuous advancements in data analytics. The importance of responsible AI deployment is emphasized, emphasizing the need for transparent and ethical frameworks to guide law enforcement agencies. The article concludes by envisioning a future where AI technologies are seamlessly integrated into law enforcement practices, fostering improved crime detection, community safety, and overall operational efficiency. The insights presented aim to contribute to informed discussions surrounding the responsible and effective use of AI in the evolving landscape of law enforcement. terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/ by/4.0/). Keywords: Artificial intelligence; Data analysis; Digital automation; Law enforcement; Facial recognition systems Proceedings of 10th Socratic Lectures 2024 121 of 165 1. Introduction 1.1. Background The rapid evolution of technology has permeated every facet of contemporary society, revolutionizing traditional approaches and introducing innovative solutions. One such transformative force is Artificial Intelligence (AI), which has progressively found its way into the realm of law enforcement. As law enforcement agencies worldwide seek to enhance their capabilities, AI emerges as a potent tool, offering unprecedented opportunities to tackle evolving challenges in crime prevention, investigation, and public safety. 1.2. Scope of the article This article explores the current state and development prospects of AI in law enforcement, aiming to provide a comprehensive understanding of the technological landscape and its implications. From predictive policing to facial recognition and data analytics, we delve into the diverse applications of AI that have reshaped the modus operandi of law enforcement agencies. 1.3. Current state of AI implementation To comprehend the present scenario, it is essential to dissect the various AI applications currently integrated into law enforcement practices. This article will scrutinize predictive policing algorithms, the use of facial recognition technologies, data-driven crime analysis, and the incorporation of AI in identifying complex crime patterns. Additionally, we`ll analyze benefits accrued and challenges encountered in the adoption of these technologies. 1.4. Challenges and ethical considerations: While AI offers immense potential, it is not without its challenges. Privacy concerns, algorithmic bias, and ethical considerations have emerged as critical issues that demand careful examination. This article will delve into the multifaceted challenges associated with AI implementation in law enforcement and discuss the ethical frameworks required to navigate this rapidly evolving landscape. 1.5. Evolution: The evolution of AI in law enforcement has been marked by technological advancements, particularly in machine learning. This segment will explore how these advancements enhance predictive capabilities, streamline investigative processes, and contribute to proactive crime prevention.Through this exploration, we aim to contribute valuable insights that foster a nuanced understanding of AI's current role in law enforcement and illuminate the path forward in harnessing its potential for future development. 2. Methods The article employs a multifaceted approach to achieve its objectives. The research utilizes both theoretical and empirical methods, combining a comprehensive analysis of specialist literature with practical investigations. The theoretical framework involves an in-depth examination of the existing literature pertaining to the problem of integrating artificial intelligence into law enforcement activities. This includes a detailed analysis of scholarly works addressing various aspects of AI implementation in the field. On the empirical front, the research incorporates interviews conducted with students, cadets, and teachers from Donetsk State University of Internal Affairs and specialists of law enforcement agencies of Ukraine. This qualitative approach aims to gather valuable insights and perspectives regarding the practical implications and challenges of incorporating artificial intelligence into law enforcement practices. This combination of theoretical and empirical methods ensures a comprehensive exploration of the current state and future prospects of artificial intelligence in law enforcement. Proceedings of 10th Socratic Lectures 2024 122 of 165 3. Results The current state and future prospects of Artificial Intelligence (AI) implementation in law enforcement represent a dynamic landscape with significant implications for the future of policing. Current state is reflected in the works of such Ukrainian scientists as Zachek O., Dmytryk Yu., Senyk V., Kovtun V., Rvachov O., Haborets O., Karchevsky M. and other researchers. Zachek O., Dmytryk Yu. and Senyk V. (Zachek O, et al. 2023) note that one of the problems that need to be solved is the lack or imperfection of legal regulation of Artificial Intelligence both in Ukraine and abroad. Kovtun and Rvachov (2020) write that in law enforcement, the use of artificial intelligence is the process of endowing law enforcement activities with systematic intellectual properties. This process should serve as a factor for intensive development and a tool for enhancing the efficiency of law enforcement through the development and implementation of new technologies in the following directions: information-analytical support for law enforcement activities; information-reference support for law enforcement activities; creation of specialized information intelligence systems for operational-search purposes; advancement of intelligent video surveillance systems; establishment of departmental specialized intelligent information systems; implementation and development of intelligent information educational systems. Most scientists note that use of AI in identifying crime patterns contributes to more effective and efficient crime-solving, leading to improved public safety. There are many examples of successful implementation of artificial intelligence in domestic and international law enforcement practice. So, for example, in the USA, the Federal Bureau of Investigation (FBI) uses artificial intelligence to recognize criminals on video from surveillance cameras in real time. With the help of artificial intelligence, FBI agents can access and analyze large amounts of data to identify criminals and predict the place and time of the next crime (Zachek et al. 2023). In Great Britain, the police use artificial intelligence to analyze social networks and other open sources of information to identify possible threats to national security. In Germany, the police use artificial intelligence to detect crimes related to financial transactions. In various countries AI-powered facial recognition systems analyze facial features to identify and verify individuals, providing law enforcement with a powerful tool for surveillance and identification. Facial recognition is used in public spaces and airports for law enforcement investigations, assisting in suspect identification and tracking. As an example, the Miami Police Department uses Clearview AI software to investigate crimes. This technology allows law enforcement agencies to check a person's photo for a match in the database, then providing links to relevant images on the Internet. But at the same time, there are controversies surround the ethical implications of mass surveillance, potential misuse of facial recognition, and the need for stringent privacy regulations. In March 2022, the company Clearview AI provided its technology to Ukraine to assist in defense against the Russian invasion. Initially, this technology was given to the Ministry of Defense of Ukraine, and later, many other agencies, including the National Police of Ukraine, joined the project (Zachek et al. 2023). Clearview AI's technology is applied for checking individuals at checkpoints, identifying deceased soldiers and prisoners of war, and searching for missing persons. Additionally, facial recognition technology can be used to investigate military crimes by identifying the faces of criminals in photos and videos that the criminals themselves proudly share on the Internet. AI algorithms process vast amounts of data, extracting valuable insights to aid law enforcement in decision-making. Data analysis helps law enforcement agencies identify trends, anticipate criminal activities, and streamline investigative processes. But striking a balance between utilizing data for crime prevention and protecting individual privacy remains a critical challenge. Current state and development prospects of Artificial Intelligence in law enforcement are considered also by Foreign scientists, such as Rademacher T, Wischmeyer T, Raaijmakers S, Apostolakis K, Dimitriou N, Margetis G, Surden H, Safdar N, Banja J, Meltzer C and other researchers. Proceedings of 10th Socratic Lectures 2024 123 of 165 Scientists Rademacher and Wischmeyer (2020) claim in their works that in certain domains, AI already fulfills the task of detecting suspicious activities better than human police officers ever could. Safdar et al. (2020) in their research pay special attention to ethical considerations in artificial intelligence. In conclusion, the contemporary landscape of AI applications in law enforcement showcases the transformative potential of predictive policing, facial recognition, data analysis, and crime pattern identification. While these technologies offer unprecedented benefits, addressing ethical concerns, ensuring privacy, and fostering public trust are imperative for responsible implementation in the evolving realm of law enforcement. 4. Discussion Let`s analyze the key aspects, including existing applications, benefits, challenges, and the anticipated trajectory of AI integration in the law enforcement domain. 1. 2. 3. 4. 5. 6. 7. Predictive policing algorithms: AI is increasingly employed for surveillance through facial recognition, object detection, and behavioral analysis. Predictive policing algorithms leverage historical crime data to identify potential hotspots, aiding law enforcement in proactive measures. Automation of routine tasks: AI technologies automate routine tasks such as data analysis, paperwork, and evidence processing. This not only enhances efficiency but also allows human officers to focus on more complex aspects of policing. Crime Pattern Recognition: AI algorithms can identify intricate patterns within large datasets, aiding law enforcement in recognizing trends, modus operandi, and potential threats. This contributes to more effective crime prevention and investigation. Virtual assistants and chatbots: police departments are integrating AI-powered virtual assistants and chatbots to enhance communication with the public, provide information, and streamline administrative tasks. Enhanced efficiency: AI streamlines processes, reducing the time and resources required for tasks such as data analysis and paperwork, allowing law enforcement agencies to operate more efficiently. Improved decision-making: AI systems, when trained on relevant data, can assist law enforcement in making data-driven decisions, contributing to more informed and objective policing. Public safety: predictive policing and surveillance technologies contribute to enhanced public safety by enabling proactive responses to potential threats and criminal activities. As for the issues open for discussion, we can include: 1. Bias and discrimination: AI systems may inherit biases present in training data, leading to discriminatory outcomes. Ensuring fairness and mitigating bias in AI algorithms poses a significant challenge. 2. Privacy concerns: the extensive use of AI in surveillance raises concerns about privacy infringement. Striking a balance between public safety and individual privacy is a com plex issue. 3. Ethical considerations: the ethical implications of AI in law enforcement, including accountability, transparency, and the potential misuse of technology, require careful consideration. As AI continues to evolve, there will be a growing emphasis on developing and adhering to ethical frameworks to address biases, privacy concerns, and other ethical considerations. Conflicts of Interest: The authors declare no conflict of interest. Proceedings of 10th Socratic Lectures 2024 124 of 165 References 1. 2. 3. 4. 5. 6. Karchevsky M. Artificial intelligence and crime prevention. The use of artificial intelligence technologies in combating crime: scientific and practical materials. 2020; 32–41. Kovtun V, Rvachov O. Overview and prospects of the use of Artificial Intelligence technologies in law enforcement activities. The use of Artificial Intelligence technologies in combating crime: scientific and practical materials. 2020; 44–51. Lunhol O, Haborets O. Innovative methods and digital technologies in operational and research activities. Current issues in modern science, 2023, 11: 602-615 Rademacher T, Wischmeyer T. Artificial Intelligence and Law Enforcement. Published in: Regulating Artificial Intelligence. Springer, Cham. 2020. Safdar N, Banja J, Meltzer C. Ethical considerations in artificial intelligence. European Journal of Radiology. 2020, 122. Zachek O, Dmytryk Yu, Senyk V. The role of Artificial Intelligence in increasing efficiency law enforcement activities. Scientific Journal of Lviv State University of Internal Affairs. Law. 2013; 3; 148–156. Proceedings of 10th Socratic Lectures 2024 126 of 165 Corporate Social Responsibility as a Key Factor for Development of Sustainable Business Tokushev Damir1,* 1. Kazakh-British Technical University, Kazakhstan * Correspondence: Damir Tokushev; d.tokusev@gmail.com Citation: Tokushev D. Corporate Social Responsibility as a Key Factor for Development of Sustainable Busines: 2024, 10, 126-129. https://doi.org/10.55295/PSL.2024.II13 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the Abstract: Responsibility is an organizational position that ensures discipline and the most effective attitude of the Company to its responsibilities to the public. Corporate social responsibility contributes to the creation of a favorable environment for business development, interaction between business and the state, where the interests of the population are represented by public organizations, which in turn turns into social partnership. Social partnership depends on the balance of three important systems, such as: social, economic and environmental. In one of the companies in Kazakhstan that takes an active part in the development of corporate social policy a program is being used within the framework of social responsibility that the company is actively pursuing. Within the framework of this program, the construction of socially significant facilities is being implemented, such as: construction of a central hospital in the city of Kulsary, construction of a central stadium in the city of Kulsary, construction of a pre-school in the Koktem microdistrict in the city of Atyrau, construction of a school named after Zhumabayev in the city of Atyrau. The company carries out partnership with higher educational institutions of the Republic of Kazakhstan, one of such educational institutions is Kazakh-British Technical University (KBTU) University. The company is actively developing a corporate social policy in the field of environmental protection, supporting social programs and improving the lives of the local population. Considering the above, we can conclude that corporate social responsibility is an integral part of the development of the company’s sustainable business in Kazakhstan. terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/ by/4.0/). Keywords: Corporate culture; Corporate social policy; Responsibility; Ecology; Social program; Environment. Proceedings of 10th Socratic Lectures 2024 127 of 165 1. Introduction 1.1. Purpose of the scientific article The purpose of this scientific article is to systematically and in-depth analyze the role of Corporate Social Responsibility (CSR) as a major factor influencing the development of sustainable business (Green Alternative Energy Assets, n.d.). Speaking about the experience of applying CSR in the company’s activities in order to sustainable development, we note that in Kazakhstan there are a number of large socially responsible companies. The benefits of CSR are practically demonstrated by such companies. The specific focus of the article is aimed at studying a particular company (Company A) in the field of CSR and identifying specific practices that contributed to the creation of a sustainable socially corporate model. 1.2. Goals of the research The key tasks to achieve the goal were: • Study of CSR practices in Company A: Assessment of current programs and strategies related to social responsibility implemented in the company's activities; • Community and Environmental Engagement Analysis: Assessing the impact of CSR on relationships with local communities, including the impact on social structures, support for education and charitable initiatives. Also considering the impact on environmental sustainability. Total Cost of Ownership (TCO) rigorously complies with legislative requirements of Republic of Kazakhstan (RoK) and performs extensive air protection activities through stateof-art technologies resulting in the continuous reduction of air emissions while oil production volumes have increased significantly over the years. It should be emphasized that the company's emissions are currently at an extraordinarily low level as a result of the execution of several significant projects aimed at improving operational reliability. These have allowed the company to lower its air emissions by 75% per ton of oil produced during the past 22 years (Figure 1). Figure 1. Air emissions of Company A with respect to time from 2000 to 2022. 2. Methods To write this article, a quantitative research method was used, based on the use of available statistical data related to the social and environmental activities of the Company from the official website. The basis was a survey method among former colleagues who are directly residents of the Atyrau region and had the opportunity to observe the changes that were described in this article. 3. Results Having analyzed open sources that disclose Company A’s social programs aimed at stimulating the potential of the region as a whole and its individual resident, the following Proceedings of 10th Socratic Lectures 2024 128 of 165 results were discovered in the field of sustainable support for the corporate social responsibility program and opinions of my colleagues who was able to provide oral feedback the following conclusions were drawn. Company A contributes to environmental protection, environmental monitoring, social investment and the development of social and infrastructure development programs. So, according to the environmental report for 2022, the main environmental indicators for 2022 are: • Reduction of emissions intensity from 2000 to 2022 by 75%; • Reduction of gas flaring over the past 5 years by 57%; • Increase of water reuse rates by 49%; • Increase of waste recycling rates by 63%. As a part of the implementation of the Roadmap for a comprehensive solution to environmental problems in the Atyrau region, TCO is carrying out landscaping work in the city of Kulsary. In 2022, spring-autumn planting of elm, ash, apple, maple, and island seedlings was carried out on an area of 15 hectares in the city of Kulsary for a total of 9,030 trees on the street. R. Balmukhanova, st. Akzhar and along the Atyrau-Kulsary road, in the microdistrict Atyrau. The survival rate averaged 69.7%. In the places where 32.3% of seedlings did not take root, an additional 3,030 seedlings were planted in October and November 2022. The company continues to plant trees in Kulsary and cares for seedlings. In 2022, under the program, Company A began to implement the construction of facilities such as: Central Hospital in the city of Kulsary, Central Stadium in the city of Kulsary, Preschool in the Koktem microdistrict, Atyrau, Pre-school in the microdistrict. Zhuldyz, Atyrau, Construction of a school named after Zhumabayev in Atyrau. The program gave a powerful impetus to the infrastructural development of the Atyrau region. New, modern buildings have appeared that have beautified the regional center and Zhylyoi district, multifunctional medical clinics and centers, well-equipped schools and other educational institutions. Decisions on which projects are to be included in the program are made jointly with the Akimat of the Atyrau region. The main criteria: sustainability, focus on the priority needs of the local population, benefits for the majority of the population, and most importantly, projects should create opportunities to unlock the potential of residents of the Atyrau region (ibid.). The Social Investment Program, which was launched in 2010, is also being implemented. It is aimed at supporting education, healthcare, community involvement in the volunteer movement and economic development. Within the framework of this program in 20102022, 108 projects were implemented for a total amount of about 13.4 million US dollars. In 2022, the budget for this program was 2.3 million US dollars. 