SOCRATIC LECTURES 10TH 9.-11.12.2023 S O C R A T I C L E C T U R E S 10TH INTERNATIONAL SYMPOSIUM, LJUBLJANA, 9. DECEMBER 2023 PEER REVIEWED PROCEEDINGS PART I EDITED BY: VERONIKA KRALJ-IGLIČ, YELENA ISTILEULOVA AND ANNA ROMOLO FACULTY OF HEALTH SCIENCES, UNIVERSITY OF LJUBLJANA Proceedings of 10th Socratic Lectures 2024 Socratic Lectures 10th International Symposium, Ljubljana, December 9, 2023 Peer Reviewed Proceedings, Part I 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: photos from Archive Zupanič, Archive Romolo, Archive Griessler Bulc, Free royalities internet; photo on page 147 by Drago Videmšek; reproduction of painting »Girl with the red can« by Ivan Vaupotič, reproductions of other paintings by Marguerite de Saint Champs Image on the front page: Silvia Romolo Garcia First digital edition. Publication is available online in PDF format at: https://www.zf.uni-lj.si/images/stories/datoteke/Zalozba/Sokratska_10_I.pdf http://ebooks.uni-lj.si DOI: 10.55295/PSL.2024.I 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 186758147 ISBN 978-961-297-266-0 (PDF) I Proceedings of 10th 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-lj- si.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 10th 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 Štuhec M, Physiotherapy approach for treating De Quervain tenosynovitis during and after pregnancy 12.30 - 12.45 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 III Proceedings of 10th Socratic Lectures 2024 13.15 - 13.30 Vauhnik R, Zuil Escobar JC, Martinez Cepa C, Functional approach to musculoskeletal injuries in physiotherapy: A COIL project in Physiotherapy 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 IV Proceedings of 10th Socratic Lectures 2024 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 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 V Proceedings of 10th Socratic Lectures 2024 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 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 VI Proceedings of 10th Socratic Lectures 2024 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 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 VII Proceedings of 10th Socratic Lectures 2024 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 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 10th Socratic Lectures 2024 Editorial 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. Roberta Schmid took her diploma in Organ and Organ Composition at the Musical Conservatoire “S. Pietro a Maiella” in Naples with the mentorship of prof. A.M.Robilotta and then improved her technique with maestros L. Ghielmi, E. Kooiman, M. Radulescu, D. Roth, K, Schnorr, L. F. Tagliavini, M. Torrent, P. Westerbrinck and W. Zerer. She specialized in Italian and German Organ Music Interpretation at the Musical Academy in Pistoia and in Gregorian Chant at the International Study Centre of Gregorian Chant in Cremona. She performed as a soloist and in chamber formations in XXVIII Festival Internazionale di Noale, Rassegna Antichi organi di Piacenza, Associazione Alessandro Scarlatti di Napoli, Associazione Studi Mezzogiorno, Associazione Ricercare, Accademia Organistica Campana, Rassegna organistica internazionale di Avezzano, Rassegna organistica di Pescara, Settembre Organistico Fabrianese, Rassegna organistica veneta “Musica nell'Agordino” , Rassegna “Musicalia” in Pavia, Festival Organistico Internazionale Città di Senigallia, Rassegna Organistica di Fiemme (Trento), Rassegna organistica della Svizzera Italiana, International festivals at Notre Dame de Compassion in Paris, Merano Cathedral, Amalfi Cathedral, S. Vitale's Basilica in Ravenna, S. Ambrogio' s Basilica in Milan and in Mexico City. She has recorded a CD of Bach and pre-Bach music sponsored by the Goethe-Institut, Italy. Presently she is Artistic Director of the Festival "Musica intorno all'organo" at the St. Maria della Rotonda Church in Naples and organist of Mascioni mechanical organ at St. Maria della Rotonda Church and at Santa Chiara's Basilica in Naples. www.robertaschmid.com Program of the organ recital of Roberta Schmid J.S. Bach: Wachet auf, ruft uns die Stimme, BWV645 N. Bruhns: Praeludium in e minor (the Great) P. A. Yon: Toccatina for Flute W. A. Mozart: Fantasia in f minor K594 J.S. Bach: Nun komm, der Heiden Heiland, BWV 659 C. Franck: Choral in b minor C. M. Widor: Toccata from Symphony V The next day, scientific symposium took place online. The plenary lecture on mesenchymal stem cell extracellular vesicles was donated by prof. Bernd Giebel from Essen, Germany, followed by 17 scientific sections. The symposium was organized by the Centre of lifelong learning of the Faculty of Health Sciences, University of Ljubljana in collaboration with Universities from Georgia, Spain, Ukraine and Kazakhstan. There were about 200 participants from 21 countries. The subjects of interest were medicine, natural sciences, green transition, digital transition, business, social sciences and education. There were two sections on extracellular particles. At the closing of the symposium Agata Angelika Sojecka and Borys Urbanowicz from Poland performed The swan (by C. Saint Saens) and Apres un reve (By G. Faure) on harp and viola, respectively. 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. Concert on December 11 at 18.00 was in person at Pallace Kazina, Ljubljana IX Proceedings of 10th Socratic Lectures 2024 Program of the Concert Classical music, Concert Hall of Julij Betteto J.S. Bach: Wachet auf, ruft uns die Stimme BWV645, organ: Roberta Schmid J.S. Bach: Toccata and fugue in d minor BWV 565, organ: Roberta Schmid D. Cimarosa: Concerto for two flutes and piano, flutes: Anita Prelovšek and Sara Brumat, piano: Elena Startseva Somun D. Cimarosa: Two sonatas for piano, piano: Matic Bogataj M. Ravel: Blues, Moderato from Sonata No.2 for violin and piano, violin: Branko Brezavšček, piano: Elena Startseva Somun J Pachelbel: Canon in D, piano: Elena Startseva Somun G. Caccini: Ave Maria, soprano: Kaya Tokuhisa, organ: Jana Jamšek Anonymus: Amazing grace, soprano: Kaya Tokuhisa, organ: Jana Jamšek Contemporary music, Club Kazina P. de Seneville, Marriage d'amour, classical ballet: Darja Eržen, Jelena Ficzko, Lara Elisabeth Lee, piano: Jana Jamšek A. von Sultanova: JC Maxwell, Moja domovina, JR Oppenheimer, voice and piano: Yelena Istileulova A. Chailleux: Andante et Allegro, saxophone: Teodor Herem F. Kuchler: Concertino for violin and piano, violin: Živa Bratko, piano: Barbara Smrke In the club Kazina, another memorable social event was held to create a network uniting scientists and artists. Wellcome to the next Socratic lectures. Contact kraljiglic@gmail.com or annaromolo@gmail.com Veronika Kralj-Iglič, Yelena Istileulova and Anna Romolo X Proceedings of 10th Socratic Lectures 2024 CONTENTS TEXTS 1. Tadel Kocjančič Špela: Extracorporeal Oxygenation (ECMO) in Patients with Acute Respiratory Failur………. 1 2. Kocjančič Ema, Kocjančič Boštjan: From Hip Arthrosis to Hip Replacement Surgery – Total Hip Endoprosthesis …………………………………………………………………………………………………………….. 6 3. Kolar Matic, Mavčič Blaž, Kralj-Iglič Veronika, Antolič Vane: Preserved Proximal Femoral Bone Stock Volume in Total Hip Arthroplasty Significantly Reduces the Risk for Periprosthetic Fractures. A Novel Modelling Technique and Preliminary Clinical Results.…………………………………………………..13 4. Šimundić Metka: Urinary Tract Infections in Dogs and Cats ……………………………………………………......23 5. Urbančič Špela, Vauhnik Renata: Physiotherapy Approach for Treating Lateral Epicondilalgia..…….………... 29 6. Vrbinc Katarina Nina, Vauhnik Renata: The Impact of Sleep Deprivation on Physical Activity……..………… 35 7. Štuhec Maša, Vauhnik Renata: Physiotherapy Approach for De Quervain Tenosynovitis Dur-ing and After Pregnancy ……………………………………………………………………………………………………... 42 8. Ošlak Andreja, Vauhnik Renata: Effects of Therapeutic Approaches in Treating Varus Malalignment of the Knee Joint …………………………………………………………………………………………………………...47 9. Berry Maxence, Arko Matevž, Romolo Anna, Brložnik Maja, Mrvar Brečko Anita, Korenjak Boštjan, Iglič Aleš, Kadunc Kos Vesna, Kruljc Peter, Nemec Svete Alenka, Erjavec Vladimira, Kralj-Iglič Veronika : Validation of Interferometric Light Microscopy for Assessment of Extracellular Particles in 250 samples of Diluted Plasma: Preparing the Path for Future Clinical Practices……………………………………… 54 10. Beletić Anđelo, Duvnjak Orešković Ivana, Pribić Tea, Krištić Jasminka, Lauc Gordan: Glycosylation Research in Bovines-the Significance and Recent Updates…………………………………………………………... 60 11. Schlosser Gitta, Molnár Adrienn, Papp Dávid, Gellén Gabriella, Virág Dávid, Ludányi Krisztina, Dalma-di-Kiss Borbála, Arko Matevž, Iglič Aleš, Svete Nemec Alenka, Erjavec Vladimira, Kralj-Iglič Veronika: Omics Mass Spectrometry Analysis of Canine Plasma………………………………………. 67 12. Arko Matevž, Hočevar Matej, Korenjak Boštjan, Iglič Aleš, Kralj-Iglič Veronika: Extracellular Particles from Equine Milk ……………………………………………………………………………………………….74 13. Mišič Jančar Jakob, Schofs Laureano, Pečan Luka Irenej, Oblak Tine, Sánchez Bruni Sergio, Kuhar Aleš, Ponikvar-Svet Maja, Tavčar Gašper, Hupli Aleksi, Jeran Marko: An Insight into the Use of Cannabis in Medical and Veterinary Dermatological Applications and its Legal Regulation…………… 79 14. Árva Zsolt, Barbulova Ani, Fiume Immacolata, Moubarak Maneea, Pocsfalvi Gabriella: A Multiplex GC-MS/MS Analysis for the Quantitative Moni-toring of Bilobalide, Ginkgolides and Ginkgotoxin in Ginkgo biloba-Derived Products and Biomaterials………………………………………………………………... 93 15. Birk Luka, Junkar Ita, Rener-Sitar Ksenija: Use of Gaseous Plasma for Dental Applications……………………110 16. Bonin Luka, Jeromen Matic, Jeran Marko: Endangered Butterflies and their Conservation: the Decline of Parnassius apollo and Phengaris spp. in Europe and Slovenia…………………………………………………...117 17. Cepec Eva, Griessler-Bulc Tjaša, Istenič Darja: Uncovering algae biomass potentials: from wastewater to biostimulants………………………………………………………………………………………………………….. 127 XI Proceedings of 10th Socratic Lectures 2024 18. Horvat Barbara, Mušič Branka: Green Transition in Slovenian Building and Civil Engineering Industry: 10 Years of Research on Alkali-Activated Materials and Alkali-Activated Foams…………………....136 19. Verbič Anja, Golja Barbara, Stres Blaž, Likozar Blaž, Novak Uroš: Unveiling PFAS-free Solutions for Hydrophobic and Oleopho-bic Textile Coatings………………………………………………………………….148 20. Hribešek Matija, Kulovec Simon: Design Optimization and Fatigue Evaluation of Wood Composite Gears …………………………………………………………………………………………………………158 21. Mušič Branka, Sever Škapin Andrijana: Weathering Effects on Cellulose Acetate Microplastics from Discarded Cigarette Butts ………………………………………………………………………………………..168 22. Protasenko Olga: Digital education in the development of the company’s human capital………...…………... 176 23. Salun Maryna, Zaslavska Kateryna: Strategies for Resilience in a Dynamic World: from VUCA to BANI …………………………………………………………………………………………………….185 24. Dybach Inna: Features of the Formation of Cross-Cultural Communication Skills in IT Business……………...191 25. Vlasenko Tetiana, Nemashkalo Karina, Shapovalova Inga: Digital Transformations: Trends and Perspectives Worldwide ………………………………………………………………………………………………...197 26. Dostiyarova Alima: Innovative Approaches in Teaching Business Courses: Start-Up Simulation in the Change Management course; students’ experience ………………………………………………………….. 204 27. Kocjančič Rok: Economic and Financial Analysis of Artificial Intelligence's Impact on Law and Legal Profession …………………………………………………………………………………………………… 211 28. Haborets Olha, Kushkovyi Artem: OSINT-Technologies: Applications and Challenges in the Dig-ital Age ……………………………………………………………………………………………………… 217 29. Nurtazina Maral, Nurseitov Azamat: Innovative Approaches to Language Learning through the Lens of Use Artificial Intelligence ………………………………………………………………………………….222 30. Prelovšek Anita: Music in the Life and Works of Boris Leonidovich Pasternak ...………………………………..227 XII Proceedings of 10th Socratic Lectures 2024 1 of 234 Invited lecture/Review Extracorporeal Oxygenation (ECMO) in Patients with Acute Respiratory Failure Tadel Kocjančič Špela1,* 1. University Medical Center Ljubljana, Slovenia; Faculty of Medicine, University of Ljubljana, Slovenia * Correspondence: Špela Tadel Kocjančič; spela.kocjancic@gmail.com Abstract: Extracorporeal membrane oxygenation (ECMO) is a method for oxygenation and removal of carbon dioxide in patients with respiratory failure in whom we cannot achieve that with Citation: Tadel Kocjančič Š. standard methods of invasive mechanical ventilation. The method works as an extracor-Extracorporeal oxygenation poreal bypass of blood, which we take from one central vein, then the blood goes through (ECMO) in patients with acute an oxigenator and returns oxygenated and free of carbon dioxide to another central vein. respiratory failure. Proceedings of Socratic Lectures . The system is run by a pump with steady non-pulsatile flow. We use two standard ECMO 2024, 10, 1-4. types. For respiratory failure alone we use veno-venous ECMO and for heart failure we https://doi.org/10.55295/PSL.2024.I1 use veno-arterial ECMO. We can also combine more types of ECMO according to the patientś needs. We started using ECMO in 2009, with the number of patients is increasing Publisher’s Note: UL ZF stays neutral with regard to jurisdictional every year. The majority of patients with the need of ECMO has influenza, pneumococcal claims in published maps and insti-pneumonia or infection with Legionella species. We also used ECMO a lot during COVID-titutional affiliations. 19 pandemic. With this new method even the patients who would otherwise (even 10 years ago) die of respiratory failure, have a greater possibility to survive. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Keywords: ECMO – extracorporeal membrane oxygenation, respiratory failure, can-Attribution (CC BY) license nulas, non-pulsatile blood flow (https://creativecommons.org/licens ______________________________________________________________________________ es/by/4.0/). Proceedings of 10th Socratic Lectures 2024 2 of 234 1. Introduction ECMO means ExtraCorporeal Membrane Oxygenation (extracorporeal life support, extracorporeal lung assist). It is a method for oxygenation and removal of carbon dioxide in patients with respiratory failure in whom we cannot achieve that with standard methods of invasive mechanical ventilation. ECMO is not a treatment and does not correct the underlying pathological insult. We use two types of ECMO: venovenous (VV ECMO) for respiratory support and ven-oarterial (VA ECMO) for respiratory and hemodynamic support. We can also combine these two methods according to the patientś needs. ECMO was developed in 1970 for respiratory support in acute respiratory failure. It was used for adding O2 and removing CO2, but it was performed with cannulation of femoral artery and vein and limited to 5 days. In 1979, the first randomized trial showed very high complication rates and survival rates no higher than mechanical ventilation alone. In early 1980s the switch to veno-venous was made, but after that the technology was largely abandoned due to bleeding and poor outcomes (Mosier et al., 2015). There was a boom of ECMO during the global pandemic of influenza H1N1 in 2009-2010 and again in 2020-2022 during global pandemic od COVID-19. Several centers reported survival benefits for ARDS secondary to influenza. Two retrospective case-control studies showed lower mortality when transferred to ECMO centers and lower mortality among younger patients who received ECMO (Schmid et al., 2015; Australia Group, 2009; Luyt et al., 2012; Zangrillo et al., 2013; Bednarczyk et al., 2014). 2. VV ECMO circuit VV ECMO is used in isolated failure of the lungs, unresponsive to optimal ventilatory support and medical treatment. It is used in children and in adults. It consists of large conduit tubing, a blood pump, an oxygenator and additional components which may include a heat exchanger, monitors, and alarms. Two ECMO cannulas are inserted percutaneously, ultrasound or diascopy guided. Usually they are inserted through the jugular or femoral vein, one for draining deoxygenated blood from the venous system (superior or inferior vena cava) to ECMO circuit, the other one for returning the oxygenated blood to the right atrium. Their size is dependant on the height of the patient. The cannulas are manufactured from biocompatible silicone polyurethane polymer, which may be coated with polymers that may reduce platelet activation and the inflammatory response at the blood-cannula interface (Pavlushkov et al., 2017; Lequier et al., 2013; Kohler et al., 2013). The cannulas are constructed with a reinforced stainless steel (SS) wire. Wire reinforcement of the cannula walls is used to prevent kinking or collapse. A rigid cannula introducer is made of polyvinylchloride with an embedded SS rod (Pavlushkov et al., 2017; Lequier et al., 2013; Kohler et al., 2013; Beckmann et al., 2011; Medtronic, 2012). Surface coatings are applied on the cannula to reduce the activation of the clotting; control of blood clotting is mandatory during the extracorporeal life support. The surfaces can be coated with heparin, bivalirudin, or tethered-liquid perfluorocarbon (Leslie et al., 2014; Wyss Institute, 2014; Yang et al., 2012). A blood pump and an oxygenator can be joined together or separate. For adult respiratory failure the largest size ECMO machine is used which includes large conduit tubing, a blood pump capable of at least 5 L/min, and an oxygenator with rated flow over 5 L/min. A blood pump has a steady non-pulsatile flow (ELSO Guidelines, 2017). In the absence of lung function, VV access can supply all metabolic oxygen requirements. Patient ‘s PaCO2 is controlled by the sweep gas flow (ELSO Guidelines, 2017). 3. Indications and contraindications for veno-venous ECMO Indication for VV ECMO is potentially reversible acute respiratory insufficiency, which may be hypoxemic, hypercapnic (pH less than 7.0), and ireversible lung damage in patients who have already been accepted for lung tranplantation as a bridge to transplantation (ELSO Guidelines, 2017). Contraindications may be absolute or relative. Absolute contraindications are pre-existing conditions, incompatible with recovery, for example advanced Proceedings of 10th Socratic Lectures 2024 3 of 234 lung disease without possibility for transplantation, other end stage diseases. Relative contraindications are uncontrollable bleeding, very poor prognosis from primary condition, age above 65, advanced septic shock, imunosuppresive treatment (ELSO Guidelines, 2017). 4. Complications Complications on ECMO are very common and are associated with a significant increase in morbidity and mortality. These complication could be related to the underlining pathology needed ECMO, or of the ECMO condition itself . Patients on ECMO are very ill patients with respiratory insufficiency, usually with multiple organ disfunction. The complications are common and include complications during insertion of the cannulas, during treatment with ECMO and during decannulation. Complications due to the insertion of cannulas are hemorrhage, venous spasm, arrhyth-mias, ruptured blood vessels, pneumothorax. During treatment with ECMO there can be adsorption and sequestration of drugs and blood cells on artificial materials, hemorrhage, blood clots, HIT (heparin induced thrombocytopenia), infections. There can also be neurologic complications, intracranial bleeding, infarction, cerebral edema. It is important to re-alize that these findings may be a consequence of the condition that prompted ECMO, rather than a complication of the ECMO process (Mateen et al., 2011; Mehta & Ibsen, 2013; Lidegran et al., 2007). These may be partially due to systemic heparinization, thrombocytopenia, coagulopathies, or systolic hypertension. Complications due to the removal of the cannulas are hemorrhage, venous spasm and ruptured blood vessels. 5. Outcome of patients on veno-venous ECMO According to the published data, 67% of patients with acute respiratory failure treated with ECMO were weaned off ECMO and 52% survived to hospital discharge (ELSO Guidelines, 2017; Hemmila et al., 2004; Noah et al., 2011; Pham et al., 2013). This was also confirmed by the CESAR study (Peek et al., 2009), which demonstrated that referral to an ECMO center significantly improves recovery and survival from severe ARDS. 6. Conclusion Introduction of ECMO in the treatment of patients with acute respiratory failure has improved survival of these patients. ECMO use has risen since H1N1 influenza outbreak in 2009 and until now ECMO centres still continue to report high survival rates for patients who are supported with ECMO. It is important to emphasize that that patients, treated with ECMO return to a reasonable quality of life, although after prolonged rehabilitation. Conflicts of Interest: The author declares no conflict of interest. References 1. Australia, New Zealand Extracorporeal Membrane Oxygenation (ANZ ECMO) Influenza Investigators. Davies A, Jones D, Bailey M, et al. Extracorporeal membrane oxygenation for 2009 influenza A(H1N1) acute respiratory distress syndrome. JAMA. 2009; 302:1888–95. DOI: 10.1001/jama.2009.1535 2. Beckmann A, Benk C, Beyersdorf F, et al. ECLS working group. Position article for the use of extracorporeal life support in adult patients. Eur J Cardiothorac Surg. 2011; 40:676–80. DOI: 10.1016/j.ejcts.2011.05. 011 3. Bednarczyk JM, White CW, Ducas RA, Golian M, et al. Resuscitative extracorporeal membrane oxygenation for in hospital cardiac arrest: a Canadian observational experience. Resuscitation. 2014; 85:1713–1719. DOI:10.1016/j.resuscitation.2014.09.026 4. ELSO Guidelines for Cardiopulmonary Extracorporeal Life Support Extracorporeal Life Support Organization, Version 1.4 August 2017 Ann Arbor, MI, USA. www.elso.org . Access 26.01.2024. Available from: https://www.elso.org/ecmo-resources/elso-ecmo-guidelines.aspx Proceedings of 10th Socratic Lectures 2024 4 of 234 5. Hemmila MR, Rowe SA, Boules TN, et al. Extracorporeal life support for severe acute respiratory distress syndrome in adults. Ann Surg 2004; 240: 595-605. DOI: 10.1097/01.sla.0000141159.90676.2d 6. Kohler K, Valchanov K, Nias G, Vuylsteke A. ECMO cannula review. Perfusion 2013; 28:114–24. DOI: 10.1177/0267659112468014 7. Leslie DC, Ingber DE, Waterhouse A, et al. A bioinspired omniphobic surface coating on medical devices prevents thrombosis and biofouling. Nat Biotechnol. 2014; 32:1134–40. DOI: 10.1038/NBT.3020 8. Lequier L, Horton SB, McMullan DM, Bartlett RH. Extracorporeal membrane oxygenation circuitry. Pediatr Crit Care Med. 2013; 14(5 Suppl 1):S7–12.DOI: 10.1097/PCC.0b013e318292dd10 9. Lidegran MK, Mosskin M, Ringertz HG, et al. Cranial CT for diagnosis of intracranial complications in adult and pediatric patients during ECMO: Clinical benefits in diagnosis and treatment. Acad Radiol. 2007; 14:62-71. DOI: 10.1016/j.acra.2006.10.004 10. Luyt CE, Combes A, Becquemin MH, Beigelman-Aubry C, et al. Long-term outcomes of pandemic 2009 influenza A(H1N1)-associated severe ARDS. Chest. 2012; 142:583–92. DOI: 10.1378/chest.11-2196 11. Mateen FJ, Muralidharan R, Shinohara RT, et al. Neurological injury in adults treated with extracorporeal membrane oxygenation. Arch Neurol. 2011;68:1543-9. DOI: 10.1001/archneurol.2011.209 12. Medtronic. Select 3D® EOPA 3D® Arterial Cannulae [online]. 