4. Discussion Supporting social corporate responsibility has given the Company A a number of advantages such as: • Enhanced Reputation: Adopting social responsibility can significantly enhance a company's reputation. This creates a positive perception in the eyes of the public and government; • Support for public policy: creating tax incentives for companies that build strong social responsibility; • Improved relationships with regulators: Companies that demonstrate transparency, ethics, and social responsibility often interact better with regulators, which can reduce the likelihood of regulatory issues arising. Thus, corporate social responsibility not only contributes to the well-being of society, but also benefits companies in their relations with the government. 5. Conclusion CSR has grown from a simple term to a fundamental principle that plays a key role in the development of sustainable business. In the course of analyzing the role of CSR using the example of the Company A, it becomes clear that the integration of the principles of social responsibility into the strategy of an enterprise has an important impact on its sustaina- Proceedings of 10th Socratic Lectures 2024 129 of 165 bility and prosperity. First of all, CSR is an important tool for establishing long-term relationships with stakeholders. Engaging with employees, customers, suppliers and the community through ethics, transparency and respect for human rights strengthens the company's reputation and creates the basis for lasting partnerships. CSR also has a significant impact on financial stability. Companies that integrate social and environmental aspects into their strategy demonstrate not only social responsibility, but also a higher degree of efficiency and innovation, which ultimately impacts their financial performance. In addition, socially responsible practices have a positive impact on the internal dynamics of the company. Participation in social and educational projects, creating favorable working conditions and supporting diversity in the team help attract and retain talented employees. Thus, CSR is not just a fashionable trend, but is becoming an integral part of the sustainable development strategy of modern companies. Company A, as an example of successful implementation of the principles of social responsibility, confirms that investing in social welfare and respect for the environment are key factors not only for sustainable business, but also for leadership in the industry. In the future, business management based on ethics and responsibility represents a promising prospect for achieving harmony between corporate interests and the needs of society. Conflicts of Interest: The author declares no conflict of interest. References 1. 2. 3. CSR, World Business Council for Sustainable Development (WBCSD) definition, Green Alternative Energy Assets (GAEA). Accessed 30.01.2024. Available from https://www.gaea.bg/about-GAEA/corporate-social-responsibility.html (internet source) Tengizchevroil official page. Social investment. Sustainability. Accessed 30.01.2024. Available from https://www.tengizchevroil.com/sustainability (internet source) Tengizchevroil overview of 2022 environmental activities and performances. Air emission reductions. Accessed 30.01.2024. Available from https://www.tengizchevroil.com/docs/default-source/default-documentlibrary/sustainability-page/en/ (internet source) Proceedings of 10th Socratic Lectures 2024 131 of 165 Challenges of Modernising European Universities Cizelj Boris1,* 1. * DOBA Faculty of Applied Business and Social Studies, Maribor, Slovenia Correspondence: Boris Cizelj, boris.cizelj@sis-egiz.eu Citation: Cizelj B. Challenges of Modernising European Universities. 2024, 10, 131-136. https://doi.org/10.55295/PSL.2024.II14 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Abstract: One of the paradoxes of the 21st century is that while most countries are striving to build knowledge societies and economies, the majority of universities remain conservative in modernizing their curricula. This lack of modernization includes issues such as balancing knowledge, competencies, and skills, implementing student-centered and interactive teaching methods, and involving practical experts in the study process. Awareness of this paradox is still limited not only within academia but also among governments, professional sectors, and the general public. The media, too, could play a more active role in promoting necessary changes. One key reason for the slow modernization of universities is the conservative interpretation of academic freedom and institutional autonomy. This stems from several factors, including the misinterpretation of academic freedom, the career patterns of university academic staff, the prevalent use of traditional lecture-based teaching methods, inadequate research funding, and limited inter-university collaboration. As a result of these conditions, countries struggle to fully develop their human capital, hindering their transition into innovation-based knowledge economies and impeding their international competitiveness. To address these challenges, all societal actors must play a role. Governments should stimulate the development of modern tertiary education systems, business leaders should clearly articulate the competencies and skills they expect from graduates, academia should advocate for sufficient funding for their modernization efforts, and NGOs and the media should raise awareness and urge governments to actively support the development of knowledge economies. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/ by/4.0/). Keywords: Modern university; Knowledge society and economy; Importance of competencies and skills; Innovation ecosystem; Inter-university collaboration _____________________________________________________________________________ Proceedings of 10th Socratic Lectures 2024 132 of 165 1. Introduction 1.1. Tertiary education´s division between the classical and higher professional education The post-industrial society based on the quality of human capital is much richer in knowledge, as well as in competencies and skills, than earlier societies. That puts much higher and qualitatively different demand on the entire educational system – including on universities. This has been properly understood by only a few countries, who have reflected pragmatically, therefore divided tertiary education into classical and higher professional education. The first remained general, knowledge-based academic upbringing of future intellectuals - expected to perform various jobs in spheres of management, administration, education, and other social services. The second, however, was designed to provide the background and skills for students who were expected to perform engineering as well as technical jobs. These programmes are usually carried out by “Technical Universities” or Colleges – normally taking 3 years (while university programmes normally run for 4 years). This is a practical answer to the issues opened, with classical universities still facing many of the issues introduced by the knowledge society. The questions being faced by universities in most countries around the world are not easy to answer, taking into account the changes having happened in modern societies, where knowledge is not any more the privilege of the few, and when diversification of knowledge has grown beyond anything, we have been used to, even only 2-3 generations ago. When looking for the main causes of this challenge, we need to go some two centuries back. Namely, that was the time, when the principal role of modern universities in Europe has been to prepare the future elite of society. Therefore, it is not surprising that in that period, less than estimated 5% of youngsters entered university, and they mostly came from the upper classes. Therefore, they were actually predetermined to become part of the elite in their respective societies. For those circumstances the elite university made perfect sense, anything else would not have been accepted. 1.2. The concept of modern university: The concept of the modern university dates back to the year 1809 when the Prussian government appointed one of its leading intellectuals, Friedrich Wilhelm Heinrich Alexander von Humboldt, to be responsible for education. He performed excellently, and his system was adopted by most European countries due to its elaborateness, systematic approach, and effectiveness in achieving objectives. This was the case two centuries ago when the majority of working people were illiterate. Now, the situation is, of course, completely different: all working people are not only able to read and write, but have also completed at least 9 years of education. About 1/3 of youngsters even graduate from university, implying that they have been educated for 17-18 years, yet they are not automatically considered the elite of their respective societies. The following quote from von Humboldt's text indicates that he himself was much more modern than generally interpreted: “Just as primary instruction makes the teacher possible, so he renders himself dispensable through schooling at the secondary level. The university teacher is thus no longer a teacher, and the student is no longer a pupil. Instead, the student conducts research on his own behalf and the professor supervises his research and supports him in it1” (Clark, 2006). This clearly indicates that von Humboldt is often misunderstood, and that he was more modern than commonly believed. In fact, this perspective taken by von Humboldt is quite different, indeed even contrary, to what is often considered the “Humboldt-type” university, which still serves as the mental background for the concept of most present-day European universities. However, this is not the case in the USA, which von Humboldt visited several times and influenced their academia. While von Humboldt operated in the period of industrial society, we are now experiencing the achievements as well as the challenges of the digital age in the mature innovative, knowledge society. Many work operations that traditionally depended on responsible 1 Clark CM, 2006. Iron kingdom: the rise and downfall of Prussia, 1600-1947, Harvard University Press, p. 333. Proceedings of 10th Socratic Lectures 2024 133 of 165 workers' reasoning and decisions are now digitized and increasingly controlled by artificial intelligence. At the same time, people's roles have shifted to primarily guiding and controlling computer-guided processes and decisions. Unfortunately, this shift is not properly reflected in the concept of present-day European and many other universities, which have not recognized the changes experienced over the last 50-60 years and still adhere to a traditional approach in fulfilling their mission of building intellectuals. In essence, they still see their mission as enriching students with scientific knowledge to make them intellectuals and a kind of societal elite. Most academics, and consequently, also governments, therefore accept the position that teaching practical competences and skills does not fit into the world of academia and should not be part of postsecondary education curricula 2. Methods In preparation for the article, observation research has been undertaken. A reduced interest in studying the Bologna experience has been observed since 2015. What could be the reasons for this unexpected trend? Probably the main reason could be that very little is happening in terms of the Bologna Process impacting university education. Namely, out of the 49 subscribing countries, even one third of them did not introduce the two-cycle university study concept, while there is very limited information about the implementation of the other Bologna targets. Therefore, our approach remain mainly interpretative: it is based on published documentation, combined with some observations of the private and public universities in Slovenia. Observations are accumulated over decades from active involvement at various Slovenian universities. 3. Results Our principal observation on the achievements of the Bologna Process is that – after 20 years since the adoption of the Declaration – the results achieved are very modest, to say the least. This qualification is justified by the fact that modern circumstances more than justify the need to update and adjust university studies, including equipping students much more with competencies and skills required in today's working environment. Obviously, one cannot blame only the universities – though their responsibility cannot be underestimated or even ignored. The more they emphasize their academic autonomy, the higher is their responsibility for failing to accept the need to change! The fundamental responsibility, however, remains with governments, which in most countries continue paying inadequate attention to education, including university education. This fits into a more general weakness preventing many governments from accepting primary responsibility for formulating policies for building a knowledge economy and society as a building block of national development strategy. Leaving the strategies and declared policies aside, there is one indication of how much they are willing to undertake to support building a knowledge economy. This is the share of GDP devoted to financing research and innovation from the budget (GERD, Gross domestic expenditure on R&D). Governments of countries which do care, maintain GERD at 3% or more, and those which do not, achieve GERD below 2%. Of course, it's not only about how much funding is available; what matters as well is how the financial support is offered and provided. If public funding is available as a source of supporting institutions' budgets – this is simpler, but not most effective. It should be available for seriously selected and promising projects, which is procedurally more demanding, but definitely much more effective and productive. Equally important for innovation performance of a country is the development of its financial market, offering support not only to interesting research projects, but also for opening new companies (start-ups) and supporting the growth of small and young companies (scale-ups). As a share of available risk capital in GDP, the US is five times stronger than the EU average. Without important changes in the entrepreneurial environment, Europe is not facing a promising future in the global innovation-based competition! This is related also to the general awareness of the critical importance of the quality of education, science, and research – which is in most countries around the globe far from sufficient. Proceedings of 10th Socratic Lectures 2024 134 of 165 4. Discussion What are the major causes for the dissatisfactory situation in implementing Bologna? Let us first look at the universities themselves, and identify the key factors responsible for the problems preventing progress along Bologna model: (1) A major contributing factor is the traditional career pattern of most European university professors, who predominantly remain at the university from entering as a student, distinguishing themselves, being invited to join the staff, and normally experiencing their entire professional career at the university, up to their retirement. Therefore, they remain encapsulated in this particular environment, depriving themselves of the opportunity to experience "real-life conditions." Being gradually recognized as a problem, in some countries they are now starting to take measures to prevent this "deformation." For example, in Germany, this is now prevented by law, as well as by university rules for professors' re-elections, according to which the candidate for re-election has to provide evidence of at least some out-of-university work experience. This approach is very slowly gaining acceptance around the world, and therefore reaching beyond the oldfashioned academic tradition will obviously take some time2. (2) Indeed, a major change from "teaching theory" to "preparing students for future work" is needed, and that is in itself rather demanding. This is not only an issue at the university level – many experts in pedagogy claim that generally and in most countries teachers are not sufficiently stimulating creativity and critical thinking in their students. This is a problem throughout the whole educational system, contradicting what is needed by people working in the 21st century in any sector. This goes particularly for workers who are surrounded by and actively assisted by high technology, including artificial intelligence. The main function of most workers nowadays is to control and guide the machines and make decisions based on options offered by the HITECH equipment. If not enabled to function in this fashion, even a highly knowledgeable and intellectually capable individual will not necessarily be very productive in a modern work environment3. In these circumstances, besides knowledge, what determines the quality of workers is their attitude towards co-workers, their ability to communicate productively and flexibly, as well as develop critical thinking – with the objective to find solutions accepted by the respective teams. In the last British Employers Association’s survey among 72,000 employers even 23% of newly engaged workers were declared to be lacking relevant skills. 3) When commenting on this result, one needs to take into account that British universities are more pragmatic than their colleagues on the continent. The government has categorized public universities into 3 categories by the quality of their performance and differentiated levels of funding accordingly: only the best could receive more money, while the worst experienced serious cuts, and some even had to merge with others or close their doors. (3) A major factor in developing relevant curricula and maintaining its quality is the ability of universities to conduct good basic and applied research, which implies receiving appropriate level of funding. The average official data for European countries' on GERD (Gross Expenditures on R&D) - where universities participate with about 1/4 to 1/3 – is around 2.2% of GDP – while many member states remain below 1.5%, demonstrate that neither public, nor the private funding come close to what is required. As a logical consequence, for example, Slovenian universities have lost over the last 20 years about a quarter of their researchers due to insufficient funding. Facing this situation – which 2 Employer Skills Survey 2022 – Research Report, December 2023, London, 216 pages 3 Nic Mitchell -- “University World News” Online Conclusions, 2023 Proceedings of 10th Socratic Lectures 2024 135 of 165 undermines also the quality of teaching -- the public universities should raise this question with governments more energetically, as it is a condition for their normal functioning. (4) A bit of the theory-practice curriculum balance can be achieved where universities invite experienced practitioners to contribute some lectures and seminars/workshops. It is an excellent opportunity for students to hear from distinguished people from companies and other practical environments about the problems experienced in real life, understand their origin, dimensions and impact. It is important that this is well integrated into the curriculum, and treated as its valuable component. Experience shows that these “guest lectures” are highly appreciated by students, which speaks for itself. (5) Connected and related to the above issues is also the modest inter-university collaboration – at national and at international level. As Swedish Professor Charles Edquist – an internationally recognized authority on innovation – has proven, the innovation contributes at least 60% to the GDP growth, and that modern innovation is not any more following the traditional linear pattern (from basic to applied research, to development, and finishing at the market). The innovation has become much more complex, involving practically all segments of society – including the government. This is particularly important through direct funding, as well as supporting domestic innovation activities through public procurement (which comes on average to 13-20% of GDP – and representing globally an impressive amount of 9.5 trillion $). The most undisputable indicator about the innovation awareness and commitment of a country is GERD. According to the Global Innovation Index reports 2018 – 2022, in the first category the following 20 countries appear on the top: Switzerland, Sweden, USA. Neither most of the European universities’ mind-frame, nor the relevant regulatory environments are very collaboration-oriented. Therefore, the modest extent, and the impact of national and international university collaboration is not fully in line with the intensity of present-day globalization - affecting also research and innovation activities. Limitations to productive collaboration are reflected also in the slow and modest achievements of the objectives of the Bologna Declaration – as recognized for example in the Rome Communique of 2020. It is even noticed that the interest for doing research on Bologna implementation seems to be decreasing after 2015. One very graphic illustration of the above assessment is also the number of European universities actively involved in the existing alliances – for example in the European University Alliance, with only 130 members from the total of 1,706 European universities. Realizing these problems, the European Commission has created in September 2020 (with some delay – compared to other domains), the European Research Area – as a logical component of the European integration process. On this basis, and with rising awareness of the importance of creating conditions for research and innovation collaboration, the next step was undertaken in November 2021 by the creation of the Pact for R&I. The European Commission seems to be aware that - in times of knowledge economy - lack of integration in R&I domain has serious negative consequences for the European international competitiveness. Therefore, the new ERA monitoring system, including a scoreboard, a dashboard and an online policy platform, has also been created in 2023. All this is rather promissing, but it is still too early to expect tangible efects. With the bulk of high quality research potential being concentrated at universities and their affiliated institutes, the prime resposibility to activate fully this potential remains with the universities, and in particular with their leadership. Fortunately, many innovation actors tend to collaborate more intensely from year to year, but too many of the European universities remain almost unaffected by this important and promising trend. There are, however, also some good examples, and they should be identified at all continents, presenting their achievements and benefits, as well as trying to identify what are the causes behind the respective universities’ collaboration orientation, and the good results achieved. Proceedings of 10th Socratic Lectures 2024 136 of 165 What are the reasons for slow adjustments by most universities? There is general understanding that the following factors have an impact on universities’ collaboration: ▪ ▪ ▪ ▪ ▪ Modestly international composition of teaching & research staff; Funding instruments promoting insufficiently international collaboration; Share of international students; Mechanisms stimulating teachers for developing international collaboration; Impact of graduates’ employers (wanting their future workers to have an internationally open mindset, and appropriate skills). Conclusions In summary, European universities are less internationalized than those in other parts of the globe – particularly in North America and in many Asian countries. In present-day circumstances exceptional achievements in training and research can hardly be achieved without intensive links among national and international colleagues (sharing and evaluating experience and challenges), and understanding wider, global trends and problems. Conflicts of Interest: The author declares no conflict of interest. References “Quality Assurance Fit for the Future”, European University Association led EU project (2023-2024); https://eua.eu/resources/projects/840-qa-fit.html: 2. “The European Higher Education Area in 2015: Implementation Report Bologna Process”, Bologna Process, European Commission, European Education Area, Brussels, 2016, 304 pages. 3. Clark CM. Iron kingdom: the rise and downfall of Prussia, 1600-1947. Harvard University Press. 2006; p. 333 4. Employer Skills Survey 2022 – Research Report, December 2023, London, 216 pages, https://assets.publishing.service.gov.uk/media/65855506fc07f3000d8d46bd/Employer_skills_survey_2022_researc h_report.pdf; Authors:IFF Research 5. Hudson RL. “AI talent is moving fast around the world, OECD database shows« Science/Business online Bulletin, 27 February 2020. 6. Ingvild M. Larsen, Peter Maassen and Bjørn Stensaker »Four Basic Dilemmas in University Governance Reform«, OECD, Paris, 2009, 18 pages 7. Kroher M, Leuze K, Thomsen SL, Trunzer J. 2021. Did the" Bologna Process" Achieve Its Goals? 20 Years of Empirical Evidence on Student Enrolment, Study Success and Labour Market Outcomes (). IZA Discussion Paper No. 14757. 2021. https://doi.org/10.2139/ssrn.4114283 8. Le-Ha Phan »Transnational Education Crossing 'Asia' and 'the West - Adjusted desire, transformative mediocrity and neo-colonial disguise, Imprint Routledge, London, 180 pages. https://doi.org/10.4324/9781315759098; 9. Mitchell N. (2023). Transformative Leadership Section at online Bulletin. Accessed 19 November 2023. “University World News – The Global Window on Higher Education”. Available from http://www.universityworldnews.com/ Bulletin – Section Transformative Ledership« 10. Simon Marginson »Higher education faces a more fragile and contensted future« University World News, Online Magazine, No.773, 7 February 2024; https://www.universityworldnews.com/post.php?story=20240215145839243&utm_source=newsletter&utm_ medium=email&utm_campaign=GLNL0773; 11. Vuokko Kohtamaki, Tambere University, and Elisabeth Balbachemsky, University of Sao Paulo, chapter Theoretical and Methodological Perspectives on Higher education Management and Transformation (pp.180-193) in the book »University autonomy from past to present, University of Tampere Press, 2018. 1. Proceedings of 10th Socratic Lectures 2024 138 of 165 STEAM Approach: SMS (Stories Based on Music about Scientists) on Artificial Intelligence Created by Jacob Bruce (1669–1735) Istileulova Yelena1 Global South Arts & Health Envoy, STEAM Ambassador, DAAD expert “Bologna Hub Peer Support”, Ljubljana, Slovenia, * Correspondence: Yelena Istileulova; yelena.istileulova@gmail.com 1. Citation: Istileulova Y. STEAM Approach: SMS (Stories Based on Music about Scientists) on Artificial Inteligence Created by Jacob Bruce (1669– 1735). Proceedings of Socratic Lectures. 2024, 10, 138-146. https://doi.org/10.55295/PSL.2024.II15 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/ by/4.0/). Abstract: The proposed theme of this paper is how "Arts" and "Science," with the inclusion of the topic of "Health", can be combined through a story based on research about innovation in the Past. The goal of this paper is threefold. First, to present a research-based story about Jacob Bruce (1669–1735), a scholar of Scottish descent born in Moscow, and his innovation – the prototype of Artificial Intelligence, humanoid robot. Second, to illustrate the new experimental interdisciplinary method of storytelling - SMS (Stories based on Music about Scientists), a novel approach that integrates Arts (Music, Poem, Digital images), Science (Historical Research about Innovations of the Past), and Culture (cultural heritage with metaphor of a philosophical soul development). Third, to explain how this method can be applied. SMS method created in 2012 uses the Avant-garde genre of Music with the integration of interdisciplinary knowledge, holistic approach, and implies a deeper level of integration of various disciplines. The theme of this SMS is a song-story about the beautiful humanoid girl-robot (but with a different technology of the 17th century), which serves as a prototype of the modern homunculus or AI created by Jacob Bruce. AI humanoid already resembled a natural human being by the end of the 17th century. The author explains and discusses how and why SMS can be used as an Innovative Teaching and Learning method, related to Science and Arts (similar to STEAM approach), with the power of Lifelong Learning (LLL), and pedagogical didactics across branches of sciences, thus stimulating innovations and discoveries. Keywords: Arts and Science; Innovations; STEAM/SMS (Stories based on Music about Scientists) method; Jacob Bruce (1669–1735); Innovative Teaching and Learning; Health Proceedings of 10th Socratic Lectures 2024 139 of 165 “Only Art and Science can raise men to the level of gods.” Ludwig van Beethoven (1770 – 1827) Music is beneficial for everyone, both to cure diseases and to maintain health. Its power should not be underestimated Al-Farabi (870-950 AD) (The Great Book of Music) 1. Introduction 1.1. Research: Story-telling about Jacob Bruce, a Scientist of Scottish descent A central figure in this “new type” of storytelling´s research is Jacob Bruce (1669-1735), who created AI in the 17th century. Jacob Bruce was born in Moscow and descended on his father’s side from the Bruces of Clackmannan in Scotland (Collis, 2012). Bruce’s biographer M.D. Khmyrov (М. Д. Хмыров) emphasizes the encyclopedic nature of Bruce’s knowledge, and writes about him1: “Astronomer and mathematician, artilleryman and engineer, botanist and mineralogist, geographer, author of several and translator of many scientific works” (Луппов, 1973). Luppov (1973) also added that Count Bruce was the first prominent figure in the field of Russian pedagogy and science of that time, and a companion of Peter the Great (Comments: this name of the tsar is commonly used in the West, while in the East, he is better known as Peter the First, - “Петр Первый“). The ratio of the number of books on different branches of knowledge in Bruce's library characterizes the area of his primary interests. Noteworthy is the large number of literature on physical and mathematical sciences (233 books), medicine (116 books), geological and geographical sciences (71 books). Bruce was at the forefront of Russian printing, and rich practice in mint reforms (after visiting Isaac Newton in England and discussing it), travelling, the literature on Arts and religions and military sciences (91 books) in artillery, and fortification (Копанева, 2020). He was a leading figure in the Russian science, and the head of mining and industry, the head of the Mathematical and Navigation School based in the Sukharev Tower. Bruce also established the first observatory of Russia on the third floor of this Tower in 1699. Figure 1 (below) demonstrates his portrait and pictures of the legendary Suharev Tower, where Jacob Bruce worked on his innovations. Bruce was also one of the leading diplomats, esteemed for his great learning, and Russia’s first Newtonian, who created a housemaid from flowers (Collis, 2012). This housemaid was an android or AI, robot-type girl, created in the Sukharev Tower, who looked like a genuine girl: she cleaned the bedroom, served coffee, but she could not speak. This bears a resemblance to a legend about the medieval alchemist Albertus Magnus, in which he reputedly created an android in his secret workshop (Collis, 2012, p. 53). In medieval and early writings, there are stories about medieval AI or an artificial construct in the shape of a human body animated for the purpose of divination, associated with Gerbert of Aurillac, Albertus Magnus, Robert Grosseteste, Roger Bacon (Kang and Halliburton, 2020, p. 72). In this story, the author refers to the 17th century time frame which was a crucial period in the development of modern science in Europe. It saw advancements in various fields such as astronomy, physics, mathematics, and biology, and this period is often referred to as the Scientific Revolution. The Royal Society of London for Improving Natural Knowledge, commonly known as the Royal Society, was founded on November 28, 1660, when a group of prominent scientists and philosophers gathered at Gresham College in London. It was granted a royal charter by King Charles II in 1662, officially establishing it as The Royal Society. Similarly, in 1666, Louis XIV established learned societies to encourage the spirit of French scientific research, promoting academic disciplines such as the arts and sciences. 1 Луппов С.П. Книга в России в первой четверти XVIII в. Л., 1973. Глава пятая. Частные книжные собрания. Translated from Russian, the original text is in the source: https://www.alib.ru/bouluppov52.phtml Proceedings of 10th Socratic Lectures 2024 140 of 165 Figure 1. From the left to the right: Photo of Suharev Tower. Portrait of J.V.Bruce by lithography of F. Mayer. XIX century. From the book: Primitive Calendar of Bruce, from the 1st edition during the lifetime of Bruce, with his portrait and biography. Kharkiv, 1875. MAE Russian Academy of Sciences. On the right: Suharev Tower in Moscow (Moscow, 1802). 1.2. Health: A healing power of Arts (Storytelling in Poetry, Music, and Virtual Arts) In this paper, the author introduces the new method of research with a story telling (based on research about Jacob Bruce) - mechanism, which embeds Arts and Science. In facts, it includes many related fields: Arts, Culture, Science, Innovations and Health. The applied SMS (Stories based on Music about Scientists) is a novel method, a STEAM approach which integrates Science (Historical Research stories about Innovations of the Past), Arts (Music, Poem, Digital images), and Culture (cultural heritage with metaphor of philosophical soul development). In order to understand these concepts we briefly define them. Stories are a universal language loaded with symbolism and significance, allowing us to unravel the mysteries of the world: they seek to discover the meaning of human existence and consider our own individual purpose within it (Rooney et al., p. 147). Kant in “Critique of the Power of Judgment” gives the definition of Art, which is defined: Art is “a kind of representation that is purposive in itself and, though without an end, nevertheless promotes the cultivation of the mental powers for sociable communication” (Adajian, 2007). “The cultivation of the mental power” in this Kant´s definition is very important, because of similarities with a broader concept of “Culture”. Culture as a word originates from the Latin root “cultura” or “cultus” meaning to "inhabit, cultivate, or honour". Thus, culture includes ideas, language, beliefs, customs, codes, institutions, tools, techniques, works of art, rituals, and ceremonies. The origins of the word "poem" trace back to the Greek word "poiema," which means "a thing made" or "a creation", and a poet, as defined in ancient terms, is essentially "a maker of things." This concept underscores the creative act of crafting poetry. Poetry has a special place and meaning in the history of healing: shamans and medicine men and women chanted poems as a part of their healing art (Chavis, 2011, p. 19). In ancient Greece, Appolo, the patron God of music and poetry (poetry: light, sun and prophecy; drama: insipration and catarsis for entire community), is also recognised as the divinity of medicine and healing (Chavis, 2011), thus, directly linked to “health” issues. Al-Farabi, the Central Asian scientist who wrote on the perfect city, logic, astronomy, linguistics, politics, mathematics, geometry, medicine, optic, philosophy, and music, known as the 'second teacher' (after Aristotle, the first teacher) discussed the importance of music Proceedings of 10th Socratic Lectures 2024 141 of 165 (Nurysheva & Tercan, 2021). Music was not only as an art form, but also as a tool for cultivating the soul and promoting physical and mental well-being in the 9th AD (Nurysheva & Tercan, 2021). Al-Farabi who raised most of his philosophy and theory on Greek philosophers such as Aristotle, Pythagoras and Plato, wrote in his Great Book of Music: Music is beneficial for everyone, both to cure diseases and to maintain health”. He acknowledges the therapeutic effects of music on the soul and its ability to induce states of calmness and relaxation. Al-Farabi believed that music had the power to influence both the body and the soul, depending on type of music: rational, ethical, and customary. Rational music (closely linked to philosophical and intellectual pursuits), had the most profound effect on the soul, leading to intellectual and spiritual development, ethical music aimed to instill virtuous qualities in listeners, and customary music was associated with entertainment and social customs. SMS incorporates the rational music qualities, and might go even beyond it due to the additional and intentional components of Arts and Poetry. Al-Farabi shared the story of 'Alqama ibn 'Abda (sixth century), the poet who was refused a hearing by the Ghassanid king Al-Harith ibn Abi Shamir (529-69) until he had melodized his verse and sung it to him (Farmer, 1929, p.18). Thus, music and arts in the Past played integral roles in promoting health and well-being by addressing the emotional, cognitive, social, and physical aspects of human experience, and contributing to a multitude of additional qualities and abilities. 2. Methods SMS (Stories based on Music about Scientists) method was developed as a music experiment on a “hobby” research in 2012-2013 years in Europe (Slovenia), with the idea to create the author project “Education for all”, when STEAM approach was not known. Following the inspiring European ideas of Leonardo da Vinci (1452-1519), an Italian Renaissance painter, scientist and inventor with “principles for the development of a complete mind”: “Study the science of art. Study the art of science. Develop your senses – especially learn how to see. Realize that everything connects to everything else” (Haesen and Van De Put, 2018, p. 9). Therefore, it was created as the European novel and pioneering approach that integrates Arts (Music, Poem, Visual Arts (digital images), Science (Research about Innovations) and Culture (Language, Ideas, and Beliefs). The author has been using SMS since 2012 year and received 13 awards from the International Music festivals. The author uses this approach with the both components of Arts and Science as a STEAM Ambassador (volunteer in the Deloitte & European Commission´s ESTEAM project) to promote the power of innovations through Arts and incorporate the digital competences of women and girls in STEM disciplines. 2.1 SMS (Stories based on Music about Scientists): Innovative Teaching and Learning method Storytelling here is present as a Methodological Approach in Research. The other key inspiring method, which is based on a storytelling, is the method of SMS (Stories based on Music about Scientists) which embeds both the Arts and Sciences. The description of SMS method is presented and explained here through: “What (1-2)-Why (3)-How (4-7)” questions. The SMS method has the following conceptual framework: 1. Research about the scientist of the Past time 2. Story about a Scientist-Polymath of the Past time (mainly, the 17th-20th centuries) 3. Key idea of Innovation, its Ethical issue or inspiring facts from the life of Scientist 4. Lyrics (a): written based on research)/or Poem (b) written by Scientist himself with some elements of phantasies (in 3a) or construction of the key shorter version (3b) 5. Music (emphasising the idea of lyrics) 6. Visual Arts (digital images /pictures, video, etc.) to explain the key ideas to those who do not understand the original languages of lyrics (English, Russian, Slovenian, or Latin). 7. Performance (live) or Music Notes or Recording (digital) placed on Youtube/Facebook There are two types of SMS, which can be used as a method of Innovative Teaching and Leaning, the interdisciplinary method with the intersection of Arts and Science: Proceedings of 10th Socratic Lectures 2024 142 of 165 First type of SMS – written based on research with the initial poem about scientist of the Past time based on research facts about the life of scientist or his innovation Second type of SMS – written based on the poem of scientist himself (his original song), and the composed music is added to the poem with the digital images The project “Heritage of Science” in “Education for All” is a personal author´s artistic nonfunded initiative, which was set up as an attempt to share the knowledge about Science through the poetic stories about scientists of the past centuries based on the volunteering author´s research. The author promotes the power of Science and Arts in the method SMS (Stories based on Music about Scientists) which can be used as Innovative Teaching and Learning method (the Bologna initiative), Life-Long Learning technique and a holistic and health approach. 2.2 SMS (Stories based on Music about Scientists) vs. STEAM approach The SMS principle was developed independently, and approximately at the same time as the STEAM approach was developed in the United States (in about 2013, described in the history of its development in “STEM to STEAM” (Istileulova, 2022)). STEAM (science, technology, engineering, the arts and mathematics approaches to education have been recently launched in the EU to improve the relevance and quality of higher education with a holistic and dynamic educational experience with integration of concepts and methods from multiple disciplines, fostering interdisciplinary learning. The European Commission is championing the STEAM approach through the work of the EU STEM Coalition and the actions outlined in the European Skills Agenda. In 2013, John Maeda, the founder of STEAM, an Asian American computer scientist, educator, and professor at the MIT Media Lab, answered the question: “What does it mean to add Art to turn STEM into STEAM?” (Lamont, about 2013) He announced that art and design transform the American economy, and the STEAM movement is an opportunity for America to sustain its role as an innovator of the world through 'problem-solving, fearlessness, and critical thinking' (Maeda, 2013). 