2012. Eopa 3D® artErial CannulaE - Find your ideal – Medtronic. E-paper. Access 26.01.2024. Available from: https://www.yumpu.com/en/document/read/4692650/eopa-3dr-arterial-cannulae-find-your-ideal-medtronic 13. Mehta A, Ibsen LM. Neurologic complications and neurodevelopmental outcome with extracorporeal life support. World J Crit Care Med 2013; 2: 40-47. DOI: 10.5492/wjccm.v2.i4.40 14. Mosier JM, Kelsey M, Raz Y, et al. Extracorporeal membrane oxygenation (ECMO) for critically ill adults in the emergency department: history, current applications, and future directions". Critical Care. 2015; 19: 431. DOI: 10.1186/s13054-015-1155-7 15. Noah MA, Peek GJ, Finney S, et al. Referral to an extracorporeal membrane oxygenation center and mortality among patients with severe 2009 influenza A (H1N1). JAMA. 2011; 306:1659e1668. DOI: 10.1001/jama.2011.1471 16. Pham T, Combes A, Roze H, et al. REVA Research Network. Extracorporeal Membrane Oxygenation for Pandemic Influenza A(H1N1)–induced Acute Respiratory Distress Syndrome. A Cohort Study and Propensity-matched Analysis. Am J Respir Crit Care Med. 2013; 187:276–285. DOI: 10.1164/rccm.201205-0815OC 17. Pavlushkov E, Berman M, Valchanov K. Cannulation techniques for extracorporeal life support. Ann Transl Med. 2017; 5: 70. DOI: 10.21037/atm.2016.11.47 18. Peek GJ, Mugford M, Tiruvoipati R, et al. Efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet. 2009; 374:1351-1363. DOI.org/10.1016/S0140-6736(09)61069-2 19. Schmidt M, Hodgson C, Combes A. Extracorporeal gas exchange for acute respiratory failure in adult patients: a systematic review. Crit Care. 2015; 19:99. DOI: 10.1186/s13054-a015-0806-z 20. Wyss Institute. Bioinspired Coating for Medical Devices Repels Blood and Bacteria [online]. 2014. Access 26.01.2024. Available from: https://wyss.harvard.edu/news/bioinspired-coating-for-medical-devices-repels-blood-and-bacteria/ 21. Zangrillo A, Biondi-Zoccai G, Landoni G, et al. Extracorporeal membrane oxygenation (ECMO) in patients with H1N1 influenza infection: a systematic review and meta-analysis including 8 studies and 266 patients receiving ECMO. Crit Care. 2013; 17:R30. DOI: 10.1186/cc12512 22. Yang Z, Tu Q, Maitz MF, et al. Direct thrombin inhibitor-bivalirudin functionalized plasma polymerized allyla-mine coating for improved biocompatibility of vascular devices. Biomaterials. 2012; 33:7959–7971. DOI: 10.1016/j.biomaterials.2012.07. 050 . Proceedings of 10th Socratic Lectures 2024 6 of 234 Invited lecture/Review From Hip Arthrosis to Hip Replacement Surgery – Total Hip Endoprosthesis Kocjančič Ema 1, Kocjančič Boštjan1,3 1. University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 2. University Medical Centre, Department of Orthopaedic Surgery, Ljubljana, Slovenia, *Correspondence: ema.kocjancic17@gmail.com Abstract: Hip arthrosis is a medical condition that is more commonly present in elderly people that is nowadays also becoming a healthcare burden among the younger, active population. Arthrosis is a process during which cartilage, normally present as a protective factor of the hip joint, gradually wears away, becoming frayed and rough. This results in limited pro-Citation: Kocjančič E, Kocjančič B. tective joint space, which leads to increased friction and rubbing between hip joint compo-From Hip Arthrosis to Hip Replace- nents; acetabulum and femoral bone. Due to arthrosis, patients experience daily pain, lim-ment Surgery – Total Hip Endopros- ited movements and decreased quality of life. Primary treatment of osteoarthritis includes thesis. informing patients of the importance of a healthy lifestyle, including physical activity. As Proceedings of Socratic Lectures . non-surgical alternatives to treating osteoarthritis, orthopaedic surgeons may relieve their 2024, 10, 6-11. patients’ symptoms with pharmacological interventions (paracetamol and NSAIDs) and https://doi.org/10.55295/PSL.2024.I2 in selected cases intraarticular infiltrations. When all this is no longer effective, surgical relief of pain is recommended, including less invasive procedures, such as hip arthroscopy, Publisher’s Note: UL ZF stays neu-hip biopsy, or simple core decompression (CD), or the most invasive, but effective hip retral with regard to jurisdictional placement surgery - total hip arthroplasty (THA). The latter is a procedure during which claims in published maps and insti-diseased and necrotic tissue from the hip joint is removed and replaced with artificial matutional affiliations. terials, the hip endoprosthesis. The aim of this procedure is to reduce pain and improve patient's mobility and quality of life. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and Keywords: hip osteoarthritis, hip arthrosis, hip endoprosthesis, surgery, quality of life. conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licens es/by/4.0/). Proceedings of 10th Socratic Lectures 2024 7 of 234 1. Introduction Hip arthrosis, also referred to as hip osteoarthritis, is a medical condition commonly present in elderly people. The condition occurs over time, as day-to-day activities gradually cause the protective cartilage of the hip joint to wear away (Swiss Medical Network, 2023). During the process of arthrosis, joint cartilage becomes frayed and rough, becoming virtually useless. Consequently, the hip joint has minimal protective joint space which would otherwise prevent rubbing between the acetabulum (socket part of hip joint) and femoral head (ball part of joint) (Foran, 2021). Friction and rubbing of bones in a joint may also cause bone spurs (irregular bone growths on edges of the bone which cause a change in the bone's shape) (Hopkins Medicine Org, 2023). Arthrosis causes patients to experience pain when engaging in daily activities, limiting them in their everyday lives. This causes patients to seek different types of orthopaedic treatment, in hopes of improving their quality of life. 2. Non-surgical treatment options for hip osteoarthritis Osteoarthritis treatment has been vastly changing over the past decades. This is a result of earlier diagnosis, patient education, and more in-depth knowledge of the biology of tissues involved. Patient education includes teaching patients, as well as their families the importance of managing a healthy lifestyle, regular physical activity, balanced diet and weight loss. All this can help the patient stay mobile, relieve pain, and reduce risk of other medical issues or at least prolong the time they live prior to developing conditions such as arthrosis (Dunkin, 2023). Knowledge of hip joint tissues has been key in developing nonsurgical treatments of hip osteoarthritis. These treatments include infiltration of joint with hyaluronic acid (viscosupplementation) or with platelet rich plasma which contains autologous growth factors (Innocenti et al., 2013). Treatment is coupled with pharmacological interventions in the form of paracetamol and non-steroid anti-inflammatory drugs (NSAID). In cases where such treatment is no longer effective, surgical relief of pain is recommended to the patient (Poulsen et al., 2011). 3. Surgical treatment options for hip osteoarthritis Surgical treatments of hip osteoarthritis may be more or less invasive, their main goal being a delay of prosthetic hip replacement surgery. The latter is, despite its effectiveness and patient satisfaction, still considered a very final surgical solution and may be the cause of patient impediments. Less invasive surgical procedures which a surgeon may offer to their patient include hip arthroscopy. Arthroscopy is a general surgical procedure during which a surgeon creates a small incision at the joint area, through which they insert a narrow tube, attached to a fiber/optic video camera. This allows for the surgeon to view the inside of a patient’s joint on a monitor (Staff, 2022). Arthroscopies may be performed on virtually any joint in the human body. Some patients may also seek orthopaedic help due to a condition called avascular necrosis. The latter is a condition where there is necrosis of bone components due to restriction of blood supply. If untreated, avascular necrosis causes osteoarthritis and bone collapse. In such cases, a surgeon may perform hip biopsy or simple core decompression (CD). The procedure is meant to decompress the femoral head in a condition called avascular necrosis (Padmawar & Landge, 2021). It was, for many years, considered highly cost-effective, minimally invasive and had low complication rate in putting off THA. Nowadays, the procedure’s results are debated and controversial, and it is considered outdated, as surgeons no longer perform it except in exceptional circumstances (Sadile et al., 2016). 4. Hip replacement surgery - total hip arthroplasty (THA) Undoubtedly more invasive, but also one of the most common and effective orthopaedic surgical treatment methods for osteoarthritis is hip replacement surgery, or total hip arthroplasty (THA) (Varacallo et al., 2017). It is a procedure during which the orthopaedic surgeon removes diseased or necrotic tissue from the hip joint. This tissue includes bone, cartilage and surrounding muscle and fat tissue. The diseased parts of bone include femoral head and acetabulum, which are replaced with artificial materials, prosthesis (Figure Proceedings of 10th Socratic Lectures 2024 8 of 234 1). The aim of THA procedure is to reduce pain and improve patient's mobility and quality of life (Staff, 2022). The hip endoprosthesis which a surgeon inserts into the patient’s joint is made of two separate components: femoral and acetabular. Femoral component is nowadays made of two separate parts – a metallic stem and femoral head. Acetabular component is made of metal acetabular cup and acetabular interface (liner) (Baura, 2021). Endoprosthesis components may be composed of several different materials, each providing its own benefits and restrictions. Femoral stem is nowadays usually fabricated with stainless steel, titanium alloys or cobalt-chromium-molybdenum alloys (Merola & Affatato, 2019). Cobalt-chromium (CoCr) alloy or aluminium alloy, while in the past the prosthetic component was also made from stainless steel (Baura, 2021). Prosthesis developments have, however, been largely targeted towards the articulating parts of prosthesis, which are the femoral head and acetabular component. In the past, femoral heads were mostly made from CoCr alloy, like the femoral stem, while acetabular cup was metal and its articulating interface either ultra-high molecular weight polyethylene (UHMWPE) (Gibon et al., 2013) or ceramic (Baura, 2021). Components of the hip prosthesis may be made from several different materials, which should be biocompatible and enable long-term survivability of the implant, which is the surgery’s main goal (Gibon et al., 2013). Attempts to reduce friction and consequent particle debris formation have caused the metal femoral heads to be replaced with either alumina and zirconia ceramics, as well as oxidized zirconium. Ceramics as prosthetics components have consistently demonstrated reduced friction and consequent longer prosthesis lifespan, but unfortunately it was found that their toughness does not match that of metal. Still, they are the most commonly used materials for femoral heads of hip endoprosthesis today (Salehi and Hunter, 2010). Figure 1. Surgeon reaming acetabular cup (left) and inserting femoral stem component of hip endoprosthesis (right). Source: Author's own archive. There are three most common approaches for THA procedure (Varacallo et al., 2023). The most common approach for primary and revision cases is posterior approach. It includes blunt dissection of gluteus maximus muscle and sharp incision of fascia lata distally, avoiding hip abductors (Hyland, 2023). This approach is also favourable because of its good exposure of both acetabulum and femur, with the option for elongation of incision proximally or distally. However, some studies have cited higher dislocation rate in posterior approach compared to other surgical approaches (Varacallo et al., 2023). Proceedings of 10th Socratic Lectures 2024 9 of 234 The second possible THA approach is the direct anterior (DA) approach. The intermuscular interval being used with this approach is between tensor fascia lata and sartorius muscle superficially, and between gluteus medius and rectus femoris deep in the leg. The advantages of approach are the avoidance of hip abductors and reduced dislocation rates following surgery. However, its DA approach’s reported disadvantages are increased wound complications, especially in obese patients, who are amongst those more at risk for arthrosis, along with limited femoral exposure and risk of paraesthesia of lateral femoral cutaneous nerve (Varacallo et al., 2023). The less commonly used THA approach is the anterolateral or Watson-Jones approach. It utilises the intermuscular plane between tensor fascia lata and gluteus medius with a partial or complete detachment of anterior fibres of abductor muscles. In the past few years, incision or detachment of muscles or tendons has been avoided when performing the procedure (Lepri et al., 2020). The anterolateral approach theoretically offers decreased dislocation rate at the cost of postoperative limp (Varacallo et al., 2023). Following incision and careful retraction of surrounding muscles, femoral neck osteotomy is performed. Usually, a reciprocating saw is used during this step, with the cutting beginning proximally to the lesser trochanter. Osteotomy (bone-cutting process) is continued in a proximal-lateral direction towards the base of the greater trochanter. Following this, surgeon removes surrounding soft tissue (Varacallo et al., 2023). Retractors are placed around the incision to provide acetabular visualization. Before acetabular component is placed, labrum (soft tissue surrounding hip joint) must be removed (Petis et al., 2015). This is done with a scalpel or an electrocautery. Then, acetabulum itself is prepared by reaming, starting with small-size machinery, gradually increasing sized for appropriate medialization of cup. This is demonstrated in Figure 1. Once sclerotic bone is removed and healthy bone is established, prosthetic acetabular component is inserted in a press-fit fashion. Then, corresponding liner is inserted (Varacallo et al., 2023). Then, the femoral canal is prepared for prosthesis insertion. This is done by broaches proximally and by cylindrical reamers distally. Both parts of the femoral endoprosthesis component are then inserted into the femur, which is demonstrated in Figure 1 (right)(Fye et al., 1998). Prosthesis components may either be "press fit" into the bone, allowing it to grow around prosthesis, or they may be cemented into the bone. The quality and strength of patient's bone is a factor in choosing the right fixation method (Baura, 2021). 5. Hip arthroplasty – an increasing problem in today’s aging population In the recent years, there has been an increase in the number of both older and younger orthopaedic patients seeking surgical treatment. This might be due to the increasing number of sports injuries, as well as higher life standards and patients wanting to perform everyday activities without limitations. As the incidence of hip osteoarthritis generally increases with age, in today’s aging population this is leading to a higher demand for surgical intervention. This presents an increased burden on healthcare resources and hospital budgets (Nemes et al., 2014). Indications for THA have been expanded to include younger patients who are usually more active and recover faster than older patients. Additionally, their postsurgical activity levels and their return to sports are expected to improve, consequently leading to overall higher patient satisfaction post-surgery (Fujita et al., 2022). As patient satisfaction is an essential indicator in measuring the quality of care, several studies have attempted to measure patient satisfaction immediately and, in the years, following total hip arthroplasty (Varacallo et al., 2018; Okafor and Chen, 2019; Freudenberger et al., 2018). Said procedure is supposed to help with decreasing pain and improving motor function, patient mobility and quality of life. Freudenberger et al (2018) cited almost 60% of THA patients being highly satisfied with their hospital experience (a rating of 9 or 10 out of 10) (Freudenberger et al., 2018). Varacallo et al. (2018) reported 78% of interviewed patients 2-4 years after surgery perceived their replaced hip as “native”, and 54% reported uninhibited function of replaced joint (Varacallo et al., 2018). Okafor and Chen (2019) found that older patients may experience greater post-operative, which might be due to their lower expectations of pain relief after having lived with the joint disease for years. Proceedings of 10th Socratic Lectures 2024 10 of 234 5. Conclusion With today’s aging population, hip arthrosis is becoming a major problem that, especially the elderly, are facing every day. Osteoarthritis is also becoming a problem amongst the younger, active population, including professional sportsmen and women. Due to the development of newer, more advanced pharmacological interventions, as well as intraarticular infiltrations, the time which passes before patients can no longer put off hip replacement surgery has been prolonged. However, with the development materials and newer, less invasive approaches to hip replacement surgery, the procedure has become one of the most cost-effective and successful orthopaedic surgeries, with patients being highly satisfied with their reduced pain levels and improved mobility and quality of life post-surgery. Conflicts of Interest: The authors declare no conflict of interest. References 1. Baura G. Total hip prostheses. 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Available from https://www.swissmedi-cal.net/en/orthopedics/hip-surgery/hip-osteoarthritis Proceedings of 10th Socratic Lectures 2024 13 of 234 Research Preserved Proximal Femoral Bone Stock Volume in Total Hip Arthroplasty Significantly Reduces the Risk for Periprosthetic Fractures. A Novel Modelling Technique and Preliminary Clinical Results Kolar Matic1*, Mavčič Blaž1,2, Kralj-Iglič Veronika3, Antolič Vane1,2 1. University Medical Centre Ljubljana, Department of Orthopaedic Surgery, Ljubljana, Slovenia 2. University of Ljubljana, Faculty of Medicine, Chair of Orthopaedics, Ljubljana, Slovenia 3. University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia * Correspondence: Matic Kolar; matic.kolar@kclj.si Abstract: Periprosthetic proximal femoral fractures (PPFFs) are one of the main causes for revision after total hip arthroplasty (THA), and are associated with some already known patient- /surgical-/implant-related risk factors. Despite the established increased risk of single and Citation: Kolar M, Mavčič B, Kralj-double-wedge femoral implants, the highest incidence in our institution has been observed Iglič V, Antolič V. Preserved Proxi-with the anatomical cementless femoral component Anatomic Benoist Girard (ABG) II. The mal Femoral Bone Stock Volume in cumulative probability of PPFFs rose from 2.1% at 1 year to 6.5% at 10 years post-implan-Total Hip Arthroplasty Significantly tation, prompting comprehensive and multidisciplinary analysis. A novel parameter of Reduces the Risk for Periprosthetic preserved proximal femoral bone stock volume around implanted ABG II femoral stems Fractures. A Novel Modelling Tech- ( V PF) and the modelling technique for its evaluation on the standing anteroposterior (AP) nique and Preliminary Clinical Re-hip radiographs were introduced and estimated for each hip. Study was designed accord-sults. ing to the standard protocol for matched case-control research. In the preliminary analysis, Proceedings of Socratic Lectures . 5 age-/sex-/implant size-/surgeon-matched stratums, each comprising a case and 2 2024, 10, 13-21. matched controls, were included. To calculate V PF, a mathematical model was constructed https://doi.org/10.55295/PSL.2024.I3 by composing parts of rotational bodies and a prism, subject to geometrical parameters of the proximal femur that were assessed from radiographs. The mean value of V PF in the Publisher’s Note: UL ZF stays neu-group of cases was 141.6 ± 36.2 cm3 and significantly lower compared to the mean volume tral with regard to jurisdictional of 254.2 ± 33.8 cm3 in the control group (P < 0.01). Based on the preliminary results, the V PF claims in published maps and insti- seems crucial for the PPFFs prevention. However, the mechanism of its effect works as a tutional affiliations. ‘black box’. At this point, it can be hypothesized that insufficient bone stock from the implantation onwards interferes with adequate osseointegration by itself acutely and with increased stress shielding in the long term. The bone stock preservation should be emphasized and considered at all steps, starting from the preoperative planning. The novel pa-Copyright: © 2024 by the authors. rameter in THA, and the method for its evaluation were introduced and are further exten-Submitted for possible open access sively analysed. publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licens Keywords: Total Hip Arthroplasty, Periprosthetic Fracture, Bone Stock, Osseointegration, es/by/4.0/). Stress Shielding Proceedings of 10th Socratic Lectures 2024 14 of 234 1. Introduction Hip osteoarthritis (HOA) is an invalidating and prevalent disease with an estimated lifetime risk of symptomatic stage at around 25% (Katz et al., 2021; Murphy et al., 2010). Annually, more than 1 million total hip arthroplasties (THA) are performed worldwide with projections of further steady increase of primary as well as revision procedures (Ferguson et al., 2018). A number of factors, including the ageing population, the generalized demand for improved quality of life and functioning, along with the expansion of indications to the younger populations are governing the upscaling demand that has been proven insensitive even to the global economic downturns. In the next decades, a 2- to as much as 6-fold increase is projected by some studies (Shichman et al., 2023; Sloan et al., 2018). As the incidence of primary THA continues to rise, the absolute burden of complications has increased with further growth being anticipated (Kurtz S et al., 2007; Schwartz AM et al., 2020). The periprosthetic fractures (PPFs) are one of the four most common reasons for revision after primary THA, the other three being infection, aseptic loosening and instability/dislocation (Smith PN et al., 2023). PPFs represent a complex orthopaedic pathology with significant patients' morbidity and mortality, and socio-economic implications. In more than 80% of cases, the mechanism of injury is a low-energy trauma, mainly fall from the standing height. Most of the PPFs affect the proximal femur (PPFFs), while acetabulum is involved in less than 10% of THA-related fractures (Abdel et al., 2015; Bozic et al., 2009; Patsiogiannis et al., 2021). PPFFs are associated with some already known patient- (age, female sex, osteoporosis/os-teopenia, neuromuscular diseases, cognitive disorders, Paget’s disease, developmental hip dysplasia, rheumatoid arthritis), surgical- (malposition, extensive broaching), and implant-related (cementless, design/type, loosening, stress shielding) risk factors (Patsiogiannis et al., 2021; Singh et al., 2013). Despite the established increased risk of single and double-wedge femoral implants for PPFFs, the highest incidence in our institution has been observed with the anatomical cementless femoral component Anatomic Benoist Girard (ABG) II (Stryker Orthopaedics, Mahwah, NJ, USA), which is in line with some other studies in the literature (Carli et al., 2017; Catanach et al., 2015; Kropivšek et al., 2023; Mulford et al., 2022; Thien et al., 2014). The cumulative probability of PPFFs rose from 2.1% at 1 year to 6.5% at 10 years post-implantation, prompting comprehensive and multidisciplinary analysis (Kropivšek et al., 2023). Given that, ultimately, it is the bone that fractures, and preserved bone stock is pivotal for revision procedures, the novel parameter of preserved proximal femoral bone stock volume around the implanted femoral stems ( V PF) has been hypothesized. The primary aim of the present study was to develop a practical and reliable method for the evaluation of this novel parameter on the widely available hip anterior-posterior (AP) radiographs, and to conduct a preliminary assessment of its validity. 