3. Results The Romance about Alchemist (with the dedication to Jacob Bruce) – is the title of SMS, and this song was written in Spring, 2015. It has the following elements: 1. Historical research about Jacob Bruce on AI of the Past time, but different technology with the additional elements of phantasy related to the “technology of creation” (which is a “black box”) suggesting that one element was made with the inclusion of cultural heritage (Slovenian Neanderthal flute, about 60-67 mln. years, the oldest instrument): https://www.youtube.com/watch?v=EaN_NLOvvbo 2. Inspiring Story about a Scientist-Polymath of the Past time who introduced innovation. 3. Key idea of Innovation (AI in its perfect form) and its Ethical issue in transformed into the SMS (song) entitled “Romance about Alchemist”. 4. Lyrics (a): written based on research with some elements of phantasies dedicated to the act of woman´s creation. For instance, in a poem, there was a metaphor to depict the creation of woman through musical performance on the ancient flute (referring to the Neanderthal flute, the oldest instrument in the world, found in Slovenia). 5. Music (emphasising the idea of lyrics) – Musical Notes (below, the introduction). 6. Visual Arts (digital images /pictures, video, etc.) – should be published in YouTube channel (author/under her other name of Aleona von Sultanova – used in Music/SMS): https://www.youtube.com/channel/UCl2j8TiTOj1cwQpHWDb8Ngg 7. Performance (live, in the hostel Celica), and/or Music Notes https://www.facebook.com/1346167803/videos/pcb.10220238261770494/10220238242370009. Typically, The link presents a live performance, dedicated to the memory of Ljuben Dimkaroski, a dear friend, and musician, who played tidldibab, as he wished to call it or the oldest Neanderthal flute (which was found in Divija baba, Slovenia). The act of creating the artificial 'Jashkina Baba' (or AI of the 17th century, a girl-android) is described in the song, where Proceedings of 10th Socratic Lectures 2024 143 of 165 one of Jacob Bruce's rituals involved playing the oldest instrument (the oldest flute), hence the numerous analogies. Although there are some elements of phantasies, but they are also linked to some legends and real stories. Ljuben Dimkaroski, shared one of the real stories. During his interview in 2015 to the author of this article, he shared a story, that after 2.5 years of having the replica of this flute in his apartment in Ljubljana, during one night, he saw himself in the night dream. In that dream, someone was showing to him how to play this ancient flute (from the bone). It should also be noted, that the concept of AI is broader than that of a robot, and the concept of a robot is broader than that of an android, which specifically refers to robots designed to resemble humans. Thus, according to these three concepts, it would probably be more precise to use the term 'android' for a humanoid robot. At the same time, the author uses the general term AI to illustrate that AI models could be more advanced in the 17th century compared to those created today, yet ethical questions remain the same. The text of SMS lyrics is provided below: Романс об Алхимике (Romance about Alchemist) В конце 17-го столетья алхимик формулу искал: Искал он формулу совершенства, чтобы создать свой идеал. Мечтал увидеть он новый облик – свой женский Евы идеал, Спускаясь с башни в пещеру, алхимик романс свой тайный напевал: Припев: Как вверху, так и внизу, как сейчас, так и тогда, При полной луне и в маске от блаженства Как Парацельс, сотвори ту алхимию любви И прошепчи ты формулу Трисмегиста. Что такое душа? Как ее сотворить? Духов ты вызывал, с ангелами говорил, книгу Еноха читал, в шар хрустальный смотрел, Мудрости меч рассекал, и душой постарел: Чернокнижником став, злато из меди создав, омолодив живою водой о сокровенном мечтал, Чтобы душу вдохнуть – это не плоть омладить: равносильно творцу то уменье творить Из рая иль ада, и что же ты создал? И формулу жизни вложил в свой идеал, Глаза - как изумруды, копна волос в цветах. Восточные глаза и совершенный стан. Но образ твой прекрасный немой и без души, И ты не знаешь точно, что чувствует внутри, И движется прекрасно, и кофе подает, улыбка ж безучастна, глаза ее грустны. Книга Соломона указала некий знак, Фиалками и розами гомунгул осыпал, И каплями росы и дуновением весны и, до сих пор, ты поисках алхимии души Маг и алхимик славянской страны, Древнюю флейту в руки возьми И совершенство ты сотвори: В танце суфиста движенье планет пробуди, прошептав: Proceedings of 10th Socratic Lectures 2024 144 of 165 Припев: Как вверху, так и внизу, как сейчас, так и тогда, При полной луне и в маске от блаженства Ты во сне увидал, как играть на флейте той, При полной луне на восток в танце суфиста. Romance about Alchemist (translated in English language). At the end of the 17th century, an alchemist sought a formula: He searched for perfection to create his ideal. He dreamed of witnessing a new manifestation - his feminine ideal of Eve. Descending from the tower into the cave, the alchemist sang his secret romance: Chorus: As above, so below, as now, so in the Past, Under the full moon wearing a mask of bliss You, like Paracelsus, create that alchemy of love And whisper the formula of Trismegistus. What is the soul? How does one create it? You called upon your spirits and conversed with angels, You delved into the book of Enoch and gazed into the crystal ball, You cleaved the air with the sword of wisdom, yet your soul aged further: You became a warlock, transmuting copper into gold, rejuvenated by living water, You dreamed of the internal secret: to 'breathe in' a soul is not to rejuvenate your flesh but to embody the Creator's ability to create Whether from heaven or hell, what have you created? And he infused the formula of life into his ideal: Her eyes akin to emeralds, a head adorned with lovely flowers, Oriental eyes and a perfect figure. Yet your beautiful image is mute and soulless, And you do not understand what resides within. It moves gracefully and serves coffee, Her smile is indifferent, her eyes are sad. The Book of Solomon indicated a certain sign, you showered the homungul with violets and roses, with drops of dew and a breath of spring and, somewhere there, you are still in search of the alchemy of the soul Magician and alchemist of the Slavic lands, take the ancient flute in your hands, and strive for perfection: In the dance of a Sufi, awaken the movement of the planets, whispering: Chorus: As above, so below, as now, so in the Past, Under the full moon and wearing a mask of bliss, In your dream, you saw how to play the ancient flute of the one With the full moon to the east in a Sufi dance. Proceedings of 10th Socratic Lectures 2024 145 of 165 Figure 2: Excerpt from the Introduction of the Musical Composition on Jacob Bruce (Romance about Alchemist). 4. Discussion In the modern higher education of the third decade of the 21st century there are a lot of attempts to integrate STEAM approaches in the inter- and transdisciplinary higher education through developing a comprehensive methodological framework through, speculative design, art thinking, process-based research and participatory practice and the methods to implement STEAM pedagogical approaches (Carter et al., 2021). The SMS method is easily associated with the STEAM approach. However, the STEAM approach does not incorporate methods similar to SMS. According to discussions by Carter (2021), HEIs refer to the ideal approach to STEAM, which is (potentially) involving: • a culture of the Arts and Sciences on an equal footing • operating within a process-driven paradigm, student-centred, holistic Proceedings of 10th Socratic Lectures 2024 146 of 165 • • • • • being collaborative, diverse and delivered through safe spaces establishing a mind of openness, reflection, experimentation and curiosity generating qualities that promote learning, cooperation and multi-modality supporting transdisciplinary practices and emphasise prototyping developing competencies of critical thinking, creativity and communication whilst investigating how to generate solutions. The SMS method easily meets all the criteria set forth by prominent HEIs. It is the Avantgarde & Experimental genre of music which embeds the key fields of knowledge: Science (History of Science/Historical research), Arts (Music, Poetry, and Digital Images), and Culture (Language, Ideas, Beliefs). Additionally, it aligns with innovative teaching and learning methods, as well as lifelong learning (LLL) practices, which can be associated with STEAM Ambassadorship. Returning to the elements of art and science cited by Beethoven, when music contributes to health benefits and the cultivation of mental faculties for sociable communication, as an element of culture, along with music and poetry, provides a holistic approach. Rational music, with its power to influence both the body and the soul through philosophical and intellectual pursuits, leads to intellectual and spiritual development. Further effects is to be studied through Al-Farabi and Avicenna, who wrote about the influence of music on health, including its effects of curiosity and stimulation for new discoveries in science. Funding: This research is not supported. Conflicts of Interest: The author declares no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Adajian T. 2007. The definition of art. Carter C, Barnett H, Burns K, Cohen N, et al. Defining STEAM Approaches for Higher Education. European. Journal of STEM Education. 2024; 6:1-16. https://doi.org/10.20897/ejsteme/11354 Chavis GG. Poetry and story therapy: The healing power of creative expression. Jessica Kingsley Publishers. 2011 Farmer HG. 1929. A history of Arabian music to the XIIIth century. Haesen S, & Van de Put E. STEAM education in Europe: A comparative analysis report. Euro- STEAM, 2018. p. 9 Istileulova Y. (October 14, 2022) From STEM to STEAM. KEN (Brussels), 21; ISSN 2593-0257 https://www.academia.edu/88516850/From_STEM_to_STEAM Kang M. and Halliburton, B. 2020. The Android of Albertus Magnus. AI Narratives: A History of Imaginative Thinking about Intelligent Machines, pp.72-94. https://doi.org/10.1093/oso/9780198846666.003.0004 Lamont T. (n/a). John Maeda: Innovation is born when art meets science. The Guardian Retrieved from : https://www.theguardian.com/technology/2010/nov/14/my-bright-idea-john-maeda Nurysheva, G. and Tercan, N., 2021. AL-Farabi Philosophy of Music:“The Legality of Music”. Central Asian Journal of Art Studies, 6(1), pp.10-26. Rooney T, Lawlor K, Rohan E “Telling Tales; Storytelling as a methodological approach in research” The Electronic Journal of Business Research Methods 14 Issue 2 2016, 147-156 available online at www.ejbrm.com Копанева Н.П. О Собрании Я. В. Брюса, наследниках и кабинете ученого. 2020; 04 (10), Kunstkamera, 5863; удк 930.1:069.51; DOI. 10.31250/2618-8619-2020-4(10)-56-64 Луппов С.П. (1973). Книга в России в первой четверти XVIII в. Л., 1973. Глава пятая. Частные книжные собрания. Перепечатывается с незначительными сокращениями. Accessed 27.01.2024. Available online at https://www.alib.ru/bouluppov52.phtml Proceedings of 10 th Socratic Lectures 2024 148 of 165 The Slovenian Model of Scientific Research Work of Secondary School Students and their Participation in Competitions as Motivation for Career Choice Lap Patricija1,#, Lipovšek Anja 2,#, Jeran Marko1,* “Jožef Stefan” Institute, Department of Inorganic Chemistry and Technology, Ljubljana, Slovenia University of Ljubljana, Faculty of Education, Department for Preschool Education, Ljubljana, Slovenia # These two authors contributed equally to this work * Correspondence: Marko Jeran, marko.jeran@ijs.si 1. 2. Citation: Lap P, Lipovšek P, Jeran M. The Slovenian model of scientific research work of secondary school students and their participation in competitions as motivation for career choice. Proceedings of Socratic Lectures. 2024, 10, 148-157. https://doi.org/10.55295/PSL.2024.II16 Abstract: Research in education plays an important role in maintaining curiosity and training skills. It is primarily used in the classroom, but can also be found in everyday life without us even realising it. The research work includes the views of young researchers from Slovenian secondary schools who participated in a research assignment within the Association for Technical Culture of Slovenia (ATCS) between the years 2010 and 2017 and won the gold award. We found that carrying out the research assignment helped the young researchers on their path in life and on their way to success. The correlation test of the chosen research pool showed that there is no statistically significant relationship between the chosen research area in secondary school and further university or professional orientation. Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2023 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/ by/4.0/). Keywords: Research work, Young researchers, Motivation, Education, Career choice, Association for Technical Culture of Slovenia Proceedings of 10 th Socratic Lectures 2024 149 of 165 1. Introduction It is common to come into contact with research or project work at some point (Petek, 2012). Research work in itself means gathering one’s own thoughts and ideas, collecting data and dealing with a specific problem. When young people decide to undertake a more extensive project work, which means additional success and progress for them in the future, it is important that they are encouraged by the surrounding society. Various sources of support include relatives, peers, but also mentors and various institutions that help young people to achieve excellent results. Through the research approach two fundamental goals of education are pursued: to maintain the individual's curiosity and continuing interest in knowledge and to develop the skills required for independent problem solving (Petek, 2012). To achieve these goals, one of the key factors is motivation, i.e. encouragement, maintenance and direction of the individual's activities to achieve the goal (Kompare, 2009). In addition to motivation, self-confidence is also necessary, which shows us what we want to achieve and how we will achieve it. A key role in motivation in research is played by the manager, who must positively encourage the researchers, listen to them and find out what the researcher wants to achieve. A good manager also encourages motivation by helping to solve problems, by acknowledging also negative results, by boosting self-confidence and by making the work enjoyable. Both, praise and reprimand are extremely important in research work (Uranič, 2008). As mentioned earlier, institutions are also responsible for a large part of motivation by supporting designers in various ways. Such method was demonstrated at a local school in Ljubljana region (Biotechnical Educational Centre Ljubljana, General Upper Secondary School and Veterinary Technician School), where a project was launched in 2015 to further develop the knowledge on water. The project enabled students to expand their knowledge in various areas and acquire new skills at the same time. General conditions for participation in the national competition are the ranking in the regional (local) competition and the maximum number of research projects (contributions/articles) from each region that can qualify for the national competition. This is determined on the basis of the average number of research papers in the regional competition in the previous three years, which is set each year in the announcement for the national competition of the current school year. The topics of the research paper s are freely selectable. Current topics, also from the local environment, are recommended, especially in cooperation with the community, the business community or other interested parties, who can also announce the topics of the research papers via the Association for Technical Culture of Slovenia (ATCS). Participants must submit their research papers on time and in the form specified in the call for proposals for the respective school year. The research papers must be written in Slovenian and in a format that meets the current standards in the respective research fields. Foreign-language research papers that are reviewed by the main committee are exempt from the use of the Slovenian language. The use of a foreign language is permitted for such research papers. A foreign-language paper that does not deal with the use of a foreign language may be rejected by the committee. The papers are assessed by the members of the panel of experts responsible for the respective area. The panel is chaired by a chairperson. In the first round, the members of each expert panel review and evaluate the research papers that have qualified for the national competition and rank them according to points based on the criteria specified in the Call for Papers for the respective school year. During the presentation in the national competition, the members of the expert panel examine the independence of the candidate in the conception of the research project, the research approach, the methods and results, the systematics and significance of the presented material as well as the general knowledge (ATCS, n. d.). At the end of the state competition, the participants receive awards and prizes depending on the results achieved. On the recommendation of the technical committees, the best-rated Proceedings of 10 th Socratic Lectures 2024 150 of 165 projects are selected by the national committee to take part in international competitions for young researchers (ATCS, n. d.). Our work involves qualitative and quantitative data analysis of research assignments conducted by secondary school students between 2010 and 2017. We focused on areas such as chemistry, biology and interdisciplinary fields. We invited former winners of the Association for Technical Culture of Slovenia (ATCS) who were secondary school young researchers to complete a questionnaire (ATCS, n. d.). Using a correlation analysis, we looked for a relationship between the chosen research area at the secondary school level and the choice of further studies at university level. 2. Methods and hypotheses The survey (using a questionnaire (Appendix)) was completed by young researchers that were awarded the Gold Award for Young Scientists of Slovenia by ATCS during their previous education in the secondary school. The survey focused on competition in the fields of chemical and biological research projects as well as interdisciplinary scientific projects. The questionnaire was created on the 1KA website (1 KA Enklik anketa, n. d.) and then distributed to the winners via social media (Facebook) and electronic messages using Gmail. After all respondents completed the questionnaire, we collected all data using the analytical tools via Microsoft Excel (Microsoft Corporation, version 2018). The collected data was then presented using pie charts created with the same programme. Before the empirical part of the work, we established hypotheses that guided us throughout the study. We hypothesized that individuals who had a natural science focus of the project (chemistry, biology or interdisciplinary project) in the secondary school would also have a natural science focus in their further studies and profession. We hypothesized that the interdisciplinary subject will be most strongly represented among the respondents. As the research work was voluntary in most cases, we hypothesized that the students would not find it difficult, but on the contrary would enjoy it and were able to gain new experiences. We hypothesized that the majority would perform the research work again if they had the opportunity to do so. 2.1. Sample Young researchers from secondary schools and grammar schools who received the ATCS Gold Award for secondary school students during their previous education between 2010 and 2017 were included. We were assisted by the organization ATCS, the main organizer of this competition. Some individuals of the researchers on our list of names were repeated and sometimes even tripled because they applied for and worked on the competition more than once during the observation period. We treated these researchers as one person who completed the questionnaire once. Of the 75 people invited, 35 successfully answered the questionnaire, some of whom are studying or working. 2.2. Instrument We composed a questionnaire and sent it out via social media platforms (Facebook and Gmail). We opted for a closed questionnaire, which means that the respondent does not write the answer to the question, but selects one of the already prepared answers that seem the most appropriate. The questionnaire comprised a total of 14 questions, which were compiled in such a way that we could confirm or refute the hypotheses after the evaluation and find out the personal opinion of the respondents. 2.3. Statistical analysis Pearson's chi-square (χ²) is a basic statistical measure that compares observed and expected frequencies and evaluates how these frequencies differ. Larger values of Pearson's χ² indicate larger differences between the observed and expected frequencies. To interpret the results, the p-value was used, which represents the value and measures the probability that the observed difference between observed and expected frequencies is purely random if there is no real relationship between the variables. If the p-value is below Proceedings of 10 th Socratic Lectures 2024 151 of 165 the selected significance level (usually 0.05), the null hypothesis is rejected and a statistically significant correlation is concluded. The correlation analysis was carried out using the SPSS programme. We analysed correlations with the χ 2-test (hi-square) by using the programme IBM Corp. Released 2010, IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp., New York, USA. The results of the analysis (Table 2) were presented in various parameters. The degree of freedom (df) indicates the number of independent variables in the analysis. With the statistical programme SPSS, the output table contains a sig. value (2-tailed). Asymptotic pvalues are useful for sample sizes where it is difficult to calculate an exact p-value. This value represents the 2-sided p-value of the test (Asympt. Sig. 2-sided p-value). It is roughly assumed that the sample size is large enough for the test statistic to converge to the corresponding marginal normal or chi-squared distribution. The p-value calculated using the approximate true distribution is called the asymptotic p-value (Analyse-it Software, n. d.). In general, the term association refers to coefficients that measure the strength of a relationship. The coefficients in this section are designed for use with nominal data. Phi and Cramer's V are based on adjusting chi-squared significance for sample size (approximate significance, Approx. Sig.). Phi is a chi-squared based measure of association. The chi-squared coefficient depends on the strength of the relationship and the sample size. Phi eliminates the sample size by dividing the chi-square by n, the sample size, and taking the square root. In purely mathematical terms, phi is the square root of chi-square divided by n, the sample size: phi = SQRT(χ2/n). Phi therefore measures the strength of the relationship, defined as the number of cases on one diagonal minus the number on the other diagonal, adjusted for the marginal distribution of the variables (Virginia Commonwealth University, n. d.). Cramer's V is the most popular of the chisquared based measures of nominal association because it gives a good normalisation from 0 to 1 regardless of table size when the row edges are equal to the column edges. V is equal to the square root of the chi-square divided by the sample size n times m, i. e. the smaller of (rows–1) or (columns–1): V = SQRT(χ2/nm). SPSS and other important programmes indicate the significance level of the calculated V value (Virginia Commonwealth University, n. d.). 3. Results With the first question of the questionnaire, we collected information about the gender of our participants. Of the 35 respondents, 24 identified themselves as male and 11 as female. The second question related to the respondents' current education, and it turned out that the largest percentage of respondents (77%) were natural science students. The smallest percentage of respondents were social science students (6%), and only a few of them (6%) were working on a secondary school degree. The third question related to the field of study in which the respondents were researching. One percent less than half of the respondents did research in an interdisciplinary field, and the rest did research in chemistry and chemical engineering or biology (Figure 1). Figure 1. Choice of research area of the young people included in the sample. Distribution of choice within the natural sciences. Proceedings of 10 th Socratic Lectures 2024 152 of 165 The fourth question, which was answered in the affirmative by 97% of respondents, was: “If you were a young researcher in secondary education, would you attend the young researchers competition again”. With the fifth question, we wanted to find out whether the respondents thought that research is a good way to gain new knowledge, and we received the answer that they all agreed. The next question related to the selection or creation of the topic for the research paper and its elaboration. Of the 35 participa nts, 11 were without record of help (31%), 6 were supported by a school-based mentor (17%), 15 had help from an external mentor (43%) and 3 had received help from a school and an external mentor (9%). With the seventh question, we wanted to find out which part of the research the respondents enjoyed the most. Most (97%) found the practical part of the research work the most enjoyable, only one found the preparation of the written documentation the best. In the eighth question we asked about their impressions of the experimental part of the work and in the ninth question about their impressions of the written part of the work. When asked about the experimental part, 33 respondents chose the answer: “I liked it, I was motivated and had fun”. The same answer was given by 20 respondents to the ninth question. In the 9th question, the participants were asked for their opinion on the role of the mentors (school or external). Most (29 respondents) felt that the role of the mentor was important as it added value to the research and the candidate gained a lot from the research process; some felt that the role of the mentor was important but that the author of the research was responsible for everything, or that the role of the mentor was not important. The 10th question related to the current relationship with their mentor. The answers showed that more than half (60%) of the researchers have remained in contact with their mentor to this day. Regarding the 11 th question: “Do you think your research has helped you develop other skills not related to the experimental part of the research (e. g. public speaking, overcoming stage fright, courage to stand by your results, personal growth, responsibility, etc.)?” 89% of respondents answered affirmative. In 12 th question, we wanted to know if respondents were aware that research can help them in their future job search, and it turned out that 60% were aware of this fact (Figure 2). Figure 2. Question: Did you know that research can increase your chances of getting a job in the future? In the 13th question, we asked the respondents whether the research work they had carried out had helped them in their choice of further education. More than half (54%) answered “yes", 8 individuals answered “no" and the same number of individuals did not specify (“I cannot define”) (Figure 3). Proceedings of 10 th Socratic Lectures 2024 153 of 165 Figure 3. Question: Was the research helpful for your further choice of education (if you are a student) or employment or to establish yourself? In the last (14 th) question, we asked where the respondents see themselves in 10 years’ time. 51% of respondents answered that they see themselves in the role of a researcher in public institutions (e. g. as a researcher at an institute or university), 23% in a company where they are involved in the development of products, 17% in a university, 9% as an independent entrepreneur and none in the role of a teacher or university professor. Once collecting the data from the questionnaire, we also performed the correlation analysis (Table 1). The correlation of statistical differences we were interested in resulted from the second (2 nd) and third (3 rd) questions. We were interested in the statistical compatibility between the choice of subjects in secondary level and the later choice of educational or professional pathway. Table 1. Corelation between responses to Question 2 and responses to Question 3. Question 3 Chemistry and Biology Interdisciplinary Total chemical field technology Question 2 Natural science Count 0 1 3 4 student Expected count 1 1 2 4 Social sciences Count 6 8 13 27 student Expected count 7 7 13 27 Employed in the Count 1 0 1 2 natural sciences Expected count 0.5 0.5 1 2 Employed in the Count 2 0 0 2 social sciences Expected count 0.5 0.5 1 2 Total Count 9 9 17 35 Expected count 9 9 17 35 The bolded values indicate the correlation of mutually exclusive results (if the quantities were completely independent of ea ch other) (i. e. there would be no correlation). The values in italic indicate the values determined by the crosstabulation. Proceedings of 10 th Socratic Lectures 2024 154 of 165 Table 2. Correlations between the choice of research topic in secondary school and the subsequent choice of studies or employment (Q2 and Q3 questions) as calculated by Chi-square test. Value df Asymptotic significance (2-sided) (Significance test) Pearson Chi-Quare 8.646 6 0.194 Likelihood ratio 9.659 6 0.140 Linear-by-linear association 5.633 1 0.018 Number of valid cases 35 Symmetric measures Phi Cramer’s V 35 Nominal by nominal Number of valid cases Value 0.497 0.351 Approximate significance 0.194 0.194 According to the results in Table 2, the p-value is greater than 0.05, which means that there is not enough evidence to reject the null hypothesis. This indicates that there is no statistically significant relationship between the choice of research topic (biology, chemistry and interdisciplinary field) and further education or employment pat h for our sample (further studies or career choice). 4. Discussion We found that the completed research work helped the young researchers finding the path of their further career, which has at the time already taken shape. The young researchers were convinced that research is an excellent way to gain new knowledge. It is therefore of utmost importance that young individuals in primary and secondary schools are introduced to the research method for solving problems. The correlation analysis (χ 2-test) of the collected sample has shown that there is no statistically significant relationship between the chosen research field and the further educational or employment path. Our results indicate that the motivation of the individuals and their interest in any field of study are crucial. However, it should be borne in mind that the population comprised only 35 people and the lack of statistical significance could be due to an insufficient number of participants. The study will be continued. Acknowledgements: The authors would like to thank the Association for Technical Culture of Slovenia for their kind support in sharing data and for their excellent cooperation. The organisation has been successfully active at the national level for many years. We were glad that we could participate in this project. Many thanks also to Darja Kozar Balek for her help. Many thanks also to all the participants who filled in a questionnaire. Funding: This research was supported by Slovenian Research and Innovation Agency (ARIS) through the core foundlings P1-0045. Conflicts of Interest: The authors declare no conflict of interest. References: 1. 1 KA Enklik anketa (n. d.), Minimalno število klikov; Ustvarite anketo brezplačno (Engl. Minimum number of clicks; Create a survey for free). Accessed 29. 12. 2023. Available from https://www.1ka.si/d/sl# 2. Analyse-it Software (n. d.), Exact and asymptotic p-values. Accessed 12. 3. 2024. Available from https://analyseit.com/docs/user-guide/101/exact-asymptotic-p-values Proceedings of 10 th Socratic Lectures 2024 155 of 165 3. 4. 5. 6. 7. Association for Technical Culture of Slovenia (ATCS) (n. d.), Vizija in poslanstvo (Engl. Vision and mission). Accessed 17.12.2023. Available from https://zotks.si/vizija-in-poslanstvo/ Kompare A, Stražiščar M, Dogša I, et al., Uvod v psihologijo (Engl. Introduction to psychology). DZS, Ljubljana. 2009. Petek D. Inquiry-based early science teaching and learning. J Elem Educ. 2012; 5(4), 101 -114. Available from https://journals.um.si/index.php/education/article/view/491 Uranič K. Zadovoljni in motivirani zaposleni (Engl. Satisfied and motivated employees). The final thesis of the college of applied sciences for economics. University of Ljubljana, School of Economics and Business, Slovenia. 2008. Available from http://www.cek.ef.uni-lj.si/vps_diplome/uranic79.pdf Virginia Commonwealth University (n. d.), Nominal Association: Phi and Cramer's V. Accessed 12. 3. 2024. Available from https://www.people.vcu.edu/~pdattalo/702SuppRead/MeasAssoc/NominalAssoc.html Proceedings of 10 th Socratic Lectures 2024 156 of 165 Appendix 1 Questionaire 1. Select your gender. a) Male b) Female 2. Please select your current level of education. a) Secondary school student. b) Student of natural sciences. c) Student of social sciences. d) Employed in the natural sciences. e) Employed in the social sciences. 3. What is your field of research? Please select the appropriate answer. a) Chemistry and chemical technology. b) Biology. c) Interdisciplinary field. 4. As a young researcher at a secondary school, would you take part in the Young Scientists Competition again? a) Yes. b) No. 5. Do you think that research is a good way to acquire new knowledge? a) Yes. b) No. 6. How did you choose or create the topic and the development of the research paper? a) I knew what I wanted to do, so I was completely independent. b) The topic and general development of the research paper was suggested to me by my school supervisor. c) The topic and general development of the research paper was suggested to me by an external mentor. d) The topic and the general development of the research paper was created jointly by the school and the external mentor. 7. Which phase of the research work did you like best? a) Carrying out the practical work. b) Preparing the written documentation. 8. What are your impressions of the experimental part of the research work? a) I enjoyed it, I was motivated and I enjoyed it. b) I did not care. c) The experimental work was superfluous, but I did it. d) I have unpleasant memories of the experimental work. 9. What are your impressions of the written part of the survey? a) I enjoyed it, I was motivated and I enjoyed it. b) It did not bother me. c) The writing was superfluous, but I did it. d) I have unpleasant memories of the written part. 10. In your opinion, what role does a (school or external) mentor play? a) Very important because the work has added value and the candidate benefits a lot from the research process. b) Important, but the author of the thesis is responsible for everything. c) Not important. d) I have never thought about that. 11. What is your current relationship with your supervisor? a) I have no contact with him/her. b) I respect him/her because he/she put me on the right path, but we do not see/hear each other often. c) I respect him/her because he/she put me on the right path, but we see/hear each other. Proceedings of 10 th Socratic Lectures 2024 157 of 165 d) I am still in contact with him/her. 12. Do you feel that you have developed other skills through your research work that are not specifically related to experimental work (e. g. fluency, overcoming fears, standing up for your own results, personal development, responsibility, etc.)? a) Yes. b) No. c) I have never dealt with this specifically. d) I do not think research work involves other skills. 13. Were you aware that your research work could lead to future employment opportunities? a) Yes. b) No. 14. Did your research help you to choose a different course of study (if you are a student) or a job or to start your own business? c) Yes. a) No. b) I can not say. 15. Where do you see yourself in 10 years? a) In a career as a researcher or research professional. b) I will be working in a company where I can help develop products. c) I see myself as a teacher or professor encouraging young people to be creative. d) I see myself as a faculty member. e) As an independent entrepreneur. Proceedings of 10 th Socratic Lectures 2024 159 of 165 Cat Facial Action Coding System (CatFACS) and Scientific Il-lustration Kovačič Pšenica1 1. Minami Kat, Centre for Cat Culture, Ljubljana, Slovenia * Correspondence: Pšenica Kovačič; psenakovacic@gmail.com Citation: Kovačič P. Cat Facial Action Coding System (CatFACS) and Scientific Illustration. Proceedings of Socratic Lectures. 2024, 159-165. https://doi.org/10.55295/PSL.2024.II17 Publisher’s Note: UL ZF stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). Abstract: This study explores the complex world of feline communication, focusing on analyzing facial expressions. It utilizes the Cat Facial Action Coding System (CatFACS) with art theory for scientific illustration, presenting a novel approach in this field. CatFACS is employed for a detailed, objective analysis of feline facial expressions. However, as CatFACS does not inherently examine the emotional component, art theory is integrated to interpret these expressions. This combination provides a comprehensive understanding of cats' emotional states by focusing on both observable physical cues and inferred emotional contexts. Integrating CatFACS with scientific illustration reveals intricate insights into feline emotions and communication. This approach uncovers complex behavioural patterns, enhancing the accuracy of behavioural interpretation. This study significantly contributes to feline welfare and understanding of human-cat relationships. Highlighting the importance of nuanced approaches in animal behaviour studies provides practical insights for pet owners and veterinary professionals, aiding in better understanding and caring for cats. Keywords: Feline; Facial expression; Communication; Illustration Proceedings of 10 th Socratic Lectures 2024 160 of 165 1. Introduction Research into feline facial expressions is pivotal in unravelling the complexities of feline behaviour and enhancing human-cat interaction. Recognized as vital members of households worldwide, cats employ a variety of facial expressions as a means of communication. Deciphering these expressions is crucial for gaining deeper insights into their emotional states and fostering closer bonds between humans and their cats. This study specifically delves into the importance of studying feline facial expressions, utilizing scientific illustrations and advanced methods, such as the Cat Facial Action Coding System (CatFACS) (Caiero et al., 2017), decoding the nuanced language of cats. The face serves as a critical tool for visual communication in felines. Facial expressions, encompassing elements such as posture, gestures, and movement, provide critical indicators of a cat's internal state. Various visual tools are employed to analyze these expressions in depth. For instance, the Facial Grimace Scale (FGS) (Evangelista et al., 2019) and Geometric Morphometrics (GM) (SLice, 2007) offer detailed assessments. In pain evaluation in felines, professionals often rely on instruments like the UNESP-Botucatu Multidimensional Composite Pain Scale (Belli et al., 2021) and the Glasgow Composite Measure Pain Scale-Feline (rCMPS-F) (Reid et al., 2017). The Feline Grimace Scale © application is also available for cat owners, providing an accessible means to interpret their pets' discomfort or distress. The primary aim of this article is to demonstrate how integrating CatFACS with scientific illustration can significantly enhance our understanding of feline facial expressions. This study advances welfare practices and deepens human-cat relationships by emphasizing the importance of nuanced approaches in animal behaviour studies, which are crucial for enhancing the well-being of domestic cats and strengthening their bonds with humans. 