2. Methods 2.1 Assessment of geometrical parameters Geometrical parameters of the bone in contact with endoprosthesis were assessed from the standard hip AP radiographs. The images were available in DICOM format and measured by software Agfa HealthCare Enterprise Imaging (Agfa-Gevaert NV, Mortsel, Bel-gium). This software enabled measurements of lengths and delimited areas. The parameters x N’, x M’, x L’, x’L LAT, x N, x M, x L, x L LAT, HN, H L and S that were used for calculation of respective volumes are depicted in Figure 1. As the magnifications of the images were not known, the parameter dimensions were scaled by considering the known diameter of the prosthesis femoral head. Proceedings of 10th Socratic Lectures 2024 15 of 234 Figure 1. Geometrical parameters used as an input for the calculation of V PF evaluation. Proceedings of 10th Socratic Lectures 2024 16 of 234 2.2 Evaluation of the VPF parameter V PF is composed of hollow cut coni (modelling the bone of the femoral shaft in contact with the stem of the prosthesis) and a prism (modelling the remnants of the greater trochanter). In general, the volume of the conus with radius R at the base and height H is V conus =  R 2 H/3 (1) (1) and the volume of the conus cut at height h where the radius is r is V cut conus =  ( R 2 h + RrH + r 2 h)/3 , (2) (2) where r = R (1 - h/H) . (3) (3) Following Equation (2), at the lower part of the shaft the volume of the hollow cut conus is V (4) 1 =  ( x N’2 HN + x’ x M’ H N + x M’2 H N)/3 -  ( x N2 HN + x N x M H N + x M2 H N)/3. (4) In the middle part at the region of the smaller trochanter, the contour of the bone is considered asymmetric with respect to the axis along the shaft. The columns of the medial and the lateral halves are therefore calculated separately taking into account the differences between respective xL and xL LAT. The respective volumes are V (5) 2 =  ( x L’2 HL + x L’ x M’ H L + x M’2 H L)/6 -  ( x L2 HL + x L x M H L + x L2 H L)/6 (5) and V (6) 2 LAT =  ( x’2L LAT HL + x’L LAT x M’ H L + x M’2 H L)/6 -  ( x L2 H L + x L x M H L + x M2 H L)/6 . (6) It is considered that only half of each rotational body is contributing to the volume. The volume of the prism is calculated by (7) V prism = 2 S x N’ , (7) where S is the area of the prism assessed by delimiting its contours. The volume of the bone in contact with the prosthesis is VPF = V 1 + V 2 + V 2 LAT + V prism . (8) (8) 2.2 Subjects The retrospective matched case-control study was conducted according to the standard protocol for this type of research. In the preliminary analysis, the patients with late PPFFs, minimum 1 year postoperatively, were enrolled from the observational cohort of all implanted primary total hip arthroplasties with uncemented ABG II femoral stem between January 1, 2012, and January 31, 2013, at a single tertiary hospital (University Medical Centre Ljubljana, Department of Orthopaedic Surgery, Ljubljana, Slovenia). Clinical investigational plan was approved by the National Medical Ethics Committee (permit No. 0120-605/2021/3). In the group of cases, 5 patients with late PPFFs were included. An example of PPFF around the ABG II femoral stem is represented in Figure 2. For each case, 2 con- Proceedings of 10th Socratic Lectures 2024 17 of 234 trols matched for age, sex, implant size, and surgeon were found from the whole observational cohort: 1531 uncemented ABG II femoral stems implanted between January 1, 2012, and December 31, 2018. As a result, the control group of 10 patients was formed, and 5 matched stratums, each comprising a case and its 2 controls, were analysed. Patients’ demographics, medical history, stress shielding (Engh Grading Scale) (Engh, et al. 1987), Canal Flare Index (CFI) (Noble PC, et al. 1988), and length of radiographic follow-up evaluation were documented (Table 1). 3.3 Surgical intervention Patients were operated under spinal or general anesthesia, in the supine position with the direct lateral approach, or in the lateral decubitus position with the posterior approach to the hip joint. The cementless ABG II femoral stems were combined with either acetabular cup ABG II or acetabulum from another manufacturer. All surgical procedures were performed in the two operating rooms of the same operating suite of a single tertiary university hospital. Perioperative antibiotic prophylaxis, thromboembolic prophylaxis and postoperative rehabilitation protocol were uniform for all patients at a given time-point, but they have been changing between 2012 and 2018 in accordance with the national guidelines. The patients were followed from the initial primary total hip arthroplasty until the eventual outcome assessment on October 31, 2023. Table 1. Demographics, medical history, and follow-up time of the radiographs’ evaluation in both groups. Categorical variables are presented as frequencies (percentages), while continuous variables as mean (standard deviation). For comparison of both groups Student t test (continuous variables) or Chi square test (categorical variables) were applied. All Cases Controls Comparison Characteristic (n = 15) (n = 5) (n = 10) (P values) Age (years) 70.2 ± 5.0 68.2 ± 4.2 71.1 ± 5.3 0.31 Sex (n) Female 9 (60%) 3 (60%) 6 (60%) 1.0 Male 6 (40%) 2 (40%) 4 (40%) Height (m) 168.6 ± 8.9 168.6 ± 9.4 168.6 ± 9.1 1.0 Weight (kg) 78.3 ± 7.4 76.8 ± 7.0 79.1 ± 7.8 0.59 BMI (kg/m2) 27.7 ± 3.0 27.0 ± 1.7 28.0 ± 3.6 0.60 Follow-up (months) 59.7 ± 32.7 59.6± 35.2 59.7 ± 33.8 0.99 Osteoporosis (n) 2 (13%) 1 (20%) 1 (10%) 0.60 Stress shielding 1.6 ± 1.0 2.6 ± 0.5 0.8 ± 0.4 0.01* (Engh Grading Scale) CFI 2.9 ± 0.4 2.8 ± 0.3 3.0 ± 0.4 0.58 CFI– Canal Flare Index, BMI – Body Mass Index, m – meter, kg – kilogram, kg/m2 – kilogram per square meter Proceedings of 10th Socratic Lectures 2024 18 of 234 Figure 2. Case of PPFF around the ABG II femoral stem. Type B2 (unstable implant, sufficient bone stock) according to the Vancou-ver classification (Duncan CP et al., 1995) . 2.4 Statistical analysis Descriptive statistical analysis was used to describe patients’ demographics, medical history, stress shielding (Engh Grading Scale) (Engh et al., 1987), Canal Flare Index (CFI) (Noble et al., 1988), and length of radiographic follow-up evaluation. Continuous variables were presented as means with standard deviations (SD), and categorical variables as frequencies with corresponding percentages. For comparison of both groups either Student t test (continuous variables) or Chi square test (categorical variables) were applied. Statistical analysis was performed with SPSS (Version 25.0; IBM, Chicago, IL, USA). The level of statistical significance was set at P < 0.05. Proceedings of 10th Socratic Lectures 2024 19 of 234 3. Results The mean value of VPF in the group of cases was 141.6 ± 36.2 cm3 and significantly lower compared to the mean value of 254.2 ± 33.8 cm3 in the control group (P < 0.01). Moreover, in all 5 age-/sex-/implant size-/surgeon-matched stratums, the mean VPF of both controls was lower than VPF of the case (Table 2). Table 2. V PF in the 5 age-/sex-/implant size-/surgeon-matched stratums. In each stratum, the VPF of the case (PPFF) and the mean V PF with standard deviation of both controls are presented. Stratum No. VPF (cm3) PPFF 90.0 1 Controls mean 241.5 ± 1.6 PPFF 180.2 2 Controls mean 2 8 0 . 1 ± 3 . 8 PPFF 118.5 3 Controls mean 2 1 7. 4 ± 2 7 .3 PPFF 154.4 4 Controls mean 239.3 ± 18.9 PPFF 165.0 5 Controls mean 2 9 2. 5 ± 1 2 .1 V PF – Volume of Preserved proximal Femoral bone stock around femoral stems, PPFF - Periprosthetic Proximal Femoral Fracture, cm3 – cubic centim 4. Discussion The present study introduces a novel method for the evaluation of preserved proximal femoral bone stock volume around the implanted femoral stems on the widely available hip AP radiographs. The preliminary results are promising. Despite the longstanding awareness of the increasing incidence and consequences of PPFFs, and the rationale protective role of the preserved bone stock, no method for its evaluation, with the potential for routine clinical application, has been available. Interestingly, the research and development have been for decades intensively focused mainly on the artificial implants, their materials, composition, design, and other characteristics, while the local host environmental factors have remained mostly unaddressed (Burchard et al., 2023; Carli et al., 2017; Glassman et al., 2006; Huiskes et al., 1992; Rivière et al., 2018; Sumner et al., 1992). The introduced modelling technique aims to tackle the increasing PPFFs problem, and proposes a novel VPF parameter, which seems to significantly influence the risk of PPFFs and could be controlled to some degree. The method utilizes readily available standing hip AP radiographs that are part of every routine diagnostic assessment of patients with the indication for the primary THA. Moreover, its simplicity, quick learning curve, and time efficiency, taking only a few minutes after some examples measured, enable the surgeon to plan and control the bone stock preservation for every patient. Based on the preliminary results, the VPF seems crucial for the PPFFs prevention. However, the mechanism of its effect works as a ‘black box’ (Mavčič et al., 2012). The interplay between the two well established risk factors, osseointegration and stress shielding, with this novel third parameter of the preserved bone stock could be proposed (Savio et al., 2022). Bone preservation supports initial stability and enhance osseointegration acutely, while reducing stress shielding and preventing loosening (ensuring stable osseointegration) in the long term. Therefore, the preserved bone stock volume may be considered as the common biological denominator of osseointegration and stress shielding. Moreover, the observed significant difference in the mean grades of stress shielding between the groups supports the proposed correlations. However, the constitutional law between these factors at interplay has not been fully established yet. Despite only the preliminary analysis being performed, the Proceedings of 10th Socratic Lectures 2024 20 of 234 results clearly indicate the importance of considering bone stock preservation at all steps, starting from the preoperative planning. 5. Conclusion The introduced method for VPF evaluation demonstrated intuitive and promising results. Its wide availability, simplicity and significance promise the implementation into routine clinical practice. The novel parameter in THA, and the method for its evaluation were introduced and are further extensively analysed. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki. Clinical investigational plan was approved by the National Medical Ethics Committee (permit No. 0120- 605/2021/3). Conflicts of Interest: The authors declare no conflict of interest. References 1. Abdel MP, Cottino U, Mabry TM. Management of periprosthetic femoral fractures following total hip arthroplasty: a review. Int Orthop. 2015; 39:2005-2010. DOI: 10.1007/s00264-015-2979-0 2. Bozic KJ, Kurtz SM, Lau E, Ong K, Vail TP, Berry DJ. The epidemiology of revision total hip arthroplasty in the United States. J Bone Joint Surg Am. 2009; 91:128-133. DOI: 10.2106/JBJS.H.00155 3. Burchard R, Graw JA, Soost C, Schmitt J. 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DOI: 10.1097/00003086-199201000-00020 27. Thien TM, Chatziagorou G, Garellick G, et al. Periprosthetic femoral fracture within two years after total hip replacement: analysis of 437,629 operations in the nordic arthroplasty register association database. J Bone Joint Surg Am. 2014; 96:e167. DOI: 10.2106/JBJS.M.00643 Proceedings of 10th Socratic Lectures 2024 23 of 234 Invited lecture /Rewiev Urinary Tract Infections in Dogs and Cats Šimundić Metka1,* 1. Veterinarske storitve, Ljubljana, Slovenia * Correspondence: Metka Šimundić; metka.simundic3@gmail.com Abstract: Urinary tract infection (UTI) is a very common health problem in dogs and cats, treated with antimicrobial drugs. As the antimicrobial resistance problem increases, clinicians Citation: Šimundić M. Urinary Tract should be more aware of how to treat patients with antibiotics. This article aims to high-Infections in Dogs and Cats. light the importance of antimicrobial susceptibility testing before antimicrobial treatment Proceedings of Socratic Lectures. and remind clinicians to follow updated guidelines of the International Society for Com-2024, 10, 23-27. panion Animal Infection Diseases (ISCAID) recommendations (2019) and to monitor their https://doi.org/10. 55295/PSL.2024.I4 local antimicrobial resistance situation. The short review includes sporadic bacterial cystitis, recurrent bacterial cystitis, pyelonephritis, and subclinical bacteriuria (Weese et al., Publisher’s Note: UL ZF stays neu-2019). tral with regard to jurisdictional claims in published maps and in- stitutional affiliations. Copyright: © 2024 by the authors. Keywords: Cystitis; Subclinical bacteriuria, Pyelonephritis; Antimicrobials; Canine; Feline Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/lice nses/by/4.0/). Proceedings of 10th Socratic Lectures 2024 24 of 234 1. Introduction Urinary tract infections (UTIs) are a prevalent health concern in dogs and cats. Various microorganisms, mostly pathogenic bacteria and rare infecting fungi, viruses or parasites, can instigate these infections. UTIs are a common health issue in dogs and cats and one of the main reasons for antibiotic prescription. It is estimated, that 14% of dogs and 3-19% of cats will develop UTI at least once in their lifetime (Ling, 1984; White et al., 2013; Dorsch et al., 2016). Bacterial cystitis occurs more commonly in female dogs and cats (Byron, 2019). UTI is more common in senior female cats, older than 10 years of age, mostly with comorbidities (Dorsch et al., 2014). The development of UTI depends on the host immune system and defenses on one side and bacterial virulence on the other. Genital, rectal and perineal bacteria are the main res-ervoars for UTI (Osborne et al., 1991; Johnson et al., 2003). The most common cause of UTI is uropathogenic Escherichia coli (UPEC) (Moyaert et al., 2017). Veterinarians often prescribe antibiotics without urinary microbial culture and antibiotic susceptibility testing. Antibiotics commonly used in UTI are fluoroquinolones and 3rd generation cephalosporines (De Bruyne et al., 2014). These two antibiotics are on the list of highest priority critically important (Lhermie et al., 2020) Antimicrobial resistance is a growing concern in veterinary and human medicine. We must also be aware of our responsibility for public health, as AMRD genes can transfer between animals and human pathogens (Ewers et al., 2012). For this reason and for better therapeutic results, new recommendations regarding antimicrobial drugs, doses, duration and frequency of treatment should be followed and the health status of each patient should be carefully monitored. 2. Sporadic bacterial cystitis Management of sporadic bacterial cystitis is based on ISCAID guidelines (2019) where bacterial cystitis results in inflammation and clinical signs such as stranguria, hematuria and pollakiuria occur. Sporadic bacterial cystitis is diagnosed when fewer than 3 episodes of cystitis in the preceding year occurred with or without comorbidities. The diagnosis includes clinical signs, urine analysis, and urine bacterial cultures. Urinalysis is the major diagnostic test and includes macroscopic (dipstick, specific gravity) examination and mi-croscopic examination (urinary sediment). Microbiological analysis is preferred, and urine for bacterial culture should be obtained by ultrasound-guided cystocentesis in cases where other pathologies of the urinary bladder can be evaluated (such as masses, uroliths, etc.) If cystocentesis is risky for the animal, another option for obtaining urine is catheterization or a voided sample. Proper urine storage and quick laboratory analysis are important for accurate results (Weese et al., 2019). Young and middle-aged cats rarely have bacterial UTIs. Feline idiopathic cystitis (FIC) and urolithiasis are the main reasons for feline lower urinary tract disease (Dorsch et al., 2014). Bacterial urine culture and antimicrobial susceptibility tests in cats are necessary to make a diagnosis of UTI and treat patients properly. Empirical antimicrobial treatment for dogs with no or limited history of antibiotic treatment without urine culture is acceptable (Weese et al, 2019). ISCAID guidelines (2019) recommend amoxicillin (with clavulanic acid in case amoxicillin alone is not available) or trimethoprim-sulphonamides for antibiotic treatment of sporadic bacterial cystitis. The duration of therapy is 3-5 days. When urine culture and sensitivity have been performed, with resistance to empirical antibiotic detected but clinical signs have resolved, there is no need to repeat the urinalysis after treatment. Even if the antibiotic was in line with the results of urine culture and sensitivity but clinical signs persist, clinicians should reevaluate the diagnosis and find the reason (Weese et al., 2019). The guidelines emphasizes that clinicians should consider local known antimicrobial resistance and presume alternate antimicrobials for better outcomes in empirical treatment. Treatment can be started with non-steroid anti-inflammatory drugs (NSAIDs) because of Proceedings of 10th Socratic Lectures 2024 25 of 234 inflammation (side effects of NSAIDs should be considered) and continued with antibiotics in case where clinical signs do not improve (Weese et al., 2019). 3. Recurrent bacterial cystitis ISCAID guidelines (2019) defines recurrent bacterial cystitis as: three or more episodes of clinical bacterial cystitis in the preceding 12 months or two or more episodes in the preceding 6 months. Recurrent UTIs include various scenarios: relapse of infection (with the same microorganism after successful treatment of the UTI, often seen in diseases like urolithiasis or pyelonephritis); reinfection (infection with a different microorganism after the initial microorganism responded to therapy, common in disease like diabetes mellitus); persistent infection (positive urine cultures with the same organism during treatment with appropriate antimicrobial agents); and superinfection (infection with new organisms that develop during antimicrobial treatment for the initial infecting organism, observed in diseases such as neoplasia or urinary catheterization) (Barsanti, 2009; Weese et al., 2019). Concomitant factors and underlying causes are mostly the main reasons for recurrent UTI. Advanced diagnostics should be performed to identify the underlying disease responsible for the recurrent UTI (endocrinopathy, kidney disease, obesity, abnormal vulvar conformation, congenital abnormalities of the urogenital tract (e.g. ectopic ureter), prostatic disease, bladder tumor, polypoid cystitis, urolithiasis, immunosuppressive therapy, rectal fistula, urinary incontinence/retention). Diagnostic imaging with ultrasound, radiog-raphy, contrast imaging and cystoscopy is warranted. In some cases, biopsy of urinary bladder mucosa for pathophysiologic and microbiological examination is necessary (Olin & Bartges, 2015; Weese et al., 2019). Proper diagnosis, treatment, and controlling diseases like Cushin’s disease, diabetes mellitus, correction of ectopic ureter, vulvar skin fold, and others can end the cycle of recurrence of UTI. The clinician must ensure adequate antibiotic concentration in the bladder, check dosage, regiment client compliance, etc. Treatment should be based on the results of urinary culture and sensitivity. While await-ing urine bacterial culture results, NSAIDs can be administrated, and if an empirical antibiotic is necessary, first-line amoxicillin or trimethoprim-sulphonamide should be initiated. In case of microbial invasion of urinary bladder wall, antimicrobials that are effective against Escherichia coli in tissue are prescribed. After obtaining urine culture and sensitivity results, clinical response to therapy should be considered. In case of clinical failure, bacterial culture-based antimicrobial changes should be indicated (Weese et al. 2019). Treatment duration in case of reinfection is short, typically 3-5 days, and monitoring is based on clinical response. In cases of persistent and relapsing infections, longer treatment durations of 7-14 days are indicated, with repeating urine culture after 5-7 days of treatment being prudent. A positive urine culture mandates checking compliance with therapy and further evaluation of the patient. Negative results of urine culture do not guarantee microbiological cure and these are also authors’ experiences. After 5-7 days post - treatment, urine culture should be repeated if clinical signs resolve. In cases of positive urine culture (relapse, reinfection, persistent) with the absence of clinical sign, subclinical bacteriuria is diagnosed, and referral to a specialist should be considered (Weese et al., 2019). 4. Upper urinary tract infections (pyelonephritis) Pyelonephritis is an inflammation of the renal parenchyma and pelvis, mostly caused by an ascending urinary infection rather than hematogenous spread (Parry, 2005). Leptospira should be considered in case of pyelonephritis (Sykes et al., 2011). Pyelonephritis can have acute or chronic course. Acute pyelonephritis is associated with fever, uremic signs (ano-rexia, lethargy, vomiting), painful kidneys, polyuria/polydipsia, anuria, lumbal pain, etc. Chronic pyelonephritis can present with mild (or even absent) clinical signs and slowly progressive azotemia, whicht can lead to kidney failure. Diagnosis of pyelonephritis is based on clinical signs, urinalysis, positive urine culture, compatible ultrasonographic Proceedings of 10th Socratic Lectures 2024 26 of 234 changes of the kidney, leukocytosis (neutrophilia) and azotemia. It is important to remember that not all patient with pyelonephritis have leukocytosis or azotemia (Olin & Bartges, 2015; Bouillon, 2018). In cases of negative urine culture, immunosuppressed animals, or those with fever, a blood culture is needed (Kim et al., 2017, Westropp & Sykes, 2017). In a study of pyelonephritis in dogs, Bouillon et al. (2018) found, that 74.5 % of dogs with pyelonephritis presented with comorbidities. Treatment should be started with antimicrobial agents effective against Enterobacteri-aceae, taking into account regional antimicrobial susceptibility. Veterinary fluoroquinolone or cefpodoxime are the first choice. Cefotaxime and ceftazidime are important for intravenous application. If results of antibiogram show resistance to the initial antimicrobial and clinical response is not sufficient, substitution with a susceptible antimicrobial is necessary (not necessary if the clinical response is good). If therapy is initiated with two antimicrobials and both are susceptible, one of them might be discontinued if there is a good clinical response. If results of urinary culture and susceptibility identify multidrug - resistant microbes, consultation with a specialist is recommended (Weese et al., 2019). The duration of the treatment is 10-14 days. One to two weeks after discontinuation of antimicrobials, a recheck is recommended (physical exam, urinalysis, bacterial culture, blood analysis). In cases of full clinical recovery and positive results of clinical culture, subclinical bacteriuria should be suspected (Weese et al., 2019). 5. Subclinical bacteriuria Subclinical bacteriuria is defined as the presence of bacteria in urine as determined by positive bacterial culture from a properly collected urine specimen, in the absence of clinical evidence of infectious urinary tract disease. It is not uncommon in healthy animals, patients with comorbidities, and those treated with immunosuppressive drugs. In a Nor-wegian study of the prevalence of subclinical bacteriuria, in a cohort of 108 healthy cats with a mean age of 4 years, 0.9% were positive (Eggertsdottir et al., 2011). With age, prevalence increased. Another study reported 67 cats older than 7 years of age with a prevalence of subclinical bacteriuria in 10-13% (White et al., 2016). Lamoureux et al. (2022) found that 32% of 62 dogs with chronic kidney disease (CKD), including 8 dogs in International Renal Interest Society (IRIS) stage 1, had positive urine culture, and only 8% showed clinical signs of urinary tract disease. Another study in cats with CKD reported a prevalence of 22-29% (Mayer-Roenne et al., 2007). Current guidelines recommend that antimicrobials should not be used for treatment in subclinical bacteriuria. There are recommendations for treatment of specific conditions, such as animals with suspected pyelonephritis, those undergoing surgical or endoscopic procedures of the urinary tract likely to cause bleeding, etc. Clinicians should evaluate each case individually and decide whether to treat or not (Weese et al., 2019). References 1. Barsanti J. Multidrug-resistant urinary tract infection. In: Bonagura J.D., Twedt D.C., editors. Current veterinary therapy XIV. WB Saunders; St Louis (MO): 2009. pp. 921–925. 2. Bouillon J, Snead E, Caswell J et al. Pyelonephritis in Dogs: Retrospective Study of 47 Histologically Diagnosed Cases (2005-2015). J Vet Intern Med. 2018; 32: 249-259. DOI:10.1111/jvim.14836 3. Byron JK. Urinary Tract Infection. Vet Clin North Am Small Anim Pract. 2019; 49: 211-221. DOI:10.1016/j.cvsm.2018.11.005 4. De Bruyne N, Atkinson J, Pokludová L, Borriello SP. Antibiotics used most commonly to treat animals in Europe. Vet Rec. 2014; 175: 325. DOI:10.1136/vr.102462 5. Dorsch R, Remer C, Sauter-Louis C, Hartmann K. Feline lower urinary tract disease in a German cat population. A retrospective analysis of demographic data, causes and clinical signs. Tierarztl Prax Ausg K Kleintiere Heimtiere. 2014; 42: 231-239. DOI: 10.1055/s-0038-1623769 Proceedings of 10th Socratic Lectures 2024 27 of 234 6. Dorsch R, von Vopelius-Feldt C, Wolf G et al. Urinary tract infections in cats. Prevalence of comorbidities and bacterial species, and determination of antimicrobial susceptibility to commonly used antimicrobial agents. Bakterielle Harnwegsinfektionen bei Katzen. Prävalenz prädisponierender Erkrankungen und bakterieller Isolate sowie Ermittlung der antimikrobiellen Resistenz gegenüber häufig eingesetzten Antibiotika. Tierarztl Prax Ausg K Kleintiere Heimtiere. 2016; 44: 227-236. DOI:10.15654/TPK-150604 7. Eggertsdóttir AV, Sævik BK, Halvorsen I, Sørum H. Occurrence of occult bacteriuria in healthy cats. J Feline Med Surg. 2011; 13: 800-803. DOI:10.1016/j.jfms.2011.07.004 8. Ewers C, Bethe A, Semmler T et al. Extended-spectrum β-lactamase-producing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective. Clin Microbiol Infect. 2012; 18: 646-655. DOI:10.1111/j.1469-0691.2012.03850.x 9. Johnson J.R., Kaster N., Kuskowski M.A. Identification of urovirulence traits in Escherichia coli by comparison of urinary and rectal E. coli isolates from dogs with urinary tract infection. J Clin Microbiol. 2003; 41: 337–345. DOI: 10.1128/JCM.41.1.337-345.2003 10. Kim Y, Seo MR, Kim SJ, et al. Usefulness of Blood Cultures and Radiologic Imaging Studies in the Management of Patients with Community-Acquired Acute Pyelonephritis. Infect Chemother. 2017; 49: 22-30. DOI:10.3947/ic.2017.49.1.22 11. Lamoureux A, Da Riz F, Cappelle J, et al. Frequency of bacteriuria in dogs with chronic kidney disease: A retrospective study of 201 cases. J Vet Intern Med. 2019; 33: 640-647. DOI:10.1111/jvim.15434 12. Lhermie G, La Ragione RM, Weese JS, et al. Indications for the use of highest priority critically important antimicrobials in the veterinary sector. J Antimicrob Chemother. 2020; 75: 1671-1680. DOI:10.1093/jac/dkaa104 13. Ling GV. Therapeutic strategies involving antimicrobial treatment of the canine urinary tract. J Am Vet Med Assoc. 1984; 185: 1162-1164. 14. Mayer-Roenne B, Goldstein RE, Erb HN. Urinary tract infections in cats with hyperthyroidism, diabetes mellitus and chronic kidney disease. J Feline Med Surg. 2007; 9: 124-132. DOI:10.1016/j.jfms.2006.09.004 15. Moyaert H, Morrissey I, de Jong A, et al. Antimicrobial Susceptibility Monitoring of Bacterial Pathogens Isolated from Urinary Tract Infections in Dogs and Cats Across Europe: ComPath Results. Microb Drug Resist. 2017; 23: 391-403. DOI:10.1089/mdr.2016.0110 16. Olin SJ, Bartges JW. Urinary tract infections: treatment/comparative therapeutics. Vet Clin North Am Small Anim Pract. 2015; 45: 721-746. DOI:10.1016/j.cvsm.2015.02.005 17. Osborne C, Caywood D, Johnston G. Perineal urethrostomy versus dietary management in prevention of recurrent lower urinary tract disease. J Small Anim Pract. 1991; 32: 296–305. 18. Parry NMA. Pyelonephritis in small animals. UK VET. 2005: 10: 1-5. 19. Sykes JE, Hartmann K, Lunn KF, et al. 2010 ACVIM small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention. J Vet Intern Med. 2011; 25: 1-13. DOI:10.1111/j.1939-1676.2010.0654.x 20. Weese JS, Blondeau J, Boothe D, et al. International Society for Companion Animal Infectious Diseases (ISCAID) guidelines for the diagnosis and management of bacterial urinary tract infections in dogs and cats. Vet J. 2019; 247: 8-25. DOI:10.1016/j.tvjl.2019.02.008 21. Westropp JL, Sykes JE. Diagnostics and management of pyelo - nephritis. ACVIM Forum; 2017: 8–10. 22. White JD, Stevenson M, Malik R, Snow D, Norris JM. Urinary tract infections in cats with chronic kidney disease. J Feline Med Surg. 2013; 15: 459-465. DOI:10.1177/1098612X12469522 23. White JD, Cave NJ, Grinberg A, Thomas DG, Heuer C. Subclinical Bacteriuria in Older Cats and its Association with Survival. J Vet Intern Med. 2016; 30: 1824-1829. DOI:10.1111/jvim.14598 Proceedings of 10th Socratic Lectures 2024 29 of 234 Invited lecture/Review Physiotherapy Approach for Treating Lateral Epicondilalgia Urbančič Špela1,*, Vauhnik Renata1 1. Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia *Correspondence: Špela Urbančič; spela.urbancic123@gmail.com Abstract: Lateral epicondylitis, a tendinopathy affecting forearm extensor muscles, results from overuse and commonly manifests as pain over the lateral humeral epicondyle. The extensor carpi radialis brevis (ECRB) is implicated, particularly during forceful gripping. The condition is prevalent in the 35-45 age group and it affects 1-3% of the population, with Citation: Urbančič Š, Vauhnik R. symptoms lasting two years and reoccurring after asymptomatic periods. Considering the Physiotherapy Approach for Treat-economic impact of lateral epicondylalgia-related sick leave, therapeutic focus must eming Lateral Epicondilalgia. phasize long-term as well as short-term efficacy. Treatment approaches include drugs, rest, Proceedings of Socratic Lectures. physiotherapy and surgery. Platelet-rich plasma proves highly effective, reducing pain 2024, 10, 29-33. and work absenteeism. Severe cases may require surgical approach, with studies question-https://doi.org/10.55295/PSL.2024.I5 ing the benefits of surgery over a placebo. Pain management involves shockwave therapy, ultrasound, friction massage, eccentric exercises, stretching, and orthoses. Shockwave therapy stands out for its rapid pain relief and long-term efficacy. The combination of ex-Publisher’s Note: UL ZF stays neu- ercises and kinesiotaping is effective, while high-intensity laser therapy has limited evi-tral with regard to jurisdictional dence. Functionality improves with manual therapy, stretching, and strengthening exer-claims in published maps and insti- cises. Orthoses may negatively impact hand function. Grip strength correlates closely with tutional affiliations. functionality, with therapeutic exercise and manual therapy showing significant long-term results. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and Keywords: Lateral epicondylalgia, tendinopathy, rehabilitation, physiotherapy approach conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licens es/by/4.0/). Proceedings of 10th Socratic Lectures 2024 30 of 234 1. Introduction Lateral epicondylitis is a tendinopathy of the forearm extensor muscles, often caused by overuse or repetitive use (Tarpada et al., 2018). Individuals suffering from tennis elbow often experience pain localized around the lateral humeral epicondyle. Pain tends to intensify during elbow flexion, particularly when the wrist is in a pronated position and extended against resistance. The consensus among many researchers is that the origin of the extensor carpi radialis brevis (ECRB) is the key contributor to the symptoms associated with lateral epicondylitis. From a biomechanical standpoint, forceful gripping places the ECRB under maximum strain, especially when the forearm is pronated, and the wrist is flexed and ulnar deviated. Lateral epicondylitis is most prevalent in the individuals aged 35-45, affecting approximately 1-3% of the general population. Symptoms usually last for 2 years, but it usually relapses after asymptomatic periods (Sandhu et al., 2020; Bisset & Vicenzino, 2015). Due to the increasing economic impact of lateral epicondylalgia-related sick leave, attention must also be given to the short-term effects of therapies that will reduce the duration of work absenteeism. Different treatment approaches have been proposed, such as drugs, rest, physiotherapy and surgery (Zhong et al., 2020). The first therapeutic step usually involves rest and administration of drugs, providing short-term pain relief but also yielding poor results for problem resolution and relapse prevention (Boden et al., 2019). Platelet-rich plasma treatment proves to be a highly effective therapy; research indicates that, in addition to reducing pain and improving function, the time of absence from work is shortened even more compared to non-invasive therapies (Alessio-Mazzola et al., 2018). But there is 2-11% of cases severe enough that need surgical removal of diseased tissue. With the surgical approach, immediate pain relief is achieved in 80-97% of cases, with more than 75% of patients with no or minimal pain after one year. Although 1.5% of patients undergo a second surgical procedure in the following 18-24 months (Degen et al., 2018; Holmedal et al., 2019). On the contrary, study by Kroslak and Murrell (2018) showed that surgical excision of the degenerative portion of the ECRB offers no additional benefit over and above placebo surgery for the management of chronic tennis elbow. Physiotherapeutic treatment is shown to be the most effective, but it should include manual therapy performed under the pain threshold to relieve pain and improve joint range of motion (Zhong et al., 2020). When assessing the effectiveness of rehabilitation techniques, focus is on pain, limb function, maximum grip strength, and wrist range of motion. 2. Pain management Research indicates that pain intensity decreases most rapidly (between 3 and 9 sesions) with therapies involving shockwave therapy, ultrasound, friction massage, eccentric exercises, stretching exercises, and orthoses (Landesa-Piñeiro & Leirós-Rodríguez, 2022). Shockwave therapy demonstrates a notable decrease in pain threshold after only three sessions (Yalvaç et al., 2018), whereas ultrasound therapy yields comparable effects after ten sessions, as evidenced by Yan et al. in 2019. The analgesic efficacy of these therapies arises from the stimulation of pain receptors and the activation of unmyelinated C fibers and A delta fibers, initiating the gate control theory of pain (Dedes et al., 2018). Notably, shockwave therapy employs higher energy waves, leading to a more pronounced stimulation of pain receptors (Yan et al., 2019). Moreover, shockwave therapy induces a localized metabolic response characterized by heightened vascularity and reduced adhesion formation. These contribute to the facilitation of the inherent healing processes (Orhan et al., 2004). The effect of shockwave therapy lasts longer than the effect of conventional therapy (thermotherapy, ultrasound, TENS). Shockwave therapy is more effective in the short term for chronic patients, but in the long term, it has a better impact on acute patients (Köksal et al., 2015). Another randomized contolled study showed the combination of exercises and kinesiotaping can be more effective than exercises alone or with sham taping for up to one month after treatment. There is some high quality evidence that shows positive effect on pain after performing all kind of strenghtening exercises combined (Eraslan et al., 2018). Proceedings of 10th Socratic Lectures 2024 31 of 234 There is only one high quality study that show positive effect of high-intensity laser therapy in comparison with placebo laser therpy, but the tickness of common extensor tendons did not change at all (Dundar et al., 2015). For long term pain relief there is evidence that shockwave therapy, manual therapy, eccentric strengthening exercises and corticosteroid infiltration have positive effects up to 1 year after therapy (Köksal, 2015; Olaussen, 2015). 3. Functionality and grip strenght The relationship between functionality and grip strength demonstrates a notable correlation, wherein grip strength serves as a quantifiable and objective metric. Interventions based on therapeutic exercise and manual therapy obtained good results in the long term and even significantly higher results compared to those based on shockwave therapy, orthoses, and corticosteroids (Landesa-Piñeiro & Leirós-Rodríguez, 2022). The effect of conventional therapy (stretching and strengthening exercises with education) showed good result but recovery time depends, to a great extent, on the frequency of their execution (Stasinopoulos & Johnson, 2004). Studies show that no kind of strenghtening exercises is superior than other, but there is lack of studies that isolate the effect of particular kind of exercises (Lepley et al., 2017; Stasinopoulos & Stasinopoulos, 2017). It is important to point out that studies show improvement in range of wrist motion after only eccentric exercises (Nowotny et al., 2018). This modality of strenght training also instigates collagen production, diminishes the incidence of inflammation and neovascularization, and mitigates pain through the augmentation of tendinous resistance and desensitization of central nervous pathways involved in pain transmission (Peterson et al., 2014). Shockwave showed good results but is less efective compared to kinesiotaping which should be used in combination with exercises for longer effect (Giray et al., 2019). Eraslan et al. (2018) assert that the combination of conservative therapy and kinesiotape application is more effective than the combination of conservative therapy and shockwave therapy. According to the three parameters of the Patient-Rated Tennis Elbow Evaluation Scale, the difference before and after therapy is statistically significant in both groups. However, the progress in the group with kinesiotape application is statistically significantly superior. Kinesiotape reduses pain and edema and facilitate motor activity by relieveing motor abnormal muscle tension. They also stimulate mechanoreceptors by applying pressure on the skin, which are effects that have a direct impact on the generation of strenght (Pieters et al., 2020). Studies show that the use of orthoses can have a negative effect on hand function, reducing mobility and preventing normal elbow movement (Stasinopoulos & Stasinopoulos, 2017). The use of orthoses for 6 weeks in combination with mobility exercises and stretching exercises daily has no positive effect (Yi et al., 2018). 4. Conclusion Taken together, researchers still haven't agreed on the most effective long-term therapy due to lack of studies showing lasting effects. Additionally, considering the economic consequences related to work absenteeism there is need to find therapies with best short term effect. The combination of manual therapy, involving deep friction massage, along with streching and eccentric strenghtening exercises yields the most favorable results for functional improvement and increased grip strength in lateral epicondylalgia, furthermore, their cost-benefit ratio is very favourable. Other therapy techniques such as shockwave and ultrasound therapy have the greatest effect on reducing pain intensity. Moreover, research indicates that shockwave therapy achieves the fastest results in pain management. According to available literature, the rehabilitation of lateral epicondylalgia should be divided into three phases. In the first and second phases, the focus is on reducing pain and improving hand dysfunction. The literature suggests therapy using shockwave therapy in combination with manual therapy (deep friction massage) under pain threshold and stretching exercises. In the second phase, isometric exercises are added to strengthen the wrist extensors and finger extensors. These exercises are then progressed in the third phase, when we focus on grip strenght, where concentric and eccentric Proceedings of 10th Socratic Lectures 2024 32 of 234 exercises are introduced for the wrist extensors, supinators, and pronators. In all three phases, the application of kinesiotaping can be added. However, it is important that it is applied by a trained person, as incorrect application may not bring positive results. Conflicts of Interest: The authors declare no conflict of interest. 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Prospective randomized controlled trial in the treatment of lateral epicondylitis with a new dynamic wrist orthosis. Eur J Med Res. 2018; 23: 43. DOI: 10.1186/s40001-018-0342-9 15. Olaussen M, Holmedal Ø, Mdala I, Brage S, Lindbæk M. Corticosteroid or placebo injection combined with deep transverse friction massage, Mills manipulation, stretching and eccentric exercise for acute lateral epicondylitis: a randomised, controlled trial. BMC Musculoskelet Disord. 2015; 16:122. DOI: 10.1186/s12891-015-0582-6 16. Orhan Z, Ozturan K, Guven A, Cam K. The effect of extracorporeal shock waves on a rat model of injury to tendo Achillis. A histological and biomechanical study. J Bone Joint Surg Br. 2004; 86:613-618. 17. Peterson M, Butler S, Eriksson M, Svärdsudd K. A randomized controlled trial of accentric vs. concentric graded exercise in chronic tennis elbow (lateral elbow tendinopathy). Clin Rehabil. 2014; 28 : 862-872. DOI: 10.1177/0269215514527595 18. Pieters L, Lewis J, Kuppens K, Jochems J et al. An Update of Systematic Reviews Examining the Effectiveness of Conservative Physical Therapy Interventions for Subacromial Shoulder Pain. J Orthop Sports Phys Ther. 2020; 50:131-141. DOI: 10.2519/jospt.2020.8498 Proceedings of 10th Socratic Lectures 2024 33 of 234 19. Sandhu KS, Kahal KS, Singh J, Singh J, Grewal H. A comparative study of activated platelet rich plasma versus local corticosteroid injection for the treatment of lateral epicondylitis: A randomised study. Int J Orthop. 2020; 6: 1274-1276. DOI: 10.1177/230949901502300101 20. Stasinopoulos D, Johnson MI. Cyriax physiotherapy for tennis elbow/lateral epicondylitis. Br J Sports Med. 2004; 38:675-677. DOI: 10.1136/bjsm.2004.013573 21. Stasinopoulos D, Stasinopoulos I. Comparison of effects of eccentric training, eccentric-concentric training, and eccentric-concentric training combined with isometric contraction in the treatment of lateral elbow tendinopathy. J Hand Ther. 2017; 30: 13-19. DOI: 10.1016/j.jht.2016.09.001 22. Tarpada SP, Morris MT, Lian J, Rashidi S. Current advances in the treatment of medial and lateral epicondylitis. J Orthop. 2018; 15: 107-110. DOI: 10.1016/j.jor.2018.01.040 23. Zhong Y, Zheng C, Zheng J, Xu S. Kinesio tape reduces pain in patients with lateral epicondylitis: A meta-analysis of randomized controlled trials. Int J Surg. 2020; 76: 190-199. DOI: 10.1016/j.ijsu.2020.02.044 24. Yalvaç B, Mesci N, Geler Külcü D, Yurdakul OV. Comparison of ultrasound and extracorporeal shock wave therapy in lateral epicondylosis. Acta Orthop Traumatol Turc. 2018; 52:357-362. DOI: 10.1016/j.aott.2018.06.004 25. Yan C, Xiong Y, Chen L, et al. A comparative study of the efficacy of ultrasonics and extracorporeal shock wave in the treatment of tennis elbow: a meta-analysis of randomized controlled trials. J Orthop Surg Res. 2019; 14:248. DOI: 10.1186/s13018-019-1290-y 26. Yi R, Bratchenko WW, Tan V. Deep Friction Massage Versus Steroid Injection in the Treatment of Lateral Epicondylitis. Hand (N Y). 