2. Methods and Results 2.1 Cat Facial Action Coding System (CatFACS) The CatFACS manual is used to analyze the intricacies of feline facial movements. Like ethograms (Stanton, 2015), CatFACS is not an emotion labelling system. It adopts an anatomically-based, standardized, and systematic approach to studying feline facial behaviour. This manual enables the meticulous coding of individual cat movements on their faces, utilizing a comparative and evolutionary framework (Caeiro et al., 2013). The Facial Action Coding System (FACS) provides a standardized coding system for individual movements or action units (AUs) involving specific muscle groups (Figure 1), generating various facial changes or cues. Action Descriptions (ADs) are also used to code broader movements or non-mimetic actions. Fifteen individual facial movements (Action Units), six miscellaneous movements (Action Descriptors), and seven Ear Action Descriptors have been identified in the domestic cat. Figure 1: Illustration of the Action Unit 143 eye closure and muscles (P. Kovačič). Proceedings of 10 th Socratic Lectures 2024 161 of 165 2.2 Observation and Measurement Our observational and measurement procedures involve estimating time and distance and constructing ethograms to delineate behaviour patterns before, during, and after specific events involving objects or subjects in various locations. Initially, we establish a baseline for what is considered normal or neutral for each cat breed and individual. The system utilizes facial landmarks for coding to account for individual differences in facial morphology, such as the cephalic index, fatty deposits, and wrinkles. We acknowledge that visual communication is limited when studied as unimodal; thus, we combine it with other communication modes like olfactory, auditory, gustatory, and tactile. Furthermore, certain forms of signalling, such as vocalization, are adapted for bimodal or multimodal communication, especially with visuals. Contextualizing the situation (Figure 2) allows us to measure various parameters, including the duration of emotions and emotional states, the time required for a cat to return to a normal state or behavioural homeostasis, and the spatial distance relative to the situation. Emotions manifest in the face; hence, the facial muscular plan is adapted to encompass muscle origin, insertion, fibre direction, and movement. We then focus on the natural face and spontaneous facial behaviour, explicitly targeting the head (neck, face, a nd ears). Spatial representation (cranial-caudal, dorsal, ventral, rostral-caudal; medial-lateral; proximal-distal) (Figure 3) mirrors planes in a spatial cross in art, albeit with slightly different terminology (frontal plane, medial plane, ocular plane, and ground plane) ( Figure 4). This approach is significant for dividing the cat's face into planes and directions, facilitating a more straightforward interpretation. Figure 2. Illustration of affiliative context. Proceedings of 10 th Socratic Lectures 2024 162 of 165 Figure 3. Illustration of spatial representation side view (left) and front view (right). Figure 4. Illustration of the spatial cross. Proceedings of 10 th Socratic Lectures 2024 163 of 165 2.3 Scientific Illustration The study employs scientific illustration for the rigorous analysis of feline facial expressions, body posture, limb gestures, movement, and spatial aspects, including location and distance (Case, 2010). This method enables a detailed focus on specific elements or a broader perspective through sketches and visual analyses. Techniques employed include highlighting, detailing, caricature, simplification, omission, cropping, enlargement, and comparison. Male (2017) states that such illustrations fall under non-fiction categories like documentary, referential, and instructional. Christiansen (2018) further describes these illustrations as both informative and metaphorical. The versatility of these methods allows for realistic, informative, and diagrammatic approaches, making scientific illustration indispensable for studying emotional states, motivational systems (Montag et al., 2017), behaviour, and communication in felines. Analyzing social behaviour and visual communication through scientific illustration enables a nuanced understanding of feline communication. Artists achieve a connection between art theory and facial anatomy, viewing facial features as visual elements adhering to foundational art principles such as point, line, shape, geometric body, light -dark, and colour. Facial attributes like ears, eyes, nose, muzzle, and whiskers (Illustration 6) encompass various visual variables, including position, distance, direction, size, shape, weight, density, abundance, importance, and texture (Butina, 2003). Wrinkles, muscle tension, piloerection, colours, and patterns are also considered for comprehensive communication analysis. Combining these facial parts and movements with emotional components and contextual situations gives us an overview of felines' expressive repertoire and emotional spectrum. This approach allows us to transition seamlessly from analyzing individual facial features to understanding their role and relationships within the social-physical environment. Through this method, we utilize art as a conduit for scientific communication, harmonizing creativity with accuracy (Gould, 2023) and making the invisible aspects of feline communication visible (Christiansen, 2018). 2.4. Integration of Methodologies Integrating the Cat Facial Action Coding System, observational and measurement techniques, and scientific illustration forms the backbone of our research approach. CatFACS provides a detailed and systematic method for coding feline facial movements. At the same time, our observational and measurement procedures offer a broader context for these expressions, considering the cat's overall behaviour and environment. Scientific illustration further enhances our understanding by visually representing these complex behaviours and expressions. Together, these methodologies create a comprehensive framework for analyzing and interpreting the multifaceted aspects of feline communication. A more complex approach is possible with this integrated approach to understanding feline behaviour, bridging the gap between scientific analysis and visual interpretation. Figure 5. Illustration of art theory and anatomy. Proceedings of 10 th Socratic Lectures 2024 164 of 165 3. Discussion Integrating CatFACS with observational techniques and scientific illustration has provided a comprehensive understanding of feline communication. This study faced challenges in standardizing assessments across different breeds, highlighting the complexity of feline behaviour. However, these challenges were mitigated through a rigorous methodology and artistic interpretation. The findings have practical applications in veterinary practice and enhance pet owners' understanding, contributing significantly to animal behaviour research. 3.1 Integration of Methodologies and Their Implications The seamless transition from observational and measurement procedures to scientific illustration underscores the comprehensive nature of our study. We have gained a profound understanding of feline behaviour by meticulously constructing ethograms and establishing baselines for normal or neutral behaviours in various cat breeds. This detailed observation and contextual analysis have allowed us to measure various parameters crucial for interpreting feline emotions and reactions. The integration of scientific illustration has significantly enhanced this process. The study has bridged the gap between cat visual communication and artistic expression by employing diverse artistic elements, variables, and compositions. This approach captures physical and physiological aspects and delves into the psychological realm of feline communication. The illustrator's role in organizing and presenting this information artistically has been instrumental in enriching the understanding of both researchers and viewers alike. This aspect of our study highlights the profound connection between scientific observation and artistic representation, as Butina (2020) emphasized, where scientific accuracy and artistic interpretation converge to provide a deeper understanding of the subject matter. 3.2 Enhancing Viewer Understanding Through Artistic Interpretation The illustrative aspect of our research plays a pivotal role in making complex scientific data more accessible and understandable to a broader audience. By translating intricate behavioural patterns into visual formats, we facilitate a better comprehension of feline behaviour among pet owners, veterinary professionals, and the general public. This approach contributes to academic discourse and has practical implications for improving human-feline interactions and welfare. 3.3 Future Directions This study's methodologies and findings open avenues for further å. Future studies could explore applying these techniques in different contexts or with diverse feline populations. The potential for developing more refined behavioural assessment tools and exploring other aspects of feline communication also presents exciting opportunities for continued research in this field. Conclusions This study highlights the integral role of CatFACS and scientific illustration in advancing our understanding of feline behaviour and communication. By integrating these methodologies, we have gained novel insights into feline communication, significantly impacting welfare and human-cat relationships. This research contributes to academic knowledge and offers practical applications for veterinary professionals, feline behaviourists, and cat owners, fostering a deeper appreciation and understanding of our feline companions. Future research directions include • applying these techniques in different contexts or with diverse feline populations, • aiming to develop more refined behavioural assessment tools and • exploring other aspects of feline communication. This study serves as a foundation for further exploration of felines' rich emotional lives, bridging the gap between scientific analysis and visual interpretation. Proceedings of 10 th Socratic Lectures 2024 165 of 165 Funding: This research was not funded. Conflicts of Interest: The author declares no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Belli M, de Oliveira AR, de Lima MT, et al. Clinical validation of the short and long UNESP-Botucatu scales for feline pain assessment. Peer J. 2021;9:e11225. DOI: 10.7717/peerj.11225 Butina M. Mala likovna teorija. 2003. Založba Debora, Ljubljana, Slovenija. Case LP. Canine and Feline Behavior and Training: A Complete Guide to Understanding Our Two Best Friends. 2010. Delmar Cengage Learning, England. Caeiro CC, Burrows AM, Waller BM. Development and application of CatFACS: Are human cat adopters influenced by cat facial expressions? Applied Animal Behaviour Science 2017; 189: 66 –78. DOI:10.1016/j.applanim.2015.04.001 Christiansen J. Visualizing Science: Illustration and Beyond. 2018. https://blogs.scientificamerican.com/savisual/visualizing-science-illustration-and-beyond/ Evangelista MC Watanabe R, Leung V .S. Y. et al. Facial expressions of pain in cats: the development and validation of a Feline Grimace Scale. Sci Rep. 2019; 9: 19128. DOI: 10.1038/s41598 -019-55693-8 Gould J. Scientific Illustration: Balancing Creativity and Precision. Nature Careers Podcast, November 2023. https://www.nature.com/articles/d41586-023-03391-x Male A. Illustration: A Theoretical and Contextual Perspective. 2017. Bloomsbury Publishing, London, UK. Montag C, Panksepp J. Primary Emotional Systems and Personality: An Evolutionary Perspective. Frontiers in Psychology. 2017; 8. DOI: 10.3389/fpsyg.2017.00464 Reid J, Scott EM, Calvo G et al. Definitive Glasgow Acute Pain Scale for Cats: Validation and Interv ention Level. Veterinary Record. 2017; 180: SP2016-EP2016. DOI: 10.1136/vr.104208. Slice DE. Geometric Morphometrics. Annu Rev Anthropol. 2007; 36: 261 -281. DOI:10.1146/annurev.anthro.34.081804.120613 Stanton L, Sullivan M, Fazio JA. Standardized Ethogram for the Felidae: A Tool for Behavioral Researchers. Applied Animal Behaviour Science 2015; 173: 88–95. DOI: 10.1016/j.applanim.2015.04.001. P1 A multiplex GC-MS/MS method for the quantitative analysis of active ingredients in Ginkgo biloba derivatives Zsolt Árva1,2, Ani Barbulova1, Immacolata Fiume1, Maneea Moubarak1,3, Gabriella Pócsfalvi1 1Laboratory of Extracellular Vesicles and Mass Spectrometry, Institute of Biosciences and BioResources, Italian National Research Council; 2Faculty of Science and Technology, University of Debrecen, Hungary; of Agriculture, Damanhour University, Egypt Contact: Gabriella Pócsfalvi: gabriella.pocsfalvi@ibbr.cnr.it 3Faculty OUTLINE AND BACKGROUND This study investigates the quantities of these primary ginkgospecific ingredients in the methanol extract of ginkgo nuts, ginkgo cells from suspension and callus cultures, micro- and nanovesicles isolated from seeds as well as a commercial supplement (Figure 2). A previous study compared different brand products and found Bilobalide content 2,8-3,2 mg and Ginkgolides (A, B, C) content 2,9-3,2 mg using LC-ESIMS/MS [1]. Another study analysed Ginkgotoxin content in Ginkgo biloba seed by LC-MS/MS and determined 174 ppm Ginkotoxin values [2]. While there have been LC-MS/MSbased studies on terpene lactones that investigates commercial pharmaceutical products containing standardized extracts, no GC-MS/MS method is available for the simultaneous analysis of terpene lactones and ginkgotoxin. Ginkgo biloba (GB) is among the most studied herbal drugs. GB leaf extract standardized to contain 24% Ginkgo Flavone Glycosides and 6% Terpene lactones represents the only herbal alternative to synthetic antidementia drugs in the therapy of Alzheimer's disease and cognitive decline. GB seeds are used in traditional Chinese medicine to heal stomach pain, asthma, tuberculosis, and cognitive dysfunction, and in everyday life as a food snack. Here, our goal was to develop a multiplex analytical method based on gas chromatography-tandem mass spectrometry (GC-MS/MS) for the simultaneous quantitation of five terpene lactones (Ginkgolide A, B, C, J and Bilobalide) and Ginkgotoxin (Figure 1) in different Ginkgo biloba extracts. Figure 1: Chemical structures of Ginkgolides A, B, C, J, Bilobalide and Ginkgotoxin EXPERIMENTAL METHODS Callus and cell suspension cultures were established using GB seed‘s embryo (Figure 2). Micro- and nanovesicles (MVs and NVs) were isolated by differential ultracentrifugation from the homogenate of GB seeds (Figure 3). GB supplement was from Gloryfeel (Figure 4). Five biological replicates were prepared of the suspension and callus cultures, seeds and tablet, and triplicates for MVs and NVs samples. Dry and wet samples from suspension culture and callus were also prepared. Sample homogenization was performed in methanol using a Tissuelyser (Qiagen) at 30 Hz, 5 x 1 min. Samples were centrifuged at 15000 x g for 20 min and the supernatants were vacuum dried and then further dried under nitrogen flow. Stable isotope labelled sugar alcohol, D-Mannitol-13C6 (600 ng) was added as internal standard and samples were derivatized using 50 µL silylation agent BSTFA:TMCS=99:1, 35 µL pyridine at 70 °C for 1 hour at 500 rpm. Figure 2: GB seed‘s embryo, callus and cell suspension cultures 200nm GC-MS/MS analysis were performed using a Thermo Scientific Trace 1300 GC coupled to a TSQ 8000 Duo mass spectrometer instrument equipped with an electron ionization (EI) ion source, triple quadrupole ion analyzer (Figure 5). Separation was performed using a TG-SQC 30 m, 0,25 mm x 0,25 µm capillary column. GC parameters; measurement time: 35 minutes, column flow: 1,2 ml/min (helium), injection volume: 1,0 µL, injection type: splitless, inlet temperature 230 °C. MS parameters; transfer line temperature: 280 °C, ion source temperature: 250 °C. GC temperature ramp; initial temperature: 70 °C hold for 1 min, ramp rate 1: 10 °C/min to 280 °C, ramp rate 2: 6 °C/min to 313 °C, ramp rate 3: 0,6 °C/min to 314 °C, ramp rate 4: 6 °C/min to 316 °C, ramp rate 5: 2,4 °C/min to 318 °C, ramp rate 6: 6 °C/min to 320 °C hold for 2 min. Figure 4: Gloryfeel GB supplement Figure 3: GB seeds RESULTS ✓We have set up a multiplex GC-MS/MS MRS quantitative analysis (Figure 5 and 7) for Compound the simultaneous determination of six GB secondary metabolites (Figure 1) related to the GB bioactivity. The results of our quantitative analysis are reported in Table 3. ✓Based on our analysis we found that the commercial supplement contained 7,77% terpene lactones per tablet. ✓The seed samples contained 50 ppm Ginkgolide A and 14 ppm Ginkgolide B, but Ginkgolide C and J were below LOQ. ✓Methanol extracts of Ginkgo biloba cells from callus and cell suspension, MVs and NVs Ginkgotoxin Mannitol 13 C6 Bilobalide Ginkgolide A contained considerably less bilobalides and ginkgolides than the seed and supplement extracts. Ginkgolide B ✓we found that microvesicles have 269 ppm, nanovesicles 61 ppm, suspension culture in Ginkgolide C dry condition 3 ppm, seed 266 ppm and tablet 37 ppm Ginkgotoxin . Ginkgolide J Table 1: Precursor / product ion’s m/z as well as collision energy values for Figure 5: GC-MS/MS Compound RT (min) Ginkgolide A Ginkgolide B Ginkgolide C Ginkgolide J Bilobalide Ginkgotoxin m/z Collision Energy Precursor / (eV) Product Ion 280,1 / 206,1 10 295,1 / 280,1 10 426,1 / 264,1 10 426,1 / 336,14 10 299,1 / 271,1 10 398,3 / 223,1 10 537,2 / 187,0 25 537,2 / 391,2 10 625,3 / 479,2 10 625,3 / 597,2 5 713,3 / 567,1 10 713,3 / 595,1 10 478,3 / 264,2 10 478,3 / 422,2 10 27,53 28,20 28,35 27,60 21,41 15,40 Calibration Range (ug) points 5 0,50-50 5 0,50-50 5 0,50-50 5 0,50-50 6 0,05-30 7 0,01-20 R2 Slope 0,9939 16,15 0,9965 14,44 0,9982 14,55 0,9804 14,76 0,9917 343,02 0,9944 13502,73 Table 2: Calibration data (retention times, ranges, R2, slopes) Figure 6: Representative MRM spectra of Ginkgotoxin, Bilobalide, Ginkgolide A, B, C, J and internal standard, the D-Mannitol-13C6 (MS quantitation) Weight Ginkgotoxin Sample (mg) (ppm) Nanovesicle (wet) 0,3 61 Microvesicle (wet) 0,3 269 Suspension culture (wet) 123,3-190,9 < LOQ Suspension culture (dry) 11,97 3 Callus (wet) 127,8-182,9 < LOQ Callus (dry) 12,41 < LOQ Seed (dry) 49,20-52,04 266 Tablet (dry) 274,05 37 Bilobalide (ppm) < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 11303 Ginkgolide A Ginkgolide B Ginkgolide C Ginkgolide J (ppm) (ppm) (ppm) (ppm) < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 50 14 < LOQ < LOQ 55525 4309 5008 1514 Table 3: Results of the analysis of nanovesicle, microvesicle, suspension culture, callus, seed and tablet – avarage values CONCLUSION The ginkgolides are known to inhibit platelet aggregation, while the bilobalide protects against neuronal death caused by brain ischemia. On the other hand, ginkgotoxin is a neurotoxin structurally related to vitamin B6, naturally occurring in GB nuts and leaves. These natural ingredients are present at very different quantities in GB derivatives (cells, MVs, NVs, seeds and supplement) and their simultaneous quantitation is neither trivial nor easy. To achieve our goals, we have improved the separation of 4 ginkgolides in the GC timescale by the careful setting of temperature ramping. We have selected and optimized the SRM precursor and product ions transitions as well as the collision energy for each analyte. We found that the terpene lactone level of in vitro grown ginkgo cells, MVS and NVs is lower than the detection limit. In MVs we detected more ginkgotoxin than in NVs. References: [1] DOI: 10.1016/j.jpba.2009.05.040 [2] DOI: 10.1093/jaoac/83.6.1313 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant acronym “greenEV”, grant agreement No 895579.. Production and morphological properties of water-based cellular particles containing biologically active molecules from European spruce (Picea abies) needle homogenates by experimental and theoretical approaches “Jožef Stefan” Institute Ljubljana, Slovenia Alvaro Iglesias Batalla1, Aina Defior Pons1, Maruša Mur2, Laureano Schofs3,4, Veronika Kralj-Iglič5, Maja Ponikvar-Svet5, Melita Tramšek6, Gašper Tavčar6, Marko Jeran6,* 1“Instituto de Education Sexundria Cap de Llevant, Menorca, Spain; 2”Jožef Stefan” Institute, Department of Condensed Matter Physics, Ljubljana, Slovenia; of Pharmacology, Faculty of Veterinary Medicine, Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, Argentina; 4Veterinary Research center of Tandil (CIVETAN), CONICET-CICPBA-UNCPBA, Tandil, Argentina; 5University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia; 6”Jožef Stefan” Institute, Department of Inorganic Chemistry and Technology, Ljubljana, Slovenia *Correspondence: M.J.; marko.jeran@ijs.si 3Laboratory EXPERIMENTAL METHODS OUTLINE Plants have the incredible ability to synthesize many of the active ingredients on which today’s medicines and therapies are based. They are able to defend themselves against external influences (enemies) by releasing the corresponding molecules in individual parts of the plant. The European spruce (Picea abies) (Figure 1) contains a different composition of biologically active and complex substances. The composition of these substances depends on various factors, e.g. the geological composition of the soil, the climate, the time of harvest, the availability of nutrients, etc. (Jeran, et al. 2021). The samples of spruce needles were collected and processed by mechanical deformation of the plant tissue. The homogenates (in distilled water (dH2O), saline (0.9% NaCl) and 9,0% NaCl) were aged at room temperature and after achieving boiling point