2018; 13:56-59. DOI:10.1177/1558944717692088 Proceedings of 10th Socratic Lectures 2024 35 of 234 Invited lecture/Review The Impact of Sleep Deprivation on Physical Activity Vrbinc Katarina Nina1,2, Vauhnik Renata1 1. University of Ljubljana, Faculty of Health Sciences, Ljubljana 2. Community Health Centre Ljubljana, Ljubljana * Correspondence: Katarina Nina Vrbinc; ninakvrbinc@gmail.com Abstract: Proper amount of sleep has a critical role in biological processes and overall health and is linked to negative health outcomes, such as diabetes, heart disease, brain stroke, weight changes, pain, mood swings, Alzheimer's disease, cognitive health, chromosome changes etc. Despite dedicating one-third of our lives to sleep, a substantial portion of the world's population faces sleep deprivation. Insufficient sleep goes beyond simple tiredness, and Citation: Vrbinc KN, Vauhnik R. it is affecting humans metabolic, cardiovascular, cognitive, and emotional dimensions. The Impact of Sleep Deprivation on When it comes to physical activity and athletic performance, sleep deprivation manifests Physical Activity. in compromised reaction time, accuracy, vigour, submaximal strength, and endurance. Proceedings of Socratic Lectures . These challenges also affect athlete’s cognitive functions such as judgment and decision-2024, 10, 35-40. making. In the context of athletic performance, even partial sleep deprivation can lead to https://doi.org/10.55295/PSL.2024.I6 significant impairments. Relationship between sleep and physical activity was found as emphasizing the importance of adequate sleep for optimal performance and shows that good sleep hygiene and recommended amount of physical activity really go hand in hand Publisher’s Note: UL ZF stays neu-contributing to overall well-being including physiological and mental health. tral regarding jurisdictional claims in published maps and institutional affiliations. Copyright: © 2024 by the authors. Keywords: Sleep deprivation; Physical activity; Athletic performance, Metabolic health; Submitted for possible open access Cognitive functions, Global prevalence. publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses /by/4.0/). Proceedings of 10th Socratic Lectures 2024 36 of 234 1. Sleep hygiene and sleep deprivation 1.1. What is sleep and why we need it Sleep is defined as natural and reversible state marked by reduced responsiveness to external stimuli and relative inactivity, accompanying a loss of consciousness (Rasch & Born, 2012). Sleeping represents the supreme support for human health, constituting a fundamental biological imperative. An average human dedicates approximately one-third of their lifespan to this crucial physiological function (Hirshkowitz et al., 2015). Furthermore, sleep serves as a key opportunity for the body's reparative processes (Bandyopadhyay & Sigua, 2019). It does not represent just a state where our responses are reduced, but it has 4 stages and they all have their own important role (Rasch & Born, 2012). Stages of sleep are presented in Figure 1. Figure 1. Stages of sleep; N1 = a stage of falling asleep/light sleep, N2 = stage of slower heart rate and decrease of body temperature, N3 = slow-wave sleep; stage of deep sleep and body repairation prosesses, REM sleep (stands for rapid-eye movement sleep) = phase of dreaming and skeletal muscle atonia (Racsch & Born, 2012). It is visible in Figure 1 that slow wave sleep (N3) predominates in the early hours of sleep and REM sleep becomes more dominant in the later hours of sleep (Rasch, Born, 2012). But sleep is not just a collection of physiologic processes that occur together at the inter-section of rest and activity. It is a non-negotiable biological state that is necessary for the maintenance of human life. Ensuring a plentiful duration of quality sleep, has numerous advantages on our health and it is the key to a good quality of life, such as elevated immunity, mental health and emotional resilience, cognitive advancements, physical fitness and energy balance, cardiovascular benefits, and metabolic regulation (Hirshkowitz et al., 2015). As such, it is important to consider developmental changes in sleep, which reflect directly on brain development. In preschool-aged children, decreases in napping have been associated with higher vocabulary and enhanced memory. Recent studies also report associations between sleep micro-structure and brain maturation in youths (Mason et al., 2021). 1.2. Sleep deprivation and recommended duration of sleep Obviously sleep deprivation occurs when a person is not able to get enough sleep. The amount of sleep needed to feel refreshed, and function well depends on the individual and varies across the ages. Manny people refer to 5 or 6 hours as good enough amount of sleep, but that is not enough time for our body to regenerate properly. As said earlier sleep is a very important part of our development, so it is expected that babies and very young kids need more time to dedicate to this important part of our 24h cycle (Mason et al., 2021). Recommended amount of sleep for every age group is visible in Figure 2. Proceedings of 10th Socratic Lectures 2024 37 of 234 Figure 2. Recommended duration of sleep (Hirshkowitz et al., 2015). 2. Prevalence of sleep deprivation and its effects on human body Sleep deprivation became a great problem, affecting a substantial portion of the world population. According to research conducted by the American Thoracic Society (Bandyopadhyay & Sigua, 2019), one-third of the US adult population fails to achieve the recommended seven hours of sleep. This prevalent problem is also noticeable across other age groups, with an approximately 50-70 million adults in the United States suffering from sleep disorders (Baranwal, 2023). The impact of insufficient sleep also represents a great problem with students, especially those in high schools and universities. A study conducted by Albqoor and Shaheen (2021) on university students in Jordan has shown poor sleep quality among the student population. Interestingly, the study found that students with lower income, those who were smokers, physically inactive and used media devices before bed, experienced the highest increase in sleep deprivation. The prevalence of sleep deprivation is alarming among high school students, with 73% receiving less than 8 hours of sleep and 58% of middle school students getting less than 9 hours of sleep per night. Obviously not getting enough sleep is the most common cause of sleep deprivation, but other causes also include poor sleeping habits, circadian rhythm disturbance (e.g. delayed sleep phase), sleep disorders like insomnia, restless leg syndrome and sleep apnoea, use of medications or drugs, jet lag when traveling across multiple time zones, late shift work etc. (Hirshkowitz et al., 2015). Reducing your sleep time by even 1 hour can impact your thought process and reaction time the following day, which was found to be in correlation with car crashes and other accidents. Lack of sleep can also lead to chronic fatigue, potentially affecting work or school performance (Lo et al., 2016). The main impact of sleep deprivation extends well beyond the fact that we can be tired after not getting enough sleep, but it lives consequences on various dimensions of health. Inadequate sleep disrupts fundamental physiological processes, such as metabolic or cardiovascular health. Insufficient nocturnal sleep leads to impaired glucose processing, which is elevating the risk for developing type 2 diabetes. Cardiovascular health can be negatively effected by consequences such as elevated blood pressure, increased inflammation, and physiological stress responses, contributing to a 48% increased likelihood of heart disease and a 15% increased risk of stroke in adults that sleep regularly less than 6 hours per night (Liew & Aung, 2021). The role of sleep in vascular restoration becomes evident, with deficiencies potentially leading to vessel damage and severe conditions such as aneurysms, atherosclerosis, and ischemia (Liew & Aung, 2021). Cognitive functions can also be effected by sleep deprivation, since sleep-deprived brain need significantly more effort to complete any tasks, which can lead to bad decisions or work mistakes. Beyond the physical outcomes, sleep deprivation can also impact mood, energy levels and irritability, which is reflecting the intricate our mental health and well-being (Killgore, 2010). Another result of sleep depri- Proceedings of 10th Socratic Lectures 2024 38 of 234 vation can be disrupted hormonal regulation, which is influencing child’s growth and affecting appetite and glucose breakdown in adults (Davies et al., 2014). On a molecular level, sleep deprivation affects chromosome telomeres, contributing to genetic instability and potentially reducing cell lifespan. Disruptions extend to brain circuits, molecules, and genes governing the sleep-wake cycle, affecting both cognition and emotional responses (Murillo-Rodriguez et al., 2012). Poor sleep has an influence on executive function and declarative memory (Lo et al., 2016) and is prospectively linked with the development and progression of Alzheimer’s disease (Grandner & Fernandez, 2021). Chronic sleep loss provides oxidative stress, which can potentially increase DNA damage and slow down reparation processes that can also negatively produce various disorders, including reproductive and nephrological issues (Liew & Aung, 2021). Since sleep deprivation has also been associated with several medical conditions, we can easily say prioritizing quality sleep should be seen as a fundamental strategy for optimal physical and mental health. 3. Impact of sleep deprivation on physical activity and athletic performance In exploring the relationship between sleep and physical activity, researchers have found compelling evidence of sleep deprivation influencing various of performance components. Sleep deprivation not only impairs reaction time, accuracy, vigour, submaximal strength, and endurance but also compromises cognitive functions such as judgment and decision-making (Vitale et al., 2019). The consequences extend to resistance training outcomes and metabolic health, with just three consecutive nights of sleep restriction proving sufficient negative impacts (Knowles et al., 2018). The significance of the postural control system in sports and physical activity cannot be overlooked. Sleep deprivation has higher impact on men, which we can explain with higher Centre of Pressure (COP) values in postural control testing, underscores the gender-specific sensitivity to sleep disruption. This heightened sensitivity translates into reduced postural stability, posing an increased risk of injury during physical activity in both genders (Olpinska Lischka et al., 2021). Research by Wilms et al. (2020) indicates that the impact of sleep loss on physical activity is more pronounced when sleep is restricted during the second half of the night compared to the first half. Taking a closer look at athletes, even partial sleep deprivation, amounting to just 4 hours of sleep, can result in subtle yet potentially significant impairments in athletic performance (Cullen et al., 2019). The heightened risk of injury in athletes due to decreased attention and impaired judgment is further highlighted, with evidence suggest-ing that obtaining less than 8 hours of sleep per night is associated with an increased risk of injury (Tsukahara et al., 2023). One such novel concept is "banking sleep," a practice involving sleep extension before a planned night of intentional sleep deprivation before a sporting event. This approach, as proposed by Vitale et al. (2019), suggests that intentionally extending sleep in anticipation of sleep deprivation may serve as a proactive strategy to enhance performance. The concept of "banking sleep" challenges conventional perspectives on the immediate consequences of sleep loss and underscores the potential benefits of prioritizing sleep in the days leading up to a crucial athletic event. Considering these findings, it becomes evident that the relationship between sleep and athletic performance is substile, not only for the consequences of sleep deprivation but also for potential advantages of strategic sleep extension. 4. Conclusion We can conclude that sleep and physical activity go hand in hand when it comes to influence of sleep deprivation on physical activity and the impact of physical activity on sleep patterns. Despite their positive interaction, maintaining even a relatively low volume of physical activity can decrease the risks for insufficient sleep duration. Engaging in physical activity in the morning has been especially beneficial to optimize sleep hygiene, as suggested by Valente et al. (2023). The results in research of Kizilkilic et al. Proceedings of 10th Socratic Lectures 2024 39 of 234 (2023) are showing the importance of physical activity as a powerful intervention for an overall well-being. Moreover, the importance of sleep as a crucial daily regulator of both physical and mental health throughout all life stages is highlighted. Sleep disorders are linked to significant morbidity and are associated with various medical and psychiatric conditions. It is established that sleep deprivation, especially when sleep duration is lower than 7 hours per night, can result in adverse health outcomes. Recognizing sleep as a vital physiological function, it is crucial to acknowledge its paramount role in facilitating effective exercise recovery. In conclusion, both adequate sleep and regular physical activity are integral components of a holistic approach to maintain optimal health and well-being. Conflicts of Interest: The authors declare no conflict of interest. References 1. Albqoor MA, Shaheen AM. Sleep quality, sleep latency, and sleep duration: a national comparative study of university students in Jordan. Sleep Breath. 2021; 25: 1147-1154. DOI: 10.1007/s11325-020-02188-w 2. Bandyopadhyay A, Sigua NL. What Is Sleep Deprivation? Am J Respir Crit Care Med. 2019; 199(6): 11-12. DOI: 10.1164/rccm.1996P11 3. Baranwal N, Yu PK, Siegel NS. Sleep physiology, pathophysiology, and sleep hygiene. Prog Cardiovasc Dis. 2023; 77: 59-69. DOI: 10.1016/j.pcad.2023.02.005 4. Cullen T, Thomas G, Wadley AJ, Myers T. The effects of a single night of complete and partial sleep deprivation on physical and cognitive performance: A Bayesian analysis. J Sports Sci. 2019; 37: 2726-2734. DOI: 10.1080/02640414.2019.1662539. 5. Davies SK, Ang JE, Revell VL, Holmes B, et al. Effect of sleep deprivation on the human metabolome. Proc Natl Acad Sci. 2014; 111: 10761-10766. DOI: 10.1073/pnas.1402663111 6. Grandner MA, Fernandez FX. The translational neuroscience of sleep: A contextual framework. Science. 2021; 374: 568-573. DOI: 10.1126/science.abj8188 7. Hirshkowitz M, Whiton K, Albert SM, Alessi C, et al. National Sleep Foundation's sleep time duration recommendations: methodology and results summary. Sleep Health. 2015; 1: 40-43. DOI: 10.1016/j.sleh.2014.12.010 8. Kizilkilic SE, Falter M, Dandale P. The power of movement: how physical activity can mitigate the risks of inadequate sleep. Eur J Prev Cardiol. 2023; 30: 830-831. DOI: 10.1093/eurjpc/zwad121 9. Killgore WD. Effects of sleep deprivation on cognition. Prog Brain Res. 2010; 185:105-129. DOI: 10.1016/B978-0-444-53702-7.00007-5 10. Knowles OE, Drinkwater EJ, Urwin CS, Lamon S, et al. Inadequate sleep and muscle strength: Implications for resistance training. J Sci Med Sport. 2018; 21: 959-968. DOI: 10.1016/j.jsams.2018.01.012 11. Liew SC, Aung T. Sleep deprivation and its association with diseases- a review. Sleep Med. 2021; 77: 192-204. DOI: 10.1016/j.sleep.2020.07.048 12. Lo JC, Chong PL, Ganesan S, Leong RL, et al. Sleep deprivation increases formation of false memory. J Sleep Res. 2016; 25: 673-682. DOI: 10.1111/jsr.12436. 13. Mason GM, Lokhandwala S, Riggins T, Spencer RMC. Sleep and human cognitive development. Sleep Med Rev. 2021; 57: 101472. DOI: 10.1016/j.smrv.2021.101472 14. Murillo-Rodriguez E, Arias-Carrion O, Zavala-Garcia A, et al. Basic sleep mechanisms: an integrative review. Cent Nerv Syst Agents Med Chem. 2012; 12: 38-54. DOI: 10.2174/187152412800229107 15. Ołpińska-Lischka M, Kujawa K, Maciaszek J. Differences in the Effect of Sleep Deprivation on the Postural Stability among Men and Women. Int J Environ Res Public Health. 2021; 18: 3796. DOI: 10.3390/ijerph18073796 16. Rasch B, Born J. About sleep's role in memory. Physiol Rev. 2013; 93: 681-766. DOI: 10.1152/physrev.00032.2012 17. Tsukahara Y, Kodama S, Kikuchi S, Day C. Athlete Sleep Behavior Questionnaire in Japanese (ASBQ-J): An Adaptation and Validation Study. J Sports Med. 2023; 14: 89-97. DOI: 10.2147/OAJSM.S431820 18. Valente HB, Morelhão PK, Andersen ML, Tufik S, et al. Is sleep barrier to physical activity practice? J Clin Sleep Med. 2023; 19: 851-852. DOI: 10.5664/jcsm.10482 Proceedings of 10th Socratic Lectures 2024 40 of 234 19. Vitale KC, Owens R, Hopkins SR, Malhotra A. Sleep Hygiene for Otimizing Recovery in Athletes: Review an Recommendations. Int J Sports Med. 2019; 40: 535-543. DOI: 10.1055/a-0905-3103 20. Wilms B, Kuhr M, Chamorro R, Klinsmann N, et al. Chronobiological aspects of sleep restriction modulate subsequent spontaneous physical activity. Physiol Behav. 2020; 1: 215:112795. DOI: 10.1016/j.physbeh.2019.112795 Proceedings of 10th Socratic Lectures 2024 42 of 234 Invited lecture/Review Physiotherapy Approach for De Quervain Tenosynovitis Dur- ing and After Pregnancy Štuhec Maša1*, Vauhnik Renata1 1. Univerza v Ljubljani, Ljubljana, Slovenia * Correspondence: Maša Štuhec; masa.stuhec@gmail.com Abstract: De Quervain’s tenosynovitis is an attritional and degenerative process. Pain results from resisted gliding of the abductor pollicis longus and the extensor pollicis brevis tendons in the fibro osseus canal. This review aims to evaluate the current literature surrounding evidence on physiotherapy approach for treating De Quervain's tenosynovitis during and after pregnancy. One third of pregnant females are suffering from the pain in their wrist. Pain in the wrist is the third most common musculoskeletal pain during pregnancy after Citation: Štuhec M, Vauhnik R. low back pain. Fluid retention, oedema, and repetitive forceful hand movements may Physiotherapy Approach for De cause stenosing tenosynovitis of the hand and wrist tendons. An overweight mother with Quervain Tenosynovitis During her first pregnancy is at highest risk for developing De Quervain’s tenosynovitis. Treat-and After Pregnancy. ment is aimed at reducing inflammation, preserving movement in the thumb and prevent-Proceedings of Socratic Lectures . ing reoccurrence. The symptoms should improve within four to six weeks. If non-invasive 2024,10,42-45. treatment is not successful, corticosteroid injections can be applied. https://doi.org/10.55295/PSL.2024.I7 De Quervain’s tenosynovitis has a good prognosis and non-surgical treatments are usually effective for pregnancy-related hand and wrist problems. Publisher’s Note: UL ZF stays neu- tral with regard to jurisdictional claims in published maps and insti- tutional affiliations. Keywords: de Quervain’s tenosynovitis, pregnancy, post-partum, wrist pain, Finkelstein test 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/). Proceedings of 10th Socratic Lectures 2024 43 of 234 1. De Quervain’s tenosynovitis 1.1. Stenosing tenosynovitis De Quervain’s tenosynovitis (DQT) is a stenosing tenosynovitis of the first extensor compartment of the wrist (Ilyas et al., 2007). It is described to be an attritional and degenerative process, triggered by stenosing inflammation of the tendon sheath in the first dorsal compartment of the wrist (Ramchandani et al., 2022). Pain results from resisted gliding of the abductor pollicis longus and the extensor pollicis brevis tendons in the fibro osseus canal (Ilyas et al., 2007). This review aims to evaluate the current literature surrounding evidence on physiotherapy approach for treating De Quervain's tenosynovitis during and after pregnancy. 1.2. Epidemiology According to previous reports, DQT occurs up to six times more frequently in women than men and is associated with the dominant hand use during middle age (Ilyas et al., 2007). Often it is noted in pregnancy and during the postpartum period (Ramchandani et al., 2022). 1.3. Etiology In a descriptive study conducted by Kesikburun et al. (2018) 61 out of the 184 pregnant females complained of musculoskeletal pain in the hand and wrist in pregnancy. These problems were the third most common musculoskeletal complaints, after low back pains (Afshar & Tabrizi, 2021). Balik et al. (2014) studied the hand and wrist problems in 383 pregnant females and reported that 125 (32.6%) of pregnant females were asymptomatic patients and 67.4% of the pregnant females were suffering from hand and wrist problems (Balik et al., 2014). This condition is also the second most common hand and wrist problem during pregnancy and during the postpartum period after carpal tunnel syndrome (Thabah & Ravindran, 2015). Fluid retention, oedema, and repetitive forceful hand movements may cause stenosing tenosynovitis of the hand and wrist tendons (Kesikburun et al., 2018). Read et al. (2000) studied the histopathological appearances. Histopathological examination of the tendon sheaths revealed that myxoid degeneration was responsible for the remarkable thickening observed in the sheath. In addition, characteristic intramural deposits of mucopolysaccha-rides were present, predominantly in the sub synovial region. However, acute, or chronic inflammatory changes were not seen (Read et al., 2000). Etiology of this condition is combination between mechanical factors, anatomical dimorphism, and hormonal-driven pathological changes (Thabah & Ravindran, 2015). 1.4. Risk factors People between the ages of 30 and 50 have higher risk of developing De Quervain’s tenosynovitis than people in other age groups (Kesikburun et al., 2018). The condition may be associated with baby care, lifting a child and moves that involve repetitive movements using the thumbs as leverage. Studies showed that women who had caesarean section and multiple gestation were at higher risk for De Quervain’s tenosynovitis, while baby weight was not associated with the condition (Bae et al., 2023). Daglan et al. (2023) concluded that an overweight mother with prolonged pregnancy and her first pregnancy is at highest risk for De Quervain's tenosynovitis. Proceedings of 10th Socratic Lectures 2024 44 of 234 2. Treatment 2.1. Symptoms Symptoms of De Quervain’s tenosynovitis include pain near the base of the thumb, swelling, difficulty moving the thumb as well as the wrist and also some sensation can be felt in the thumb while moving it (Ilyas et al., 2007). The pain gets worse while grasping, lifting and during any motion where ulnar deviation of the wrist is present. Tenderness over the radial styloid can be felt as well as thickened first extensor retinacullum (Read et al., 2000). 2.2. Physiotherapy examination We ask the patient to perform Finkelstein test in which patient bends their thumb across the palm and bend their fingers over the thumb. Then they bend their wrist towards little finger. If that manevour causes pain on the radial side of the wrist, then the patient most likely has De Quervain’s tenosyovitis. 2.3. Physiotherapy treatment Possible treatment is application of the brace, especially during the night, in worse cases steroid injection is possible. On rare occasions that the non-surgical treatments fail to resolve the symptoms within 4-6 months, surgical release of the first extensor compartment can be offered (Ramchandani et al., 2022). Pidgeon (2022) reported that 50 % - 80 % of patients are successfully treated nonsurgically. Treatment is aimed at reducing inflammation, preserving movement in the thumb and preventing recurrence. The symptoms should improve within 4-6 weeks if the treatment starst early. But it is more likely that the condition will get better around the end of either pregnancy or at the end of the breast-feeding. (Balik et al., 2014). If any of ther intervations are not successfull, the corticosteroid injection can be applied, (Peters-Veluthamaningal et al., 2009). With safe dose of Methylprednisolone 10 mg (Avci et al., 2002) no contraindications during pregnancy or while lactating were seen (Larsen et al., 2021). 3. Conclusion De Quervain's disease of pregnancy and lactation is self-limited condition and non-surgical treatments are usually effective for pregnancy-related hand and wrist problems. In general, pregnancy-related De Quervain’s tenosynovitis has a good prognosis and usually resolves after reducing inflammation and movement preservation. Conflicts of Interest: The authors declare no conflict of interest. References 1. Afshar A, Tabrizi A. Pregnancy-related Hand and Wrist Problems. Archiv Bone Jt Surg. 2021; 9: 345–349. DOI: 10.22038/abjs.2020.50995.2531 2. Avci S, Yilmaz C, Sayli U. Comparison of nonsurgical treatment measures for de Quervain's disease of pregnancy and lactation. J Hand Surg Am. 2002; 27: 322–324. DOI: 10.1053/jhsu.2002.32084 3. Bae KJ, Baek GH, Lee Y, Lee J, Jo YG. Incidence and Risk Factors for Pregnancy-Related de Quervain's Tenosynovitis in South Korea: A Population-Based Epidemiologic Study. Clin Orthop Surg. 2023; 15: 145–152. DOI: 10.4055/cios22099 4. Balık G, Sabri Balık M, Ustüner I, Kağıtcı M, Sahin FK, Güven ES. Hand and wrist complaints in pregnancy. Arch Gynecol Obstet. 