using microwaves. The formation of the resulting particles was investigated (Figure 3). Due to the accelerated development of medical diagnostics and the development of delivery systems, such as vesicles (liposomes), researchers began to look for alternative sources in the field of natural sources (Figure 2). Since cell particles of smaller size can also be found in plant samples and represent an alternative to biological fluids, research today also focuses on the development of cost-effective and reproducible methods for the isolation and characterization of these particles. (a) (b) Figure 2: (a) Lipid content of the foam after homogenization of spruce needles (Jeran, et al. 2023). (b) A phospholipid bilayer as the main component of the cell membrane forming cellular vesicles (de Reggi, et al. 2021). Figure 1: Example of structures of biologically active compounds Figure 3: Flow chart of the experimental design for the preparation in European spruce (Jeran, et al. 2021). of homogenates from European spruce needles. RESULTS Figure 5: (a)-(d): dH2O-Based homogenates: larger particles, aggregates and agglomerates were observed both during homogenization at room temperature and under microwave conditions. (c)-(d): A higher content of smaller cell particles of different sizes and shapes was observed under microwave homogenization conditions. (e)-(h): The homogenate in saline solution (0.9% NaCl) still contains particle clusters and agglomerates. (h): Some cellular particles with a double membrane resembling a cellular vesicle can also be seen. (i)-(j): The 0.9% and 9.0% NaCl solution allowed the opening of larger agglomerated particles and aggregates. (h): Some cellular particles with a double membrane resembling a cellular vesicle can also be seen. (i)-(j): The 0.9% and 9.0% NaCl solution allowed the opening of larger agglomerated particles and aggregates. (e)-(f): A lower concentration of cell particles and many particle clusters and agglomerates with the formation of mainly spherical particles with small dimensions can be seen. (k)-(l): A lot of material with smaller dimensions was formed under microwave conditions, slightly less if physiological conditions are included. We discovered mainly morphologically heterogeneous structures of different sizes and (mathematical) shapes. Figure 7: Cellular particles with smooth contours: (a)-(c) in a spruce needle homogenate on a model of a 9.0% NaCl solution, using microwaves, and (d)-(f) models of theoretical shapes corresponding to minimal energy membrane shapes (Jeran et al., 2023). The shapes of the particles formed in the homogenates of the spruce needles, which correspond to the theoretical shapes, were particularly pronounced in the homogenates prepared with the aid of microwaves. With appropriate addition of salt (0.9 and 9.0% NaCl), the above particles were also formed at room temperature. In general, several stomatocytes and discocytes were observed. CONCLUSION Figure 6: Spruce needle homogenate "aged" for 1 week at room temperature ((a)-(b)) showed an increased number of small cellular particles. An increased concentration of membrane-enclosed cell particles was found in the “aged” homogenate sample that was treated under microwaves before aging ((c)-(d)). They exhibited different morphological shapes and heterogeneous dimensions. REFERENCES: 1. Gazvoda de Reggi M, Malavašič U, Jeran M, Penič S. Open science: Development of open platform for giant unilamelar vesicles electroformation. Proc. Soc. Lect. 2021; 6: 99-113. DOI: https://doi.org/10.55295/PSL.2021.D.014 2. Jeran M, Pečan LI, Barrios-Francisco R. Interdisciplinary insight on European Spruce (Picea abies): Biologically active compounds and their usage. Proc. Soc. Lect. 2021; 6: 64-70. DOI: https://doi.org/10.55295/PSL.2021.D.009 3. Jeran M, Romolo A, Spasovski A, Hočevar M., et al. Small cellular particles from European spruce needle homogenate. Int. J. Mol. Sci. 2023, 24, 4349. DOI: https://doi.org/10.3390/ijms24054349 The salt concentration has a decisive influence on the formation and morphology of the formatted cell particles. Compared to NaCl media, the dH2O media shows the formation of aggregates in the form of particle agglomerates, which can be easily broken down into smaller units with the assistance of thermal energy via microwaves. The formation of particles with a double-membrane was observed in all media used as well as in a oneweek “aged” sample prepared in dH2O. Spruce needle homogenates contain particles with a minimal membrane energy that are similar in shape to particles of blood cells. This research was supported by the Slovenian Research and Innovation Agency (ARIS) through the Core Findings no. P1-0045 and P3-0388. The authors are thankful to Anja Jančič, Frida Benedek, Polonca Krašovec, and Milena Žohar for their technical support. P2 P3 MINIMALLY INVASIVE SURGERY : ENDOSCOPIC SPINE SURGERY Amar Handanagic, Faculty of Medicine, University of Ljubljana, prof. dr. Oguz Karaeminogullar, Head of Department : Handanagić Amar1, Orthopedics Karaeminogullar Oguz2 Clinic Bayındır Hospital and Traumatology 1University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia,2Bayindir Hospital, Orthopedic and Traumatology Clinic, Bayindir, Turkey INTRODUCTION PROCEDURE BENEFITS • Minimally invasive surgery (MIS) of the spine is an essential component of spine care. One type of MIS is endoscopic spine surgery (ESG), which allows surgeons to use a percutaneous approach to treat spinal pathology rather than the wide-open surgical exposure. The main objective of ESG is to minimize tissue trauma while achieving equivalent results to open spine surgery. • The approach should be carried out percutaneously with a small working cannula, which serves two important purposes -This allows the use of local anesthesia or conscious sedation, thereby minimizing anesthesia risks and minimizes tissue damage. Second, access to spinal pathology should be gained via a working window to permit the endoscope and cannula to pass through to the spinal canal. Additionally, endoscopic visualization can provide a clear view of the spinal pathology. • First, the procedure involves less tissue trauma due to a smaller skin incision, less tissue dissection, and less bone removed to access the spinal canal. Second. reduced operative time, quicker recovery due to less tissue damage, less use of narcotic medication after surgery, fewer wound complications, and quicker return to work. The mean hospital stay was 12 hours and average time to return to work was 6.79 weeks. FIGURE 1: Intraoperative view of an endoscopic disectomy at L5-S1 below a previous L5-S1 fusion FIGURE 2: A large disc fragment is removed through a working sheat FIGURE 3: Anatomical details are well demonstrated including decompressed NR, the PLL and the maternal disc FIGURE 4: Anteroposterior ﬂuoroscopic image of the area of the decompressed exiting nerve GRAPH 1: Visual analogue pain score for leg pain GRAPH 2: Visual analogue pain score for back pain REFERENCES Ahn, Y. M., Lee, U. M., Kim, W.-K. M., & Keum, H. M. (November 2018). Five-year outcomes and predic ve factors of transforaminal full-endoscopic lumbar disectomy. PubMed(Observa onal study). Yeung, C. M., Yu-Po, L. M., Oh, M. M., & Bha a, N. M. (28. February 2022). Endoscopic Spine Surgery. Contemporary Neurosurgery, 3. P4 Decoding the facial expressions of cats: Insights through scientific illustration Pšenica Kovačič Minami Kat, Centre for Cat Culture, Ljubljana, Slovenia; Contact: Pšenica Kovačič: psenakovacic@gmail.com METHODS OUTLINE The prospective doctoral thesis delves into the intricate realm of feline visual communication. This poster centers on the analysis of facial expressions employing scientific illustrations and advanced research methods. The objective is to elucidate the complexities associated with how cats convey emotions and intentions through facial cues. The examination of feline facial expressions (Illustration 1)contributes to the fields of neuroethology and psychobiology, fostering a more nuanced comprehension of feline behavior. By addressing the scientific facets of feline visual communication, the endeavor aims to bridge the divide between empirical research and practical insights into interactions with cats. Preparation for Illustration: Blending Art and Science in Capturing Cat Expressions Engaging in scholarly discussions, collaborating closely with scientists and peers, and understanding the intricacies of feline behavior lay the foundation for this artistic endeavor. Before illustrating cat facial expressions, the imperative step involves gathering photo-video references curating a comprehensive repository capturing cats in various emotional states. Illustrators employ resources like anatomy books, 3D models, and animation apps, complemented by ﬁrsthand observation of real cats (Illustration 2) or exploration of taxidermied specimens. This amalgamation ensures the sustained vitality of the project over months or even years. From Cat Visual Communication to Art Representation The focus can be on shapes (2D), bodies (3D), or forms, representing the highest level of organization of visual elements or how something is expressed. Visual variables provide constant sensory characteristics and emotional qualities relating to size, weight, position, direction, and the relationship between position and weight. These elements assist in examining and interpreting observations, facilitating the transformation of these observations into visual expressions (Illustration 3). They are crucial in simplifying complex information and identifying parallels and patterns, enabling an effective explanation through suitable visual language. Illustration 1: Portrait of domestic cat (P. Kovačič). Example: The cat's face is a form composed of visual elements (Illustration 4) or facial organs: ears, eyes, nose, muzzle, whiskers, fur, and wrinkles, interconnected in relationships and distances. Each element possesses its position, distance, shape, size, thickness, direction, weight, structure, texture, contrast, and gestures or expressions. These elements transform and move in response to external stimuli or internal emotional states, revealing the cat's soul's manifestation in the body. By sketching, simplifying, or using diagrams, we interpret expressions, deciding how to present a part (Illustration 5) or the entire face based on our study's essential aspects. 200nm CONCLUSION Our research signiﬁcantly contributes to understanding feline behavior, emphasizing the critical role of advanced methods and scientiﬁc illustrations, particularly in exploring facial expressions. Thorough research is imperative due to the complex nature of feline communication. Achieving compassionate coexistence requires an interdisciplinary approach, merging science and art to educate and foster harmony among individuals passionate about felines. In conclusion, our study deepens the understanding of feline behavior, highlighting the urgency for in-depth exploration, especially in facial expressions, utilizing advanced analytical methods and scientiﬁc illustrations. Feline communication, 500nm with its captivating yet intricate nature, calls for a nuanced approach to comprehending their subtle nonverbal signals. Illustration 4: Visual elements (P. Kovačič). Illustration 5: Nose texture and form study (P. Kovačič). RESULTS ARE ILLUSTRATIONS 1µm 200nm Illustration 3: Cat facial expression (P. Kovačič). Illustration 2: Cat in motion (P. Kovačič). Sodium disufonate salt as a water-soluble fluorescence-active fluorescein analogue and its activity in labelling urothelial urinary bladder cancer cells Patrik Pečavar Nežmah1, Mateja Erdani Kreft1, Marko Jeran2,* 2“Jožef 1Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Slovenia; Stefan” Institute, Department of Inorganic Chemistry and Technology, Ljubljana, Slovenia *Correspondence: M.J; marko.jeran@ijs.si “Jožef Stefan” Institute Ljubljana, Slovenia EXPERIMENTAL DESIGN OUTLINE Bladder cancer is the ninth most common cancer in the world. Its detection is difficult, as the clinical symptoms can be very similar to other not so severe bladder diseases. Often it is detected too late, and can consequently prove fatal. In addition to the latter, bladder cancer cells appear multifocally, i.e. in many separate spots in the bladder, so it is difficult to completely remove them by operation. The described fact indicates a high probability of a further recurrence of the disease (Zupančič, 2011). Some cases include in situ and non-invasive papillary carcinoma (the Ta and Tis stages). The Register of Cancer of the Republic of Slovenia (RRRS) for all years follows a rule that T1T4 bladder cancers are included in the incidence of cancer, especially in situ cases with non-invasive papillary carcinoma (Zupančič, 2011). In the urinary bladder, urine is only temporarily stored until being conveniently excreted. Since its composition should not change during rest, the bladder has a specifically built epithelium – urothelium. Urothelium covers most of the lower part of the urinary tract, i.e. proximal urethra, urinary bladder, ureter and renal pelvis (Romih et al., 2005). Its task is to form a bloodurine barrier that prevents the returning of toxins, water, ammonia and ions from the urine into the blood (Erdani Kreft, et al. 2010). The aim of this study was to synthesize the fluorescent active substance SUF (sodium 3',6'dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-4',5'-disulfonate (according to reference (Kostaskova, 2010)) and to test its application in medicine. The aim of the work is to find new methods and reagents that could replace expensive and time-consuming procedures for obtaining fluorescent dyes in the field of cell biology. According to the innovative synthetic approach, SUF is suitable for large-scale production because the production route is fast, simple and very efficient (high yield). We believe that the proposed method could improve efficiency in tumor detection and advance cell biology in general. We hypothesised that the compound SUF could be used to distinguish cancerous urothelial cells from healthy (normal) cells by differences in fluorescence intensity and/or distribution (Jeran, et al. 2019). Synthesis of fluorescence activity SUF Characterization data of SUF ✓ 1H- & 13C-NMR (300 MHz, DMSO-d6) ✓ λmax (UV/Vis, 10-4 M in EtOH) = 485 nm ✓ Fluorescence = 504 nm ✓ TLC (CH2Cl2/ MeOH, 9:1), Rf = 0.72 Figure 1: Urinary bladder. R E S U L T S Figure 2: SUF is soluble in polar and non-polar media. (a) SUF solution in water-based (polar) media before application to the cells, and (b) its fluorescence under UV-light. The fluorescence of SUF was only detected in polar solvents and was stronger at lower concentrations (0.1 mg/mL or 100 μg/mL). The light emission was stronger under UV-light at 366 nm. Figure 3: Comparison of the viability of normal (NPU) and cancer cells (T24) treated with SUF dye. Each set represents an independent experiment. A repetition of the viability test. We repeated the counting with the hemocytometer method and the analysis sixty times, as NPU cells grow slowly and are more demanding to cultivate. Due to the relatively small number of cells, the deviation of the results can be very large, which in our case was solved by calculating the mean values of several repetitions. The average values of all four cell viability analyses indicate that the SUF dye at a concentration of 100 µg/mL does not affect the viability of the urothelial cells and may be suitable for further in vitro and in vivo applications. Figure 4: Fluorescence microscopy. Cancer and normal urothelial cells labelled with SUF. A: Cells labelled with DAPI (blue nuclei), fragmented nuclei (yellow thin arrows; chromosomes of cells in mitosis (yellow thick arrows). B: Cells labelled with DAPI (blue nuclei) and SUF (green). Cancer urothelial cells with fragmented nuclei (yellow thin arrows) and membrane compartments with the compound SUF (red arrows), a cancer urothelial cell with normal nucleus and diffuse labelling with SUF (orange thin arrow). C: The same field of view as in A and B, but only SUF is visible. D and E: Differentiated normal urothelial cells labelled with SUF (green). In E, the area within the white box is enlarged. The white arrows show the SUF labelling of the apicolateral part of the cells. Scale bar 50 μm in A, B, C and E, and 100 μm in D. SUF mostly accumulated in the areas between the apical and basolateral cell membranes, resulting in a contrast between the fluorescent light emitted from inside and outside the cell. Since SUF does not fluoresce in non-polar media, we ruled out the possibility of binding to the lateral cell membranes. In cancer cells, we assumed that the dye enters their interior by endocytosis and causes labelling of the endocytotic compartments. From this we concluded that under in vitro conditions it is possible to distinguish between cancer cells and normal urothelial cells. REFERENCES: 1. Erdani Kreft M, Hudoklin S, Jezernik K, Romih R. Formation and maintenance of blood–urine barrier in urothelium. Protoplasma 2010, 246 (1–4), 3–14. DOI: https://doi.org/10.1007/s00709010-0112-1. 2. Jeran M, Pečavar Nežmah P, Erdani Kreft M. Synthesis of a water-soluble fluorescent active compound and its potential use for labeling of cancerous urothelial bladder cells. Proc Soc Lec 2019, 1, 49–62. ISBN 978-961-6808-94-1. 3. Kotaskova M. Synthesis of new xanthene derivatives. Doctoral Disertation, Am Fachbereich Chemie, Pharmazie und Geowissenschaften der Johannes Gutenberg-Universität, Mainz, 2012. 4. Pečan LI, Pečavar Nežmah P, Jeran M. Fluorescenca kot pomembno orodje v aplikativni naravoslovni znanosti (Engl. Fluorescence as an important tool in applied natural science). Okapi 2022, 2, 18–19. ISSN 2784-5850. 5. Romih R, Korosec P, de Mello W, Jezernik K. Differentiation of epithelial cells in the urinary tract. Cell Tissue Res 2005, 320 (2), 259–268. DOI: https://doi.org/10.1007/s00441-004-1005-4. 6. Zupančič M. Rak sečnega mehurja (Engl. Urinary bladder cancer); Splošna bolnišnica Slovenj Gradec, Oddelek za urologijo: Velenje, 2011. CONCLUSION The compound showed potential for use in the field of medical applications. Due to its chemical structure, it was found to be non-toxic and can be used as a cell marker. We have shown experimentally that the compound does not damage the cells and can differentiate between normal and cancerous urothelial cells. The results of the viability tests showed no differences between the controls (cells grown without SUF) and the samples (cells grown in the presence of SUF). Examination of the cells with a fluorescence microscope, which showed cells at different stages of cell division, confirmed that SUF does not inhibit cell division. Based on the results obtained, we assume that the compound SUF is not cytotoxic. However, further studies are required for a definitive answer. This research was supported by the Slovenian Research and Innovative Agency (ARIS) through the Core Findings no. P1-0045 and P3-0108. P5 P6 Lithium-ion battery project: ESTEAM perspectives Aleksandra Roganović1, Yelena Istileulova2 1Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia; 2Ambassador of STEAM (European Commission, Deloitte, EISMEA) Contact: Yelena Istileulova: yelena.istileulova@gmail.com EXPERIMENT CONCLUSION This poster was initiated within the framework of the ESTEAM Mentorship programme, with the objective of fostering coachability, self-awareness, and allocating time to understand each other´s needs. OUTLINE The participants were engaged in discussions on various topics of lithium through the lens of Mendeleev´s table of elements that are mutually beneficial for the mentor and mentee's development. Throughout the experiment, a fascinating revelation emerged: just as Mendeleev's Periodic Table delineates the elements in Chemistry, a comparable structure can be identified in Music. ESTEAM stands for Entrepreneurship, Science, Technology, Engineering, Arts and Mathematics) have been and will be organised in 19 EU Member States with the aim to boost women and girls’ competences, inspire them, and give them the chance to connect with likeminded peers. ESTEAM online communities for women and girls are being built to sustain the momentum of the events and to allow them to keep learning and connecting with their peers online. Throughout the communities’ workshops and activities, participants learn about ESTEAM career paths and are inspired by women role models who thrive in these domains, while developing not only their digital and entrepreneurial competences but also their collaboration, creativity, problem-solving and public speaking skills. The inception of the experiment was driven by the aspiration to uncover connections between Science and Art. Lithium-ion batteries, ubiquitous in our daily lives, powering smartphones and laptops that have transformed modern society, are now poised to revolutionize the transportation sector through electric cars, buses, and bikes. Upon closer examination of these systems, it becomes apparent that, by adhering to certain principles, Mendeleev's lithium corresponds to a measure in the Periodic Table of Music Notation. The research focused on a pivotal component of batteries – lithium – tracing its origins and discovery back to Mendeleev's Periodic Table. RESEARCH Figure 1. How lithium-ion battery works? 