2014; 90: 479–483. DOI: 10.1007/s00404-014-3244-2 5. Daglan E, Morgan S, Yechezkel M, Rutenberg TF, Shemesh S, Iordache SD, Kadar A. Risk Factors Associated With de Quervain Tenosynovitis in Postpartum Women. Hand (NY). 2023; 24:15589447221150524. DOI: 10.1177/15589447221150524 6. Ilyas AM, Ast M, Schaffer AA, Thoder J. De quervain tenosynovitis of the wrist. J Am Acad Orthop Surg. 2007; 15: 757–764. DOI: 10.5435/00124635-200712000-00009 7. Kesikburun S, Güzelküçük Ü, Fidan U, et al. Musculoskeletal pain and symptoms in pregnancy: a descriptive study. Ther Adv Musculoskelet Dis. 2018; 10: 229–234. DOI: 10.1177/1759720X18812449 Proceedings of 10th Socratic Lectures 2024 45 of 234 8. Larsen CG, Fitzgerald MJ, Nellans KW, Lane LB. Management of de Quervain Tenosynovitis: A Critical Analysis Review. JBJS Rev. 2021; 10: 9. DOI: 10.2106/JBJS.RVW.21.00069. 9. Peters-Veluthamaningal C, van der Windt DA, Winters JC, Meyboom-de Jong B. Corticosteroid injection for de Quervain's tenosynovitis. Cochrane Database Syst Rev. 2009; 3: CD005616. DOI: 10.1002/14651858.CD005616.pub2 10. Ramchandani J, Thakker A, Tharmaraja T. Time to Reconsider Occupation Induced De Quervain's Tenosynovitis: An Updated Review of Risk Factors. Orthop Rev (Pavia). 2022; 14: 36911. DOI: 10.52965/001c.36911 11. Thabah M, Ravindran V. Musculoskeletal problems in pregnancy. Rheumat Int. 2015; 35: 581–587. DOI: 10.1007/s00296-014-3135-7 Proceedings of 10th Socratic Lectures 2024 47 of 234 Invited lecture/Research Effects of Therapeutic Approaches in Treating Varus Malalign- ment of the Knee Joint Ošlak Andreja1,*, Vauhnik Renata1 1. University of Ljubljana, Faculty of Health Sciences, Department of Physiotherapy, Ljubljana, Slovenia * Correspondence: Andreja Ošlak, andreja.oslak99@gmail.com Abstract: Knee malalignment is shown to be an independent risk factor for osteoarthritis progression. The knee adduction moment is directly correlated with varus malformation and can be decreased with changes in gait pattern, external foot rotation and external support. The aim of this literature review was to determine the effects of different therapeutic approaches in treating varus malalignment of the knee joint. The literature search was con-Citation: Ošlak A, Vauhnik R. ducted in the PubMed and EBSCO databases. We used a combination of English key-Effects of Therapeutic Approaches in words. Studies were screened regarding the inclusion and exclusion criteria. We included Treating Varus Malalignment of the five studies investigating the effects of therapeutic approaches in participants with or Knee Joint. without osteoarthritis onset. Statistically significant decrease in knee adduction moment Proceedings of Socratic Lectures . was reported in one study, which was implementing modified gait pattern with real time 2024, 10, 47-52. feedback. Other outcome measures were also indicative of potential efficacy in different https://doi.org/10.55295/PSL.2024.I8 therapeutic approaches. There is a bigger potential for treating varus malalignment before osteoarthritis (OA) onset. The results indicate that weight-bearing exercise and gait mod-Publisher’s Note: UL ZF stays neu- ification in combination with a corrective training protocol provide a potential useful ap-tral with regard to jurisdictional proach to reduce varus malalignment. claims in published maps and institu- tional affiliations. Keywords: Copyright: © 2024 by the authors. Varus malalignment, knee joint, knee adduction moment, therapeutic approaches Submitted for possible open access publication under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https://cre- ativecommons.org/licenses/by/4.0/). Proceedings of 10th Socratic Lectures 2024 48 of 234 1. Introduction 1.1. Mechanical forces of the lower extremity The lower extremity is in the frontal plane aligned through a mechanical axis which is in a neutral positioned extremity running from the hip joint, medially or through the middle of the knee joint to the ankle joint (Tetsworth and Paley, 1994). A malalignment is present when the axis is shifted medial or lateral of the knee joint, creating a moment arm and causing a disturbance in the load bearing of the joints (Sharma et al., 2010). Those changes can be visualized in the frontal plane as a dynamic worsening of the varus alignment as the lower extremity accepts weight in the stance phase (Chang et al., 2010). In young individuals the changes in the frontal plane are shown to be directly associated with changes in the transverse plane including increased internal foot placement and increased tibia rotation (Stief et al., 2014). 1.2. Varus malalignment and OA Knee malalignment is shown to be an independent risk factor in OA progression (Tanamas et al., 2009). Where greater knee varus at baseline is associated with greater odds of medial compartment OA progression (Sharma et al., 2010). OA is among others one of the most prevalent diseases in older adults and is limiting independence and functional activities, such as stair climbing, home chores, carrying bundles (Guccione et al., 1994). The knee adduction moment (KAM) is directly corelated with varus malalignment (Foroughi et al., 2009) and is consequently together with other mechanical forces, such as varus thrust associated with higher chances of OA progression in the medial compartment of the knee joint (D'souza et al., 2022). 1.3. Treatment of varus malalignment The KAM and the load on the medial knee compartment can be reduced by changing the gait pattern, external foot rotation or external support (Tetsworth and Paley, 1994). Conservative approaches can include knee bracing. Considering the results of the systematic review Yan et al. (2022) valgus braces could reduce symptoms of OA through decreasing the KAM and the varus angle and with it the improper distribution of load forces. In patients with unicompartmental OA in early degenerative stages high tibial osteotomy can be indicated to correct the varus deformity. The surgical procedure can decrease the symptoms and delay the need for knee replacement (Dowd et al., 2005). 1.4. Purpose The aim of this literature review was to determine the effects of different therapeutic approaches in treating varus malalignment of the knee joint. 2. Methods The literature search was conducted in the PubMed and EBSCO databases. The last review was carried out on the December 5, 2023. Following combinations of keywords were used: varus AND knee AND treatment; knee adduction moment AND varus AND treatment; knee adduction moment AND varus AND gait. Inclusion criteria were articles in English language, populations of subjects with varus malalignment with or without OA, interventions addressing the varus malalignment directly or through changes in KAM. Studies that investigated effects of orthotic interventions and studies that didn’t measure the change in varus malalignment or KAM were excluded. 3. Results After screening and eligibility assessment, five studies published between 2010 and 2021 were included in the review (Barrios et al., 2010; Bennell et al., 2010; Bennell et al., 2014; Choi & Shin, 2021; Jafarnezhadgero et al., 2018). The characteristics of the participants in the included studies are presented in Table 1. In three of the included studies (Barrios et al.,2010; Choi & Shin, 2021; Jafarnezhadgero et al., 2018) the mean age of participants was between 11.21 and 23.0 years. In two studies (Bennell et al., 2010; Bennell et al., 2014) the mean age of participants was between 62.2 and 64.6 years. Proceedings of 10th Socratic Lectures 2024 49 of 234 Table 1: Characteristics of the participants in the included studies. Study Pathologies Samples (n) F M Barrios et al. (2010) Varus, no OA 1 7 Bennell et al. (2010) Varus, with OA 43 46 Bennell et al. (2014) Varus, with OA 52 48 Choi & Shin (2021) Varus, no OA 4 4 Jafarnezhadgero et al. (2018) Varus, no OA 0 28 OA: osteoarthritis; F: female; M: male In three of the studies (Bennell et al., 2010; Bennell et al., 2014; Jafarnezhadgero et al., 2018) the participants were divided in to an experimental and a control group which enabled a between group comprehension. In two studies (Barrios et al.,2010; Choi & Shin, 2021) only an experimental group was present and comparison of the outcome measures before and after the implementation of the intervention was performed. The interventions and outcome measures used in the included studies are presented in Table 2. Table 2: Interventions and outcome measures and time of assessment used in the included studies. Study Intervention Outcome measure Time of assessment Barrios et al. (2010) Gait retraining with real-time Gait analysis Baseline feedback. Effort After intervention 8 weeks After 1 month Bennell et al. Hip strengthening exercise Gait analysis Baseline (2010) 12 weeks Knee pai, physical function After 13 weeks Strength measures Other Bennell et al. Neuromuscular vs quadriceps Gait analysis Baseline (2014) strengthening exercise Pain, physical function After 13 weeks 12 weeks Strength measures Physical performance Health related quality of life Other Choi & Shin (2021) Medial foot loading in gait Gait analysis Baseline 8 weeks Adverse effects After initial training After 8 weeks Jafarnezhadgero et Corrective training protocol Gait analysis Baseline al. (2018) 16 weeks Joint kinematics 6 days after intervention Quadriceps angle In the gait retraining study (Barrios et al., 2010) there was a statistically significant reduction in peak KAM in comprehension of the baseline and after intervention modified gait (p = 0.027). In comprehension of the baseline and after 1 month follow up modified gait a reduction in KAM was again present (p = 0.019). When comparing the baseline and both post intervention natural gaits no differences were observed. In the hip strengthening exercise study (Bennell et al., 2010) there were no between group differences for the KAM found (p = 0.193). In the neuromuscular vs quadriceps strengthening exercise study (Bennell et al., 2014) there was no withing group differences for changes in peak KAM from baseline for both groups. There were also no between group differences in changes of peak KAM (p = 0.23). The primary outcome measure used in the medial foot loading study Proceedings of 10th Socratic Lectures 2024 50 of 234 (Choi & Shin, 2021) was the knee adduction angle which served as an indicator of KAM. A significant change in the knee adduction angle was found at initial contact, mean angle during gait cycle and maximal abduction and adduction angle (p < 0.01). Similarly, the KAM wasn’t directly measured in the corrective training protocol study (Jafarnezhadgero et al., 2018). Within and between group differences were found for some of the hip, knee, and ankle joint angles for the dominant and nondominant lower extremity compared to baseline (p < 0.05). Statistically significant differences in other outcome measures used in the included studies are presented in Table 3 and Table 4. Results for within group differences were also presented for the outcome measures in the studies Bennell et al. (2010) and Bennell et al. (2014) although the statistical significance was not determined. Table 3: Within group differences for other outcome measures - before and after intervention. Study Other outcome measures Barrios et al. Modified Post SS ↑ peak hip IR (2010) gait intervention Follow up SS ↑ peak hip IR Natural Post No difference gait intervention Follow up No difference Choi & Shin SS ↑ walking speed, step length (2021) SS ↓ knee ADD angle (initial contact, mean, maximum) SS ↓ knee ABD angle (stance phase) SS ↑ hip ADD angle (initial contact, mean, maximum) SS ↑ ankle eversion (initial contact) SS ↑ foot ER (initial contact, mean, maximum) Jafarnezhadgero EG Dominant SS ↓ peak DF, foot IR, knee IR, hip ABD and ER et al. (2018) extremity SS ↑ peak knee ER Nondominant SS ↓ peak ankle inversion and eversion, foot IR, knee IR, hip extremity ABD and ER SS: statistically significant; ↑: increased; IR; internal rotation; ↓: decreased; ADD: adduction; ABD: adduction; ER: external rotation; EG: experimental group; DF: dorsal flexion Table 4: Between group differences for other outcome measures. Study Other outcome measures Bennell et al. (2010) SS ↑ ipsilateral pelvic drop – EG SS ↓ pain – EG SS ↑ physical function – EG SS ↑ strength (hip ABD and ADD, knee EXT) – EG Bennell et al. (2014) SS ↑ single leg stance – NS Jafarnezhadgero et al. (2018) Dominant extremity SS ↑ knee ER – CG SS ↓ peak foot IR, knee IR, hip ER and ABD – EG Nondominant ex- SS ↑ peak ankle inversion – CG tremity SS ↓ peak ankle eversion, foot IR, hip ER – EG SS: statistically significant; ↑ higher; EG: in favor of the experimental group; ↓: lower; ABD: abduction; ADD: adduction; EXT: extension; NS: in favor of the neuromuscular group; ER: external rotation; CG – in favor of the control group; IR: internal rotation Proceedings of 10th Socratic Lectures 2024 51 of 234 4. Discussion The results are indicating that effectiveness of the therapeutic approaches is greater when they are implemented to younger participants with no OA present (Barrios et al.,2010; Choi & Shin, 2021; Jafarnezhadgero et al., 2018). This coincides with the fact that the loss of bone and cartilage as a result of OA can also contribute to malalignment progression (Tanamas et al., 2009). Another possible explanation for more encouraging results in these studies could be usage of load bearing therapeutic approaches. Those are directly addressing the varus trust present as worsening of the varus malalignment when the lower extremity bears weight (Chang et al., 2010). However, it needs to be considered that the number of included participants was low (Barrios et al. (2010); Choi & Shin (2021) n = 8; Jafarnezhadgero et al. (2018); n = 28). As seen in the study by Barrios et al. (2010) the gait retraining program caused changes in the modified gait through time with gait becoming a less difficult task to perform. Since changes didn’t transfer into the natural gait pattern, this indicates that a 8-week training program was too short for a permanent change to occur. Nevertheless, the potential of this results is supported by the systematic review Richards et al. (2016) where gait retraining with real-time biofeedback was shown to be useful to reduce KAM in healthy controls. To improve the motor learning of gait modification, it’s also important to consider the practice structure in a blocked or random manner, the amount and timing of feedback provided and social-cognitive-affective aspects of learning such as motivation and attention (Charlton et al., 2020). The mayor limitation of this review is a small number of studies eligible for inclusion. Heterogeneity between the studies was limiting a direct comparison of their results. There were also limitations of the included studies such as low number of participants (Barrios et al., 2010; Choi & Shin, 2021; Jafarnezhadgero et al., 2018), no control group (Barrios et al.,2010; Choi & Shin, 2021; Jafarnezhadgero et al., 2018; Bennell et al., 2014) and no follow-up assessment except in one study (Barrios et al., 2010). An important question to consider is how the change in the gait pattern would affect not only the knee but also the other joints of the lower extremity. An impact reduction walking used in the study Tajima et al. (2018) was shown to decrease the ground reaction force, the external joint moments and loading rate. This approach of gait changing could potentially be useful for individuals with varus malalignment. Different therapeutic approaches have shown to have different impact on treating varus knee alignment. There is a bigger potential for treating varus malalignment before OA onset. Where weight-bearing exercise and gait modification in combination with a corrective training protocol provide a potential useful approach to reduce varus malalignment. Future research on bigger samples with lengthy protocols and direct varus alignment measures is needed to determine the effectiveness of this approaches. Conflicts of Interest: The authors declare no conflict of interest. References 1. Barrios JA, Crossley KM, Davis IS. Gait retraining to reduce the knee adduction moment through real-time visual feedback of dynamic knee alignment. Journal of biomechanics. 2010; 43(11): 2208-2213. DOI: https://doi.org/10.1016/j.jbiomech.2010.03.040 2. Bennell KL, Hunt MA, Wrigley TV, et al. Hip strengthening reduces symptoms but not knee load in people with medial knee osteoarthritis and varus malalignment: a randomised controlled trial. Osteoarthritis and cartilage. 2010; 18(5): 621-628. DOI: 10.1016/j.joca.2010.01.010 3. Bennell KL, Kyriakides M, Metcalf B, et al. Neuromuscular versus quadriceps strengthening exercise in patients with medial knee osteoarthritis and varus malalignment: a randomized controlled trial. Arthritis & Rheumatology. 2014; 66(4) :950-959. DOI: 10.1002/art.38317 4. Chang A, Hochberg M, Song J, et al. Frequency of varus and valgus thrust and factors associated with thrust presence in persons with or at higher risk of developing knee osteoarthritis. Arthritis Rheum. 2010; 62(5): 1403-1411. DOI: 10.1002/art.27377 5. Charlton JM, Eng JJ, Li LC, Hunt MA. Learning Gait Modifications for Musculoskeletal Rehabilitation: Applying Motor Learning Principles to Improve Research and Clinical Implementation. Phys Ther. 2021; 101(2): pzaa207. doi: 10.1093/ptj/pzaa207 Proceedings of 10th Socratic Lectures 2024 52 of 234 6. Choi S, Shin G. Effect of medial foot loading self-practice on lower limb kinematics in young individuals with asymptomatic varus knee alignment. The Knee. 2021; 30: 305-313. Doi: 10.1016/j.knee.2021.04.018 7. Dowd GS, Somayaji HS, Uthukuri M. High tibial osteotomy for medial compartment osteoarthritis. Knee. 2006; 13(2): 87-92. DOI: 10.1016/j.knee.2005.08.002 8. D'souza N, Charlton J, Grayson J, et al. Are biomechanics during gait associated with the structural disease onset and progression of lower limb osteoarthritis? A systematic review and meta-analysis. Osteoarthritis and Cartilage. 2022; 30(3); 381-394. DOI: 10.1016/j.joca.2021.10.010 9. Foroughi N, Smith R, Vanwanseele B. The association of external knee adduction moment with biomechanical variables in osteoarthritis: a systematic review. Knee. 2009; 16(5): 303-309. DOI: 10.1016/j.knee.2008.12.007 10. Guccione AA, Felson DT, Anderson JJ, et al. The effects of specific medical conditions on the functional limitations of elders in the Framingham Study. Am J Public Health. 1994; 84(3): 351-358. DOI: 10.2105/ajph.84.3.351 11. Jafarnezhadgero AA, Majlesi M, Etemadi H, Robertson DG. Rehabilitation improves walking kinematics in children with a knee varus: Randomized controlled trial. Annals of physical and rehabilitation medicine. 2018; 61(3): 125-134. Doi: 10.1016/j.rehab.2018.01.007 12. Richards R, van den Noort JC, Dekker J, Harlaar J. Gait Retraining With Real-Time Biofeedback to Reduce Knee Adduction Moment: Systematic Review of Effects and Methods Used. Arch Phys Med Rehabil. 2017; 98(1): 137-150. doi: 10.1016/j.apmr.2016.07.006 13. Sharma L, Song J, Dunlop D, et al. Varus and valgus alignment and incident and progressive knee osteoarthritis. Ann Rheum Dis. 2010; 69(11): 1940-1945. DOI: 10.1136/ard.2010.129742 14. Stief F, Böhm H, Dussa CU, et al. Effect of lower limb malalignment in the frontal plane on transverse plane mechanics during gait in young individuals with varus knee alignment. Knee. 2014; 21(3): 688-693. DOI: 10.1016/j.knee.2014.03.004 15. Tajima T, Tateuchi H, Koyama Y, et al. Gait strategies to reduce the dynamic joint load in the lower limbs during a loading response in young healthy adults. Hum Mov Sci. 2018; 58: 260-267. doi: 10.1016/j.humov.2018.03.002 16. Tanamas S, Hanna FS, Cicuttini FM, et al. Does knee malalignment increase the risk of development and progression of knee osteoarthritis? A systematic review. Arthritis Rheum. 2009; 61(4): 459-467. DOI: 10.1002/art.24336 17. Tetsworth K, Paley D. Malalignment and degenerative arthropathy. Orthopedic Clinics of North America. 1994; 25(3): 367-377. DOI: https://doi.org/10.1016/S0030-5898(20)31921-0 18. Yan Y, Liu G, Zhang L, et al. Biomechanical Effect of Valgus Knee Braces on the Treatment of Medial Gonarthrosis: A Systematic Review. Appl Bionics Biomech. 2022; 2022:4194472. DOI: 10.1155/2022/4194472 Proceedings of 10th Socratic Lectures 2024 54 of 241 Invited lecture/Research Validation of Interferometric Light Microscopy for Assessment of Extracellular Particles in 250 samples of Diluted Plasma: Pre- paring the Path for Future Clinical Practices Berry Maxence1,2, Arko Matevž2, Romolo Anna 2, Brložnik Maja 2,3, Mrvar Brečko Anita 4, Korenjak Boštjan2, Iglič Aleš 5,6, Kadunc Kos Vesna7, Kruljc Peter 7, Nemec Svete Alenka 8, Erjavec Vladimira 8, Kralj-Iglič Veronika 2 1. University of Poitiers, Poitiers, France 2. University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia 3. University of Veterinary Medicine Vienna, Clinic for Small Animals and Horses, Department of Diagnostic Imaging, Vienna, Austria 4. University Medical Centre, Ljubljana, Slovenia 5. University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana, Slovenia 6. University of Ljubljana, Faculty of Medicine, Laboratory of Clinical Biophysics, Ljubljana, Slovenia 7. University of Ljubljana, Veterinary Faculty, Clinics for Reproduction and Large Animals, Ljubljana, Slovenia 8. University of Ljubljana, Veterinary Faculty, Small Animal Clinic, Ljubljana, Slovenia * Correspondence: Veronika Kralj-Iglič; kraljiglic@gmail.com Abstract: In recent years, the analysis of extracellular particles (EPs) has become instrumental in de- ciphering intercellular communication and disease biomarkers. Interferometric light mi-Citation: Berry M, Arko M, Romolo croscopy (ILM) has emerged as a powerful label-free technique for real-time characteriza-A, Brložnik M, Mrvar Brečko A, Ko- tion of nanoscale particles. This contribution presents for the first time measurements of renjak B, Iglič A, Kadunc Kos V, EPs directly in diluted plasma by ILM on populations of samples. The study involves the Kruljc P, Nemec Svete A, Erjavec V, measured number density of EPs, the corrected number density of EPs (taking into account Kralj-Iglič V. Validation of Interfero-dilution of the sample) and the size of EPs. Analysis involves plasma of multiple species— metric Light Microscopy for Assess- canine, equine, and human. Most of the D h in all three species were confined to the interval ment of Extracellular Particles in Di-between 130 nm and 200 nm. We found no statistically significant correlation between the luted Plasma: Preparing the Path for corrected n and D h indicating good performance of the method. The correlation between Future Clinical Practices. the measured n and D h was statistically significant indicating hindered movement of EPs Proceedings of Socratic Lectures . due to their higher number density. These results showed that high throughput measure-2024, 10, 54-58. ment of number density and size of EPs in plasma is feasible. https://doi.org/10.55295/PSL.2024.I9 Publisher’s Note: UL ZF stays neu- tral with regard to jurisdictional claims in published maps and insti- Keywords: Extracellular vesicles; Interferometric Light Microscopy; Plasma analysis; Natutional affiliations. noparticle characterization; Videodrop technology; Nanoparticles 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/). Proceedings of 10th Socratic Lectures 2024 55 of 241 1. Introduction In recent years, the study of EPs has emerged as a crucial avenue for understanding intercellular communication and disease biomarkers (Welsh et al., 2024). Among various analytical techniques, ILM has proven to be a powerful tool for real-time, label-free characterization of nanoscale particles of different types (Romolo et al., 2022). This technology already proved its utility by quantifying viruses in a river (Roose-Amsaleg et al., 2017) or by analysing aquatic biotic nanoparticles (Boccara M et al., 2016). In this contribution, we present the application of ILM to the measurement of extracellular particles in diluted plasma. ILM enables assessment of number density and size of particles in the size range of 80 to 500 nm. A few microliter sample is needed. The aim of this contribution is to analyse populations of samples of plasma from different species by ILM. 2. Material and methods 2.1. Preparation of the samples Animal blood samples were taken from expired transfusion bags. Three bags of canine blood and 1 bag of equine blood were used. Human blood samples were donated by the authors. Collection was established in the morning after fasting for a minimum of 12 h overnight. A G21 needle (Microlance, Becton Dickinson, Franklin Lakes, NJ, USA) and 2.7 mL evacuated tube with trisodium citrate (BD Vacutainers, 367714A, Becton Dickinson, Franklin Lakes, NJ, USA) were used. Blood samples were centrifuged (centrifuge Centric 400/R, Domel, Železniki, Slovenia) at 18°C and different centripetal accelerations (between 50  g and 1000  g) for different times (from 5 min and 30 min) to obtain plasma. We analysed 250 samples (81 canine, 113 equine and 56 human). For assessment with ILM, plasma was diluted to such extent to enable saturation of the interferred light. As the medium we used saline for injections 9 mg/mL (B Braun, Melsungen AG, Melsungen, Germany). The probe dilution was 50 ×. 2.2. Interferometric light microscopy The number density of EPs in the sample ( n) and the average hydrodynamic diameter of EPs in the sample ( D h) were determined by ILM using Videodrop (Myriade, Paris, France). Signals from the medium (saline for injections) was under the detection limit. The threshold value of 4.2 was used. Seven microliters of sample were placed between cover glasses and illuminated by 2 W of blue LED light. The light scattered on the particle was imaged by a bright-field microscope objective and allowed to interfere with the incoming light. The image was recorded by a complementary metal-oxide-semiconductor high-resolution high-speed camera. The number density of the particles is the number of detected particles within the detected volume, which depends on the microscope characteristics and the particles’ size. The typical detection volume was 15 pL. D h was estimated by tracking the position of the imaged particle within the recorded movie. It was assumed that particles undergo Brownian motion due to collisions with surrounding particles. The diffusion coefficient D of the motion of the particle is taken to be proportional to the mean square displacement d of the particle between two consecutive frames taken in the time interval ∆ t, < d 2(∆ t)> = <4 D ∆ t>, while D h was estimated by assuming that the particles were spherical and using the Stokes–Einstein relation D h = kT/3πη D. Each particle that was included in the analysis was tracked and processed individually, and the respective incident light signal was subtracted from each image. Processing of the images and the movies was performed by using the associated software, QVIR 2.6.0 (Myriade, Paris, France). More details are given in Romolo et al., (2022). 2.3. Design of the study We assume that centrifugation of blood within the above range of centripetal accelerations and times of centrifugation would not sediment EPs. However, larger centripetal accelerations and longer times more effectively sediment erythrocytes than smaller centripetal accelerations and shorter times of centrifugation. Motion of mutually interacting erythrocytes pushes plasma carrying EPs upwards into the compartment that is gathered for analysis (Božič et al., 2022). We do not expect changes in the average size of the EPs but we could expect that there would be different number densities of EPs in plasma obtained Proceedings of 10th Socratic Lectures 2024 56 of 241 by different centrifuge settings. Recommendations of the producer of the ILM (Myri-adelab, Paris, France) indicate that measurements of number density between 5×108 /mL and 5×109 /mL are reliable. We therefore hypothesized that the dependence of n on D h in the range where the measurements are reliable would be constant. We considered measured n of the diluted plasma sample and n corrected for dilution. The corrected values give information on the EP content of plasma while the measured values are of interest to analyse the performance of the instrument. 3. Results Figure 1 shows dependence of EP D h on n in canine, equine and human plasma . Different colors represent different species (orange – dog, blue – horse, black – human). In upper panels (A and B) n were corrected for dilution and lower panels (B and D) show measured n. Most of the D h in all three species were confined to the interval between 130 nm and 200 nm (Figure 1). There were four points considerably out of this range in Panels A and C, therefore we performed analysis also by excluding these 4 outliers (Panels B, D). While corrected n of EPs in human samples extended more or less evenly over a wide interval, clusters of blue and orange points pertaining to a particular animal can be distinguished in Panels A and B. Figure 1. Hydrodynamic diameter of EPs in plasma ( D h) in dependence on the number density of EPs ( n). Panels A and B: corrected n. Panels C and D: measured n. In Panels B and D, 4 outliers with D h over 230 nm have been removed from respective Panels A and C. Orange circles: canine samples, blue circles: equine samples, black circles: human samples. Table 1 shows Pearson correlation coefficient between n and D h. We found no statistically significant correlation between the corrected n and D h while the correlation between the measured n and D h was statistically significant. Removing 4 outlier points from the graph did not considerably affect the statistical analysis results. Proceedings of 10th Socratic Lectures 2024 57 of 241 Table 1. Pearson coefficients of the correlation between the corrected n and D h and between the measured n and D h. All data 4 outliers removed Corrected D h/ n Measured D h/ n Corrected D h/ n Measured D h/ n Pearson coefficient <0.001 0.38* 0.08 0.41* p >0.05 <10-4 >0.05 <10-4 Asterisk denotes statistically significant result. 4. Discussion We have for the first time performed measurement of EPs directly in diluted plasma in a large number of samples. We observed that the hydrodynamic diameter of EPs was in all species between cca 130 and 200 nm (Figure 1). The number densities of EPs in the samples varied and seemed to be characteristic to a donor (Figure 1). We observed no correlation of the corrected n of EPs in plasma and D h (Table 1) which we assumed to be in agreement with the dynamics of the sedimentation of particles during centrifugation. In contrast, we observed a statistically significant positive correlation between measured n and D h (Table 1). As Dh is determined by using the Einstein equation and the diffusion coefficient of the medium is assessed from the record of the movement of the particles, it is possible that the structure of plasma may hinder the movement of the particles depending on their concentration. If there are more particles in the plasma, they are restricted in their motion which is reflected in an overestimated size. However, in practice we are interested in concentration of EPs in blood or in plasma, both of which are relevant for therapeutic purposes. ILM performed well (Figure 1) as D h and corrected n showed no correlation. It was reported that the number density of extracellular vesicles in ex-vivo samples of human blood plasma were estimated at 1010 particles/mL but may be increased as a result of various pathological conditions (Božič et al., 2019). We have obtained n values up to the order of 1011 EPs per mL. Estimating viscosity of plasma by the viscosity of water, hydrodynamic diameter D h remained between 130 and 200 nm, which agrees with the results on extracellular vesicles isolated from plasma obtained by dynamic light scattering (150 nm) (Božič et al., 2019). 5. Conclusions We report on the first measurements of EPs in diluted plasma by ILM. The estimation of size and number density are in agreement with the data from the literature. It is indicated that high throughput measurement of number density and size of EPs in plasma is feasible. The hydrodynamic diameter of EPs in plasma did not depend on the concentration of EPs in the sample while number density of EPs measured in diluted plasma positively correlated with the hydrodynamic radius indicating hindered movement of EPs due to higher number density. Further research is needed to explore the potentials and limitations of ILM, yet our results indicate that it is a method that should be reckoned with in the study of EPs and extracellular vesicles. Funding: This research was supported by Slovenian Research Agency grant numbers P2-0232, P3-0388, P4-0053, J3-3066, and J2-4447, the Lendület (Momentum) Programme of the Hungarian Academy of Sciences, Project SNN 138407 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the SNN_21 funding scheme. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, blood was donated voluntarily by the authors of the study. Conflicts of Interest: The authors declare no conflict of interest. Proceedings of 10th Socratic Lectures 2024 58 of 241 References 1. Boccara M, Fedala Y, Vénien-Bryan C, et al. Full-field interferometry for counting and differentiating aquatic biotic nanoparticles: From laboratory to Tara Oceans. Biomed Opt Express 2016; 7: 3736–3746. DOI: 10.1364/BOE.7.003736 2. Božič D, Sitar S, Junkar I, et al. Viscosity of plasma as a key factor in assessment of extracellular vesicles by light scattering. Cells. 2019; 8:1046. DOI: 10.3390/cells8091046 3. Božič D, Vozel D, Hočevar M, et al. Enrichment of plasma in platelets and extracellular vesicles by the counterflow to erythrocyte settling. Platelets. 2022;33:592-602. DOI: 10.1080/09537104.2021.1961716 4. Romolo A, Jan Z, Bedina Zavec A, et al. Assessment of Small Cellular Particles from Four Different Natural Sources and Liposomes by Interferometric Light Microscopy. Int J Mol Sci. 2022; 23:15801. DOI: 10.3390/ijms232415801 5. Roose-Amsaleg C, Fedala Y, Vénien-Bryan C, et al. Utilization of interferometric light microscopy for the rapid analysis of virus abundance in a river. Res. Microbiol. 2017; 168: 413–418. DOI: 10.1016/j.resmic.2017.02.004 6. Welsh JA, Goberdhan DCI, O’Driscoll L, et al. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J Extracellular Ves. 2024, e12404, DOI: 10.1002/jev2.12404. Proceedings of 10th Socratic Lectures 2024 60 of 234 Invited lecture/Review Glycosylation Research in Bovines-the Significance and Recent Updates Beletić Anđelo1*, Duvnjak Orešković Ivana1, Pribić Tea1, Krištić Jasminka1, Lauc Gordan1,2 1. Genos Ltd, Glycoscience Research Laboratory, Borongajska cesta 83H, 10000 Zagreb, Croatia 2. Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb * Correspondence: Anđelo Beletić; abeletic@genos.hr Abstract: Glycosylation is an enzymatic process of attaching carbohydrate chains, glycans, to biomolecules, thereby influencing their biological features. Understanding the glycosylation Citation: Beletić A, Duvnjak patterns and mechanisms in bovines ( Bos taurus) has the potential to bring improvements Orešković I, Pribić T, Krištić J, in various fields, aspects such as reproduction, herd health management, and the quality Lauc G. Glycosylation research in bo-and safety of milk and meat products. The article, starting with a glimpse into glycobiol-vines-the significance and recent up- ogy, will continue with overviewing the previous 5-year achievements of glycosylation in dates. bovines, collated during a recent PubMed search. Hereafter, more details about the four Proceedings of Socratic Lectures . studies will follow as the selected examples and go along with the concluding remarks 2024, 10, 60-65. and general future research directions. https://doi.org/10.55295/PSL.2024.I10 Publisher’s Note: UL ZF stays neu- tral with regard to jurisdictional claims in published maps and insti- Keywords: data mining, glycosylation, bovines tutional 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/). Proceedings of 10th Socratic Lectures 2024 61 of 234 Glycosylation represents the enzymatic formation of a glycoconjugate, which consists of carbohydrate chains, or glycans, covalently linked to different biomolecules such as proteins, lipids, or RNA (Reily et al., 2019; Trbojević Akmačić et al., 2022). Glycobiology is a comprehensive science focusing on glycans, with the research spanning their biological chemistry, biosynthesis, evolution, (patho)physiological roles, analysis, and biotechnology aspects. It initially appeared among natural sciences and rapidly attracted interest from many other fundamental, biomedical, and biotechnology disciplines (Varki et al., 2022). The article will start with a brief explanation of glycobiology key postulates. It will continue with an insight into the reasons for researching glycosylation in bovines. After, four examples illustrating the relevance will follow. Concluding the article will provide a summary of the current findings' relevance and future research directions. 1. A glimpse into glycobiology Mammals are estimated to possess trillion different branched glycan structures, formed through the combination of 17 monosaccharides with multiple glycosylation sites, numerous enzymes involved, and the potential for α or β stereochemical conjugations (Reily et al., 2019). Various biomolecules serve as substrates for glycosylation, resulting in the formation of glycoproteins, proteoglycans, glycosphingolipids, glycosaminoglycans, or gly-coRNA. In addition, glycobiology also grants interest for free oligosaccharides, adding further complexity to the field (Trbojević Akmačić et al., 2022). Macro- and microheterogeneity are crucial concepts in understanding glycobiology. For example, in glycoproteins, multiple glycosylation sites on the polypeptide “backbone” constitute macroheterogeneity, while microheterogeneity indicates the presence of different glycans on the same glycosylation site. Finally, if only the N-glycan supplement distinct glycoproteins, they represent glycoforms (Varki et al., 2022). In eukaryotes, N- and O-glycans are two major types of glycans. The N-glycans attach via the side chain of an asparagine (Asn) residue within the peptide sequence Asn-X- serine (Ser)/threonine (Thr), where X stands for any amino acid except proline. Their structural core consists of two acetylglucosamines (GlcNAc) conjoined with a mannose structure (Man), which holds two additional mannoses lined at positions 3 and 6 (Varki, Cummings et al., 2022). According to biosynthetic maturation, indicated by the structures extending the core, four glycan types exist: pauci-mannosidic, oligo-mannosidic, complex, and hybrid. In pauci-mannosidic extensions are lacking, while in the oligo-mannosidic, they form only of Man residues. The characteristic of the complex glycans is the presence of up to five antennae, whose structures start with the GlcNAc and continue with the repeating N-acetyllactosamine residues. Hybrid glycans have oligomannose extensions at core position 6, while up to two antennae appear at position 3. More precise biosynthesis phase annota-tion comes with additional derived traits like antennarity, sialylation, galactosylation, fucosylation (core/antennary), or bisection (de Haan et al., 2022). The attachment sites for Oglycans are Ser or Thr residues. The O-glycans (patho)biological contexts typically diverge into features related to O- N-Acetylgalactosamine (GalNAc) and O-GlcNAc glycans. For O-GalNAc glycans, four core structures are available, each with GalNAc bound on Ser or Thr residue, differing by the presence of Gal and GlcNAc. The structure of O-GlcNAc glycans is less complex because they contain only GlcNAc attached (Varki et al., 2022). The endoplasmatic reticulum and Golgi complex are intracellular locations of the three-step N-glycosylation process, involving the generation of the lipid-linked oligosaccharide donors, co-translational glycan shifts onto glycosylation sites on polypeptide chain, and further processing of the glycans. Regulation of this comprehensive process relies on modifying the activity of enzymes involved, glycosyltransferases and glycosidases, at the transcriptional or posttranscriptional level (phosphorylation, glycosylation, availability of chaperones). Besides, the availability of their substrates also contributes to the regulation (Esmail et al., 2021). At the molecular level, N-glycans play a substantial role in proper protein folding, assembling of multimeric proteins, and stabilizing their structures. In the physiological context, Proceedings of 10th Socratic Lectures 2024 62 of 234 N-glycosylation is involved in a network of comprehensive mechanisms such as cell adhesion, recognition of foreign pro- and eucaryotic cells, or receptor activation. N-glycosylation also attracted the attention of pathophysiology and clinical disciplines. Congenital disorder of glycosylation syndrome Type I, an autosomal recessive multi-system disorder, has a depleted N-glycosylation as the cause. Changes in cell membrane N-glycome are characteristic of malignancy, thus raising the potential for advanced oncological biomarkers discovery. Terminal sialylation and fucosylation usually change, while increased branching relates to growth, infiltration, and metastasis. N-glycome alterations are frequent in chronic diseases. The best example from that field is rheumatoid arthritis (RA), an autoimmune arthritis associated with changes to the galactosylation of Ig G, where the disease severity relates to these changes in antigen-specific IgGs. Research advancements prompt additional exploration into efforts on N-glycosylation as a potential therapeutic target (Esmail et al., 2021). 2. What is the importance of researching glycosylation in bovines? Understanding the glycosylation patterns and mechanisms in bovines ( Bos taurus) has the potential to bring improvements in various fields, aspects such as reproduction, herd health management, and the quality and safety of milk and meat products (Beletić et al., 2023a). For example, the results of a recent proteomic study in cows with the retained placenta (Beletić et al., 2023b) suggested as potential biomarkers lipopolysaccharide-binding protein and haptoglobin, both of which are glycoproteins (https://www.uniprot.org). A proteomic study of milk from dairy cows with subclinical mastitis (Beletić et al., 2022) indicated that a higher abundance of thrombospondin-1, a glycoprotein (https://www.uniprot.org), could differentiate between samples with Staphylococcus spp. and Streptococcus spp. as the causative agents. Recently, we have performed a PubMed search for “glycosylation” and “B. taurus” using the following filters: full text available, the publication date of five years, and the preprints excluded. From the 244 initially retrieved results, the content analysis identified 88 as eligible, among which only one was a review article. These studies primarily focused on functional aspects and glycan profiles, with milk and tissues being the most common sample types. Among them, ten studies provided data on the total glycome of milk or tissue samples, while many others analyzed glycosylation of individual proteins, with fetuin being the most frequently studied (Beletić et al., 2023a). Apart from the associated (patho)biological relevance, which is still challenging, bovine fetuin is also worth mentioning in the analytical context as a frequent testing analyte during the development of glycoproteomics analytical approaches (Achim et al., 2023). Milk-related studies brought the data about total glycome, or individual proteins, like casein or IgG (Beletić et al., 2023a), which are of particular relevance in the assessment of milk quality and nutritive value (O'Riordan et al., 2014). 