200nm 200nm 1mm Figure 2. Metal content by battery chemistry There's antimony, arsenic, aluminum, selenium And hydrogen and oxygen and nitrogen and rhenium And nickel, neodymium, neptunium, germanium And iron, americium, ruthenium, uranium Europium, zirconium, lutetium, vanadium And lanthanum and osmium and astatine and radium And gold protactinium and indium and gallium And iodine and thorium and thulium and thallium There's yttrium, ytterbium, actinium, rubidium And boron, gadolinium, niobium, iridium And strontium and silicon and silver and samarium And bismuth, bromine, lithium, beryllium, and barium There's holmium and helium and hafnium and erbium And phosphorus and francium and fluorine and terbium And manganese and mercury, molybdenum, magnesium Dysprosium and scandium and cerium and cesium And lead, praseodymium and platinum, plutonium Palladium, promethium, potassium, polonium And tantalum, technetium, titanium, tellurium And cadmium and calcium and chromium and curium There's sulfur, californium and fermium, berkelium And also mendelevium, einsteinium, nobelium And argon, krypton, neon, radon, xenon, zinc and rhodium And chlorine, carbon, cobalt, copper, tungsten, tin and sodium These are the only ones of which the news has come to Piggy And there may be many others but they haven't been discovered Tom Lehrer – The Elements (Live Film from Copenhagen in 1967) Figure 3. Mendeleev's Periodic Table. References: How does a lithium battery work. Credits: Sarah Harman and Charles Joyner https://www.energy.gov/eere/articles/how-does-lithium-ion-battery-work https://youtu.be/AcS3NOQnsQM?feature=shared https://www.classicfm.com/discover-music/periodic-table-of-musical-notation/ https://www.nstgroup.co.uk/music-resources Kresse, C., Bastian, D., Bookhagen, B., Frenzel, M. (2022). Commodity top news: Recycling of lithium-ion batteries in Germany and Europe Manthiram, A. (2020). A reflection on lithium-ion battery cathode Chemistry. https://doi.org/10.1038/s41467-020-15355-0 Periodic Table. Image by Google. The Lithium Voice. Volume 1, 2023 ISSN 2755-354X. https://lithium.org/lithium/ Figure 4. Periodic Table of Music Notation This study has been financially supported by CEEPUS student exchange program and by the Ministry of Education, Science and Technological Development through the project no. 451-03-68/2020-14/200156: “Innovative scientific and artistic research from the FTS (activity) domain“ P7 20mm P8 New Approaches for Testing the (Geno)Toxic Activity of Nanoparticles In Vitro Iza Rozman1, Alja Štern1, Bojana Žegura1 National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Večna pot 121, 1000 Ljubljana, Slovenia 1 OUTLINE In Vitro Cell Models In the last decade, the production and use of nanomaterials has grown tremendously, and as a result, so has human exposure to these materials. Since human exposure to nanoparticles is inevitable much attention has been drawn to nanoparticle toxicology – especially to the potential acute and chronic adverse effects that nanoparticles may cause on humans – mostly because nanoparticles due to their high surface-to-volume ratio, high reactivity, and unique physical, chemical, and biological properties exhibit a greater risk of toxicity than the corresponding bulk material. Advantages of a 2D cell model: ● ● ● ● The study of nanoparticle adverse effects and toxicity is referred to as nanotoxicology and even though exposure to nanoparticles is increasing, information on their toxicological properties remains inadequate, especially when it comes to the question which in vitro model would be most appropriate for nanoparticle toxicity assessment. inexpensive well established model easier maintenance easy performance of functional tests Disadvantages of a 2D cell model: lacking complex cell-cell and cell-environment interactions unrestricted access to essential compounds ● no cellular microenvironment ● different cell morphology and molecular mechanisms compared to in vivo ● ● Within the Department of Genetic Toxicology and Cancer Biology at the National Institute of Biology and as part of my PhD research related to the H2020 project NESTOR (GA: 101007629) and J1-4395 project NaNoZymSafe we develop 3D cell models as a new in vitro methodological approach for nanoparticle (geno)toxicity assessment to better understand the impact nanomaterials have on environmental and human health. Methods The in vitro cyto- and genotoxicity of tested iron-based core-shell nanoparticles were assessed in the HepG2 cell line, using the CellTiter-Glo® assay (left) and the comet assay (right). HepG2 cells in a monolayer. Advantages of a 3D cell model: ● ● ● ● ● ● 24 or 96 h 24 or 96 h production of a matrix that promotes tissue-specific cell binding direct cell-to-cell interactions cell-to-extracellular matrix interactions preservation of natural morphology preservation of high viability for several weeks gene and protein expression levels better resemble levels found in vivo Disadvantages of a 3D cell model: CellTiter-Glo reagent ● ● ● ● ● slower culture formation more complex procedures higher reagent cost fewer commercially available tests more demanding analysis Results The results cannot be published due to still ongoing testing and certain reservation about data publishing since the data gathered form this research are part of a still unpublished article. Spheroid in cell media (no exposure to nanoparticles). Spheroid exposed to nanoparticles at a concentration of 10 µg/mL. Funding: The H2020-MSCA NESTOR project (101007629) and ARIS P1-0245 and J1-4395 Created with BioRender Poster Builder P9 Discovery of a New Candidate Type I Toxin-Antitoxin System in the Cyanobacterium Microcystis aeruginosa PCC 7806 Matija Ruparčič1, Marko Dolinar1 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia 1 Introduction Aim of the study Toxin-antitoxin (TA) systems are genetic elements that encode a stable toxin and a labile antitoxin. In type I systems, the antitoxin is a short non-coding RNA that prevents toxin translation by forming base pairs with the toxin mRNA (Fig. 1). The majority of type I toxins are short membrane proteins. Some disturb membrane integrity through pore formation while others cause nucleoid condensation1. skupna tarča Since M. aeruginosa contains the highest number of type II TA systems of all bacteria, we set out to figure out whether it also contains type I systems. Our aim was to: Protein 1. Perform a bioinformatic search for hypothetic type I TA systems in M. aeruginosa PCC 7806. The notorious bloom-forming cyanobacterium Microcystis aeruginosa is especially interesting in terms of TA systems as it contains the highest number of type II TA loci of all bacteria2. Despite this, only one locus has been experimentally studied up to now3 while type I TA systems have not been found in this species so far. antitoxin toxin RNA 2. Test the effect of expression of predicted type I toxins on Escherichia coli cell growth. DNA 3. Indirectly confirm the existence of the predicted cognate type I antitoxins by confirming the biologic activity of their promoters. Figure 1: Schematic representation of toxin translation control in type I TA systems. Bioinformatic search Our bioinformatic analysis (Fig. 2) revealed fifteen candidate type I TA loci. We chose the six most prospective loci for experimental characterisation: BH695_0311, BH695_0320, BH695_3336, BH695_4017, BH695_4989, and BH695_5020. Step 1: Finding hypothetical type I toxins 5364 annotated proteins from M. aeruginosa PCC 7806SL (GenBank: CP020771.1) hyp. T filter by type I toxin characteristics Step 2: Finding hypothetical cognate type I antitoxins Step 3: Assessing the predicted type I TA locus predict antitoxin promotersc and terminatorsd predict toxin promotersc and terminatorsd hyp. T (1) length < 90 aa, (2) at least one membrane helixa, (3) no similarity to non-toxic proteinsb hyp. T hyp. A hyp. A Does the predicted antitoxin RNA overlap with the predicted toxin mRNA? Figure 2: Schematic representation of our bioinformatic search for hypothetic type I TA systems in M. aeruginosa PCC 7806. using TMHMM 2.0, busing BLASTP, cpredicted using bTSSfinder and BPROM, dpredicted using ARNold and FindTerm. apredicted Expression of six predicted type I toxins Expression of BH695_0320 and BH695_4017 has a negative effect on E. coli cell growth. Expression of the remaining four predicted type I toxins has no effect (Fig. 3). 1,5 1,5 OD600 2 OD600 2 IPTG added 0,5 0 0 0 60 120 180 240 300 360 420 480 → MsoA1 is expressed under the control of PmsoA1. 2 IPTG added 1,5 IPTG added 1 0,5 0 0 540 60 120 180 240 300 360 420 480 540 600 0 60 120 180 t [min] t [min] empty pET28b(+) pET28b(+)-SrnB pET28b(+)-BH695_0311 pET28b(+)-BH695_0320 pET28b(+)-BH695_3336 empty pET28b(+) pET28b(+)-SrnB pET28b(+)-BH695_4989 pET28b(+)-BH695_5020 240 300 360 Biologic activity of MsoA1 antitoxin promoters B) positive control 1,5 OD600 1,5 OD600 Reporter: beta-lactamase (grants cells resistance to the antibiotic ampicillin). 2 A) negative control 1 1 0,5 0,5 0 0 0 120 240 360 480 600 720 840 0 120 240 360 480 600 720 840 t [min] C) test sample OD600 1,5 1 Recombinant E. coli BL21(DE3) were cultured in LBC medium with different concentrations of ampicillin. t [min] Beta-lactamase is under the control of: A) no promoter B) native Pbla promoter C) predicted PmsoA1 promoters Cells carrying the plasmid where beta-lactamase is under the control of the predicted PmsoA1 antitoxin promoters grow in media containing high concentrations of ampicillin (Fig. 4). Legend (ampicillin concentration in medium): → PmsoA1 antitoxin promoters are biologically active. 2 0,5 0 0 120 240 360 480 600 720 840 0 mg/mL 1 mg/mL 5 mg/mL 15 mg/mL t [min] Figure 4: Growth curves of E. coli BL21(DE3) in LBC media with different concentrations of ampicillin. 420 480 t [min] pET28b(+)-MsoT1 empty pET28b(+) pET28b(+)-srnB/srnC pET28b(+)-MsoT1 pET28b(+)-SrnB pET28b(+)-msoT1/msoA1 Figure 3: Growth curves of E. coli BL21(DE3) pLysS expressing different predicted type I toxins under the control of the inducible PT7 promoter. Cells carrying empty pET28b(+) served as a negative control (light blue). Cells expressing the SrnB type I toxin from E. coli under the control of PT7 served as a positive control (red). 2 Expression of MsoT1 has a delayed toxic effect when the plasmid also contains MsoA1 under the control of its predicted PmsoA1 promoter (Fig. 4). 2,5 1 0,5 Focus on BH695_4017→ renaming to MsoT1 and its predicted cognate antitoxin to MsoA1. OD600 Recombinant E. coli BL21(DE3) pLysS were cultured in M9CK minimal medium. IPTG was added at OD600 0.4 – 0.6 to induce predicted type I toxin expression. 1 Further characterisation of MsoT1/MsoA1 540 Figure 4: Growth curves of E. coli BL21(DE3) pLysS expressing MsoT1 under the control of the inducible PT7 promoter with the plasmid also containing MsoA1 under the control of its predicted PmsoA1 promoter (dark green) and without the presence of MsoA1 (light green). Cells carrying empty pET28b(+) served as a negative control (light blue). Cells expressing the SrnB type I toxin from E. coli under the control of PT7 served as a positive control (red). Cells expressing SrnB under the control of PT7 with the plasmid also containing its cognate antitoxin SrnC under the control of its native PsrnC promoter served as an additional positive control (pink). Conclusion Our results suggest MsoT1/MsoA1 to be a novel type I TA system as well as the first type I TA system discovered in cyanobacteria. Future prospects Test the effect of expression of predicted type I toxins on cyanobacterial cell growth → compare the results with the ones from E. coli. Confirm the existence of the predicted cognate type I antitoxins directly (RT-PCR, in vitro translation). References 1. D. Jurėnas, N. Fraikin, F. Goormaghtigh, L. Van Melderen: Biology and evolution of bacterial toxin–antitoxin systems. Nat Rev Microbiol 2022, 20, 335-350. 2. Y. Xie, Y. Wei, Y. Shen, X. Li, H. Zhou, C. Tai, Z. Deng, H.-Y. Ou: TADB 2.0: an updated database of bacterial type II toxin–antitoxin loci. Nucleic Acids Res 2018, 46, D749–D753. 3. M. Klemenčič, A. Halužan Vasle, M. Dolinar: The cysteine protease MaOC1, a prokaryotic caspase homolog, cleaves the antitoxin of a Type II toxinantitoxin system. Front Microbiol 2021, 12, 1–13. P10 Characterization of EVs Subpopulations From CIMmultus® EV Using PATfix® System Vrabec Katja1*, Goričar Blaž1, Božič Darja1, Mavri Ana1, Raspor Andrej1, Novak Valentina1, Petrović Koshmak Ivana1, Leskovec Maja1 1 * Sartorius BIA Separations d.o.o, Ajdovščina, Slovenia Corresponding author: katja.vrabec@sartorius.com 4. Determination of EV size using PATfix MALS Introduction Cells release extracellular vesicles (EVs) of different sizes and intracellular origin. Due to their heterogenicity, the isolation of the target EV population from a mixture of supernatant-derived particles can be challenging. Anion exchange chromatography (AEX) exploits the negative charge on EV surface molecules for binding to the positively charged solid phase. CIMmultus® EV, an AEX chromatography monolith column, can separate EVs in subpopulations based on charge and offers insight into the heterogenicity of particles. Besides the availability of preparative tools for separation, combining multiple orthogonal and complementary characterization tools is crucial for defining the EV product of interest. In this work, we used a multiple-detector PATfix® system for the analysis of CIMmultus EV-fractionated samples. Samples were analyzed for the presence of EV-related tetraspanins using the fluorescence detector. PATfix MALS 3609 detector was used for the analysis of particle-containing samples and calculation of particle sizes. The radius of particles was determined from SEC chromatograms in PATfix software (Figure 4). Additionally, NTA analysis was performed on Nanosight 300 (Malvern Panalytical) (Figure 5). Despite the fact that fractions vary in particle charge and surface antigens, all fractions contained particles in the size range of EVs, emphasizing the importance of using multiple approaches for the characterisation these particles. A) 60 nm bead standard 1. Sample preparation Native HEK293T cells and engineered HEK293T CD63-eGFP cells were cultivated on FACT III (Sartorius) microcarriers in Ambr250 bioreactor (Sartorius) in DMEM growth media containing 10% FBS, then switched to production media without FBS for 2 days. For harvesting, the vessels were removed from the system and microcarriers were allowed to settle by gravity. Conditioned media was collected and filtered through Sartopure PP3 capsule 1.2 µm (Sartorius). 100 mL of each conditioned media sample was treated with nuclease and re-buffered, then applied to a CIMmultus EV column with a column volume (CV) of 1 mL and 2 µm channels. The chromatographic separation was performed on an Akta Pure 25 M system (Cytiva), equipped with a MALS detector. The loading buffer composition was 25 mM BTP 100 mM NaCl 2% sorbitol pH 7.5 and the elution buffer 25 mM BTP 2 M NaCl 2% sorbitol pH 7.5. The columns were loaded at 10 CV/min flow rate and eluted at 2 CV/min. EVs were eluted at different salt concentrations and collected as shown in Figure 1. 3 1 2 3 4 HEK293T CD63-eGFP EVs 2 HEK293T EVs 1 B) HEK293T CD63-eGFP Harvest HEK293T CD63-eGFP harvest 5 C) HEK293T CD63-eGFP fraction 2 HEK293T CD63-eGFP fraction 2 Figure 1: Preparative chromatograms from CIMmultus EV column. Vesicles were separated in salt gradient and collected in fractions labeled HEK293T EV fraction 1-3 and HEK293T CD63-eGFP EV fraction 1-5. 2. PATfix analytical setup Figure 4: Light scattering chromatograms of the SEC void volume and corresponding LS charts. The particle radii were measured at the LS 90° peak. (A) SEC chromatogram of 60 nm polystyrene spherical bead standard (Postnova, Part No. Z-PS-POS-000-0,06) and the corresponding LS chart using form factor sphere. The standard was used for detector normalization. B) SEC chromatogram of HEK 293T CD63-eGFP EV Harvest and the corresponding LS chart using Zimm plot. All chromatographic samples were characterized using this plot. C ) SEC chromatogram of HEK 293T CD63-eGFP fraction 2. LS Peak Area (mV*s*mL) FL peak area (mV*s*mL) CD9 CD63 CD81 60 100 80 R2=0.9987 R2=0.9974 R2=0.9960 R2=0.9974 120 R2=0.9961 80 R2=0.9973 140 R2=0.9970 R2=0.9996 R2=0.9994 R2=0.9995 160 Particle diameter in HEK293T CD63-eGFP samples 60 40 SEC chromatograms were analyzed in PATfix software. Fluorescence (FL) and light scattering (LS) peak area were calculated from SEC void volume peak area, sample volume, and dilution. The distribution of FL and LS peaks in chromatographic samples was plotted in Figure 3. Light scattering shows the particles are distributed in two main peaks in both the native and engineered cell lines. The EVs with the highest fluorescence signal response were eluted in the first fraction. CD9, CD63 and CD81-positive particles in B) Total particles in HEK293T samples A) HEK293T samples 350000 120000 300000 250000 200000 150000 100000 50000 0 100 180 Diameter (nm) Diameter (nm) 3. Evaluation of EV distribution in chromatographic samples R2=0.9995 120 R2=0.9918 140 Figure 2: PATfix system setup used in these experiments (Created with BioRender.com). Samples are contained in an autosampler, before being injected into a SEC column. Absorbance, fluorescence, and light scattering are monitored and the data is interpreted in PATfix software. An example SEC chromatogram shows the void volume peak where particles are eluted. B) Particle diameter in HEK293T samples 160 R2=0.9916 A) void volume peak R2=0.9919 Samples were analyzed using the PATfix system (Sartorius BIA Separations) and size exclusion chromatography (SEC). HEK EVs, engineered to express CD63-eGFP on the surface were analyzed unlabeled. Native HEK EVs were labeled with anti-CD9, anti-CD63, and anti-CD81 FITC-conjugated antibodies (BioLegend). Each sample was injected to a SEC column TSKGEL G4000SWXL (Tosoh Bioscience) where the labeled EVs were separated from the excess antibody. The analysis was performed on the PATfix system with the following detectors: UV cell with 50 mm optical path length, fluorescence detector, and MALS 3609 detector. Absorbance was monitored at 260 and 280 nm, fluorescence was set to excitation and emission peaks of the used fluorophores, and light scattering was monitored at 9 angles. The running buffer was 50 mM MES, 150 mM NaCl, 0.05 % Poloxamer, pH 6.5, and the flow rate was 1 mL/min. 40 20 20 0 0 MALS Diameter (nm) NTA Mean Diameter (nm) MALS Diameter (nm) NTA Mean Diamater (nm) NTA Mode Diameter (nm) NTA Mode Diamater (nm) 100000 80000 60000 40000 Figure 5: Particle diameter determined by MALS and NTA for A) HEK293T samples and B) HEK293T CD63-eGFP samples. 20000 0 5. Conclusions CD63-eGFP particles in HEK293T CD63eGFP samples 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 CD63-eGFP D) LS Peak Area (mV*s*mL FL Peak Area (mV*s*mL) C) Total particles in HEK293T CD63-eGFP samples 250000 200000 ▪ CIMmultus EV chromatographic column fractionates supernatant-derived vesicles in multiple populations that differ in size and surface antigen composition ▪ PATfix triple detector setup enables rapid sample characterization for both native and engineered EV samples 150000 ▪ Fluorescence detector allows for relative quantification of fluorescent EVs or EVs labeled with a fluorescent antibody 100000 ▪ Combining MALS detector with SEC analytics enables relative quantification of particles in samples and determination of particle size 50000 0 6. Acknowledgements Figure 3: Distribution of FL and LS peak area in representative samples LOAD (total sample loaded to CIMmultus EV column), FT (flow-through, the unbound fraction), Fr. 1-5 (elution fractions containing EVs) and CIP (cleaning-in-place): A) and B) HEK293T samples, C) and D) HEK293T CD63eGFP samples. © Sartorius BIA Separations 2023, all rights reserved Acknowledgments: We would like to thank Bernd Giebel and his group from University Hospital Essen, Institute of Transfusion Medicine, for kindly providing the HEK293 CD63-eGFP cell line. P_2023_08

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0TH INTERNATIONAL SYMPOSIUM, LJUBLJANA, 9. DECEMBER 2023 PEER REVIEWED PROCEEDINGS, PART II EDITED BY: VERONIKA KRALJ-IGLIČ, YELENA ISTILEULOVA AND ANNA ROMOLO FACULTY OF HEALTH SCIENCES, UNIVERSITY OF LJUBLJANA

0TH INTERNATIONAL SYMPOSIUM, LJUBLJANA, 9. DECEMBER 2023 PEER REVIEWED PROCEEDINGS, PART II EDITED BY: VERONIKA KRALJ-IGLIČ, YELENA ISTILEULOVA AND ANNA ROMOLO FACULTY OF HEALTH SCIENCES, UNIVERSITY OF LJUBLJANA

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