3. The recent updates-selected examples In a recent paper, Dilimulati et al. (2023) presented intriguing findings about the involvement of N-glycosylation in the interaction between sperm and oocyte proteins in bovines. In mammalian species, a layer called zona pelucida (ZP) surrounds oocytes and has essential functions in oogenesis, fertilization, and preimplantation. The ZP structure has long interconnected fibrils harboring ZP glycoproteins (ZPGs), and ZP thickness, protein content, and N-/O-glycosylation represent a species specificity (Wassarman, 2008; Yonezawa, 2014). ZP in bovines contains three ZPG3 annotated bZP2, bZP3, and bZP4. In vitro results identified the N-terminal domain of bZP4 (ZP-N1) and the middle region of bZP4 as substantial for sperm binding but without evidence for N-glycosylation of ZP-N1 as necessary for this purpose (Dilimulati et al., 2022). Further research of the bZP4 middle region (including the hinge domain crucial for the bZP3-bZP4 complex formation, which is essential for sperm binding) showed species-specific sperm binding. Additional analyses associated the bZP4 middle region function with N-glycosylation at Asn-314 and marked N-glycosylation sites at Asn-314 (near the hinge region) and Asn-146 (within the hinge of the bZP3-bZP4 complex) as required for the unhampered sperm binding (Dilimulati et al., 2023). These findings significantly contribute to a better understanding of between- Proceedings of 10th Socratic Lectures 2024 63 of 234 species differences in ZP functions. Identification of sperm-associated factors responsible for sperm-ZP interactions and validation via in vivo bovine fertilization research represents the primary research efforts to achieve reliability in bovine reproduction practice (Dilimulati et al., 2022). The review by Zlatina and Galuska (2021) comparatively assessed N-glycosylation traits of the bovine and human lactoferrin (Lf), primarily guided by the fact that the bovine variant often serves as a model for developing innovative therapeutic strategies. Lf is a whey protein present in most mammals. Human Lf (hLf) also occurs in other organs and fluids such as kidneys, lungs, liver, prostate, saliva, plasma, and immune cells, having numerous effects: antimicrobial defense, immunomodulation, antioxidant protection, or microbiome homeostasis (Kowalczyk et al., 2022). The primary structures of hLf and bovine Lf (bLf) have a 69% analogy. Five N-glycosylation sites exist on bLf (two more than hLf). Four sites have 100% occupancy, and the fifth varies between 15% in mature milk and 30% in colostrum. Consequently, two bLf variants appear, differing in glycosylation pattern, molecular mass, and colostrum/mature milk abundance. Structural analyses of bLf and hLf suggested the N-glycans' significance with potential effects on their functionality. Their removal prominently reduces the iron-binding properties, the primary Lf function, and the “weapon” against bacteria. The site-specific presence of N-glycans on bLf is a potential inhibitor of proteases. The large amount of high-mannose N-glycans, rare among extracellular proteins of mammalian species, also differentiates bLf from hLf and might induce a quick clearance of the gastrointestinal tract and bloodstream mediated by mannose-recognizing receptors on macrophages. Another feature of bLf glycome is the presence of N-Glycolylneuraminic acid, a non-human form of sialic acid, which, besides enhancing the clearance of dietary bLf, also links to antigenicity and potential adverse effects on the efficiency of the immune response. Notwithstanding the relevance of the described advances for deciphering the bLf effects in humans, prudence is necessary in their interpretation, originating from the comprehensive network of molecular (like amino acid sequence), physiological (breed, lactation time, or diet), and methodological features (Zlatina et al., 2021). Fat, casein, and whey represent the three milk components (Haug et al., 2007). According to their amount, the whey proteins belong to highly (α-lactalbumin, β-lactoglobulin, serum albumin, IgG, glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1), and Lf), moderate (IgA, IgM, lactoperoxidase, and osteopontin) and minor (lysozyme and folate binding protein) abundant group (O’Riordan et al., 2014; Tacoma et al., 2016). Whey proteins are ingredients in numerous food products, including infant formulas. Considering N-glycans' relevance for the glycoprotein functional properties, an assessment of the bovine whey total glycome profile would be significant for predicting the quality and safety of whey-containing food products. Valk-Weeber et al. (2020) analyzed the role of individual glycoproteins in determining the comprehensive N-linked glycoprofile of bovine whey. In accordance with the protein abundance, approximately 95% of N-glycans in the total glycome originated from Lf, IgG, and GlyCAM-1. As expected, Lf dominantly participated with oligomannose-type glycans. IgG was the source of fucosylated di-antennary glycans with N-Acetyllactosamine domains. GlyCAM-1 was the only whey glycoprotein carrying highly fucosylated and sialylated tri- and tetra-antennary glycans. Analyzing differences between early and late colostrum and mature milk revealed the complex interactions between the glycoprofile alterations (dominanly higher sialylation and fucosylation degree in early colostrum) and dynamics of glycoproteins concentrations. Food fraud is a growing general issue in food safety and quality and is particularly common among animal products. Species identification is one of the key tasks in preventing adulteration problems with foods of animal origin (Smaoui et al., 2023). Tai et al. (2023) developed a glycopeptide-based analytical pipeline to identify six meat species: pork, beef, mutton, chicken, duck, and turkey. The first phase of their experiment was the sarcoplas-mic proteome analyses using one-dimensional gel electrophoresis, which showed that enolase could differentiate livestock and poultry origin. Applying ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry confirmed the species specificity of enolase glycoprofiles. The validation procedure, aiming to Proceedings of 10th Socratic Lectures 2024 64 of 234 identify livestock, poultry, and mixed meat, yielded promising results, which might be even superior to the currently available analytical platforms. 4. Conclusions The pioneer insight provided in this article allowed for the conclusion on the availability of reliable data about glycosylation in the bovines and the incarnated challenges. As such, they are eligible as the starting point for further scientific efforts on their continuous ap-pending, systematization, and multidisciplinary analyses. 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DOI: 10.1007/978-1-4939-0817-2_6 Proceedings of 10th Socratic Lectures 2024 67 of 241 Invited lecture/Research Omics Mass Spectrometry Analysis of Canine Plasma Schlosser Gitta1*, Molnár Adrienn1,2, Papp Dávid1, 2, Gellén Gabriella1, Virág Dávid3, Ludányi Krisztina3, Dalmadi-Kiss Borbála3, Arko Matevž5, Iglič Aleš5, Svete Nemec Alenka6, Erjavec Vladimira6, Kralj-Iglič Veronika5,* 1. MTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, Faculty of Science, Institute of Chemistry, ELTE Eötvös Loránd Uni versity, Budapest, Hungary 2. Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, H-1117 Budapest, Hungary 3. Department of Pharmaceutics, Semmelweis University, H-1092, Budapest, Hungary 4. University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia 5. University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Clinical Biophysics, Ljubljana, Slovenia 6. University of Ljubljana, Veterinary Faculty, Clinics for Small Animals, Ljubljana, Slovenia * Correspondence: Gitta Schlosser; gitta.schlosser@ttk.elte.hu; Veronika Kralj-Iglič, veronika.kralj-iglic@zf.uni-lj.si Abstract: Blood derived products, such as autologous plasma, have high clinical importance and Citation: . Schlosser G, Molnár A, are applied in numerous therapeutic fields. The preparation of autologous plasma from Papp D, Gellén G, Virág D, Ludányi the patient's own blood is easy to perform by centrifugation, however, the preparation K, Dalmadi Kiss B, Arko M, Iglič A, procedure can significantly affect the blood cells, platelets and vesicles in the sample. Svete Nemec A, Erjavec V, Kralj-Iglič Therefore, it is of utmost importance to understand the impact of sample processing on V. Omics Mass Spectrometry Analy-the chemical composition of plasma preparations as well as on their biological activity. sis of Canine Plasma. Here, we present a mass spectrometry-based plasma profiling method in which three Proceedings of Socratic Lectures. compound groups: lipids, proteins and glycoproteins are analysed in a single workflow. 2024, 10, 67-72. Analysis of the chemical composition of plasma samples prepared by different centrifu-https://doi.org/10.55295/PSL.2024.I11 gation protocols revealed differences in the lipid and glycoprotein profiles, demonstrating the importance of standardized protocols for the preparation of plasma products. Publisher’s Note: UL ZF stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Keywords: Plasma; Lipids; Proteins; Glycoproteins; Mass Spectrometry, Ion Mobility 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/). Proceedings of 10th Socratic Lectures 2024 68 of 241 1. Introduction Analytical characterisation of complex biological samples is still a challenging and time-consuming task, especially if a comprehensive analysis is planned. Mass spectrometry (MS) is a leading analytical method for the chemical analysis of biological samples, as MS-based measurements can be easily combined with other analytical and separation techniques. For this reason, mass spectrometry has a key role in "omics" research, where the characterisation of different sets of biomolecules is performed. Plasma can be produced from blood collected in tubes treated with an anticoagulant. The blood is then processed with centrifugation to remove cells and the autologous plasma is often used for therapeutic purposes (Troha et al., 2023). For example, the eradication of a wound in the middle ear was demonstrated after the application of autologous plasma (Božič et al., 2020). Nevertheless, the exact composition of human plasma varies from individual to individual and is largely affected by the production and storage process. It is therefore important to develop methods to characterise the molecular composition of plasma-derived products. The first step, the centrifugation procedure by which blood plasma is prepared has not yet been standardised. In this work, the lipid, glycoprotein and protein content of blood plasma was investigated using a newly developed multi-step sample preparation combined with mass spectrometry. Dog (Canis lupus familiaris) plasma samples, subjected to two different centrifugation steps, were compared to detect possible chemical differences due to differences in the preparation. 2. Methods 2.1 Blood sampling Blood was from expired transfusion bag (Figure 1). The donor was a 2 years old female dog without record of the disease. Before pouring the blood the content of the bag was homogenized by gently squeezing the bag. Blood was aliquoted into 3 ml tubes by gently pushing it through attached plastic tubes. Figure 1. A bag of expired canine transfusion blood. 2.1 Preparation of plasma samples Two different canine plasma sample sets were prepared by centrifugation at 2000 g for 5 min (at 30 °C), and by centrifugation at 4000 g for 30 mins (at 30 °C). 4-4 technical replicates were prepared for both samples. 2.2 Sample preparation for mass spectrometry In the first step, lipids were extracted with the following protocol: 150 μL methanol was added to 20-20 μL plasma samples. After mixing, 500 μL methyl- t-butyl ether (MTBE) was added and the samples were incubated at room temperature for 1 hour under shaking at 350 rpm. 125 μL MilliQ water was added, and the incubation was repeated for 10 mins. Samples were centrifuged for 10 mins at 14800 rpm. The upper organic phases were collected, and the extraction of lipids was repeated by 500 μL MTBE. The combined organic Proceedings of 10th Socratic Lectures 2024 69 of 241 phases were vacuum dried. The lower aqueous phases, containing the proteins were further processed in the next steps. 6-6 μL of 0.2%-os RapiGest SF detergent solution were added to 100-100 μL aqueous phases, as well as 2 μL 10 mM dithiotreiol (DTT) solution. Samples were reduced at 60 °C for 30 mins. After cooling to room temperature, 15 μL 200 mM ammonium hydrogen carbonate solution and 2 μL 20 mM iodoacetamide solution were added and the proteins were alkylated for 30 mins in the darkness. Then, 1-1 μL of 0.1 mg/ml trypsin was added and the samples were incubated overnight 37 °C under gentle shaking. 3 μL of formic acid was added to quench the enzymatic reaction and the samples were vacuum dried. In the last step, glycopeptide enrichment was performed. Briefly, the samples were reconstituted in 50 μL of 1% formic acid and 500 μL ice-cold acetone was added to the samples and the samples were then kept overnight at -20 °C. Samples were centrifuged for 10 mins at 12000 rpm to isolate to pellet enriched in glycopeptides. The pellet and the supernatant were then separated and vacuum dried. Peptide solutions were further purified using PierceTM C-18 Spin columns (the glycopeptide fractions were analysed without additional clean-up) 2.3 Mass spectrometry Chromatographic separations were performed using a Waters Acquity I-Class UPLC System, while MS experiments were performed on a high resolution and high mass accuracy hybrid Q-TOF equipped with cyclic ion mobility separation cell (Waters Select Series Cyclic IMS, Waters Corporation) and an electrospray ionization Z-spray source. Single Lock Mass (leucin-enkephalin) was used for mass correction. Lipid fractions were dissolved in 100 μL 50% A eluent / 50% B eluent mixture ( V/ V). Lip-idomic LC-MS measurements were performed on an ACQUITY UPLC CSH C18 column (1.7 μm, 2.1x100mm) at 55 °C column temperature and 0.35 mL/min flow rate. Eluent A was 600/390/10 acetonitrile/water/1 M ammonium formate solvent mixture containing 0.1% formic acid ( V/ V). B eluent was 900/90/10 isopropanol/ acetonitrile /1 M ammonium formate mixture containing 0.1% formic acid ( V/ V). Gradient elution profile was the following: 0 min: 50% B, 0.5 min: 53% B, 4 min: 55% B, 7 min: 65% B, 7.5 min: 80% B, 10 min: 99% B. MS analysis was performed in negative ionization mode in the m/z 50-1200 range. Collision induced dissociation (CID) was performed in the 25-45 V collision voltage range. Single pass cyclic ion mobility separation was included in the measurements. Data were evaluated and lipids were identified by the Progenesis QI software. Peptide fractions were dissolved in 25 μL 2% acetonitrile, 0.1% formic acid ( V/ V) solvent mixture. Proteomic LC-MS measurements were performed on an ACQUITY UPLC Peptide CSH C18 column (1.7 μm, 1x150mm) at 45 °C column temperature and 20 μL/min flow rate. Eluent A was 0.1% formic acid in water, B eluent 0.1% formic acid in acetonitrile ( V/ V). Gradient elution profile was the following: 0 min: 5% B, 1 min: 5% B, 45 min: 35% B, 46 min: 85% B. MS analysis was performed in positive ionization mode in the m/z 50-2000 range. Collision induced dissociation (CID) was performed in the 19-45 V collision voltage range. Data were evaluated and the proteins were identified by the ProteinLynx Global Server software. 3. Results The objective of this work was the characterisation and comparison of blood plasma samples in a comprehensive way by a multi-step sample preparation workflow. The sample preparation was divided into three main steps: first, the lipid content of the plasma was isolated using MTBE as organic solvent. Then the lipid-depleted samples containing the plasma proteins were subjected to enzymatic cleavage by trypsin. The resulting peptide mixture was further fractionated. A fraction enriched in glycopeptides was isolated by ice-cold acetone precipitation and the peptide fractions depleted in glycopeptides were also isolated. All three fractions were analysed by UPLC-MS(/MS) and the compounds were identified by database search. Lipids were analysed using cyclic ion mobility separation3 (cIM) to increase the number of compounds (e.g. isomers) that could be differentiated. Figure 2. shows the ion mobility heat map (drift time vs. retention time) of the lipids by LC- Proceedings of 10th Socratic Lectures 2024 70 of 241 MS under the optimized gradient elution. Using a single pass cyclic ion mobility separation, we were able to resolve several isomeric lipids, which otherwise elute at the same or highly similar retention times. Figure 2. 2D LC-cIM-MS heat map (mobilogram) of the extracted lipids from canine plasma centrifuged at 2000 g for 5 min. Main lipids were identified by database search. Analytical data and possible structures of the most intensive lipid species are summarized in Table 1. Note that in many cases, the exact structure of the lipids is ambiguous, due to identical masses and similar fragmenta tion patterns. Here, the collision cross sections (the drift times) are different. The most intensive species correspond to phosphatidylcholines (PC), sphingomyelins (SM) and ether-linked phosphatidyl-ethanolamines (PE). Under these conditions, formate adducts ([M+FA-H]-) were dominant in the mass spectra. Table 1. The 15 most intensive plasma lipids identified by LC-cIM-MS from canine plasma centrifuged at 2000 g for 5 min. m/z Retention time (min) CCS (Å) Formula Possible lipids Adduct type 747.5681 4.03 286.1 C39H79N2O6P SM(34:1) [M+FA-H]- 750.5459 6.64 278.3 C43H78NO7P PE(O-38:5) [M-H]- 802.5631 4.48 291.2 C42H80NO8P PC(34:2) [M+FA-H]- 804.5766 5.48 291.2 C42H82NO8P PC(34:1) [M+FA-H]- 826.5615 3.66 294.3 C44H80NO8P PC(36:4) [M+FA-H]- 826.5624 4.23 295.4 C44H80NO8P PC(36:4) [M+FA-H]- 828.5776 4.58 296.4 C44H82NO8P PC(36:3) [M+FA-H]- 830.5932 5.86 297.5 C44H84NO8P PC(36:2) [M+FA-H]- 832.6086 6.79 299.6 C44H86NO89 PC(36:1) [M+FA-H]- 854.5922 5.24 299.5 C46H84NO8P PC(38:4) [M+FA-H]- 854.5932 5.6 300.5 C46H84NO8P PC(38:4) [M+FA-H]- 856.6079 6.16 301.6 C46H86NO8P PC(38:3) [M+FA-H]- 857.6759 7.81 307.8 C47H93N2O6P SM(42:2) [M+FA-H]- 882.6243 6.53 308.7 C48H88NO8P PC(40:4) [M+FA-H]- 885.5517 4.32 300.3 C47H83O13P PI(38:4) [M-H]- CCS: Collision Cross Section Proceedings of 10th Socratic Lectures 2024 71 of 241 The type of the major lipids was identical in the samples, however, comparison of the samples prepared by different centrifugation revealed differences in the intensity of specific lipid species. Figure 3 shows the intensities of two lipid ions in the LC-MS chromatograms. While the intensity of PC(O-36:4) did not change, there is a remarkable decrease in the intensity of PE(O-38:5) due to centrifugation with elevated spin and longer time. Figure 3. Effect of the centrifugation on plasma lipids. A.) Intensity of m/z 750.5459, PE(O-38:5); B.) Intensity of m/z 812.5822, PC(O-36:4). Similarly, the protein and glycoprotein content of the samples were also compared. Protein contents of the samples were analysed after tryptic digestion. Fractions enriched and depleted in glycopeptides were prepared and proteins were identified by MS/MS peptide sequencing and database search. Database search revealed the presence of the most typical and abundant plasma proteins, including for example Apolipoproteins, Hemoglobin, Serum albumin, Haptoglobin and Fibronectin. While the peptide fraction did not show significant differences between the two sample groups, the intensity of the glycopeptides was increased in the plasma sample set which was centrifuged at higher spin and longer time (Figure 4). Based on glycopeptide marker ion intensities, larger spin and longer centrifugation time increased the intensity of glycopeptides, demonstrating that the relative ratio of glycoproteins increases in the prepared plasma. Figure 4 shows the total intensity of the glycopeptide MS/MS fragment ions at m/z 366.14 for the samples prepared by different centrifugation protocols. Figure 4. Effect of the centrifugation on plasma glycoproteins. Intensity of m/z 366.14 glycopeptide marker fragment ion in the MS/MS spectra of the LC-MS/MS chromatograms. Proceedings of 10th Socratic Lectures 2024 72 of 241 4. Discussion LC-MS-based comparison of plasma samples produced by different centrifugation methods revealed differences in their chemical compositions. In the case of lipids, certain species showed significant decrease while other species showed increase based on their LC-MS intensities. Differences were also observed in the glycopeptide fraction obtained by tryptic digestion of differently centrifuged samples: longer centrifugation times resulted in a more intense glycopeptide pattern. Our results demonstrate that the centrifugation steps, used for the preparation of plasma products, changes the chemical composition. Therefore, it is essential to obtain more information on the effect of preparation protocols and the composition of plasma products in the future. Funding: This research was funded by the Lendület (Momentum) Programme of the Hungarian Academy of Sciences, by the Slovenian Research Agency, grant numbers P2-0232, P3-0388, J3-3066, and J2-4447. Project SNN 138407 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the SNN_21 funding scheme. The authors are grateful for Antóna Patkó for her kind help in sample preparation and in data analysis. Conflicts of Interest: The authors declare no conflict of interest. References 1. Troha K, Vozel D, Arko M, et al. Autologous Platelet and Extracellular Vesicle-Rich Plasma as Therapeutic Fluid: A Review. Int J Mol Sci. 2023; 24: 3420. DOI: 10.3390/ijms24043420 2. Božič D, Hočevar M, Kononenko V, et al. Pursuing mechanisms of extracellular vesicle formation. Effects of sample processing. Advances in Biomembranes and Lipid Self-Assembly. 2020; 32: 113-155, DOI: 10.1016/bs.abl.2020.09.003 3. Giles K, Ujma J, Wildgoose J, et al. A Cyclic Ion Mobility – Mass Spectrometry System. Anal Chem. 2019; 13: 8564–8573. DOI:10.1021/acs.analchem.9b01838 Proceedings of 10th Socratic Lectures 2024 74 of 234 Invited lecture/Research Extracellular Particles from Equine Milk Arko Matevž1, Hočevar Matej2, Korenjak Boštjan1, Iglič Aleš3,4, Kralj-Iglič Veronika1,* 1. University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia; 2. Institute of Metals and Technology, Ljubljana, Slovenia 3. University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana, Slovenia; 4. University of Ljubljana, Faculty of Medicine, Laboratory of Clinical Biophysics, Ljubljana, Slovenia; * Correspondence: Veronika Kralj-Iglič veronika.kralj-iglic@zf.uni-lj.si Abstract: Equine and bovine milk extracellular particles were investigated by scanning electron mi-Citation: Arko M, Hočevar M, croscopy, interferometric light microscopy and flow cytometry. Scanning electron micros-Korenjak B, Iglič A, Kralj-Iglič V. copy revealed micro-sized globular particles and numerous nano-sized particles. Higher Extracellular particles from equine concentration of micro and nano-sized particles was found in skimmed milk when com-Milk. pared to the whole milk and the differences were statistically significant. Proceedings of Socratic Lectures. 2024, 10; 74-77. https://doi.org/10.55295/PSL.2024.I12 Publisher’s Note: UL ZF stays neu- tral with regard to jurisdictional Keywords: Extracellular vesicles; Milk; Interferometric light microscopy; Flow cytometry; claims in published maps and institu-Scanning electron microscopy; Concentration of particles tional affiliations. Copyright: © 2024 by the authors. Submitted for possible open access publication under the terms and con- ditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/li- censes/by/4.0/). Proceedings of 10th Socratic Lectures 2024 75 of 234 1. Introduction 1.1. Milk as a complete food Milk is a complete food with all nutritionally important components required for a new-born mammal – it comes in liquid form and contains proteins (corpuscular casein and dissolved whey proteins), fat (fat globules) and carbohydrates (milk sugar, i.e., lactose) (Musaev et al., 2021, Malacarne et al., 2002). 1.2. Milk as a source of a variety of particles Milk is also a source of a variety of small particles including molecules, their complexes, and extracellular particles (Ong SL et al., 2021). It contains bioactive components derived from various cellular sources (Musaev, A et al., 2021) and immunologically active cells (Palmeira P et al., 2016). Here, we investigated the extracellular particles from equine milk. The concentration was measured by interferometric light microscopy and flow cytometry. To observe the morphology of particles, scanning electron microscopy micrographs were obtained from equine milk. 2. Methods 2.1. Milk Sampling For flow cytometry and interferometric light microscopy animal was milked by hand from a Posavje (breed) mare on 91st day of lactation. Milk was collected into tubes VACUETTE® TUBE 3 ml Z No Additive 13x75 white cap-black ring, non-ridged (Greiner AG, Krems-münster, Austria). To obtain skimmed milk, whole milk was centrifuged at 300 g for 15 min and the cream was removed from the top using a pipette with the tip shortened for 2 mm by scissors. The procedure was repeated twice. For scanning electron microscopy, the milk samples were obtained at different days of lactation from Posavje (breed) mare. 2.2. Scanning electron microscopy (SEM) The milk samples were incubated with added 1% OsO4 for 1 hour, washed 3 times for 10 minutes with dH2O, dehydrated in graded ethanol (EtOH): 30%, 50%, 70%, 80% and 90% for 10 minutes at each concentration and in absolute EtOH 2-times for 10 minutes, incubated with added absolute EtOH and HMDS (ratio 3:7) for 10 minutes, incubated in added absolute EtOH and HMDS (ratio 1:1) for 10 minutes, incubated in added 100% HMDS for 10 minutes and depleted of HMDS by evaporation in exicator with silica gel for 12 hours. After fixation, samples were gold-sputtered and observed by the scanning electron microscope (SEM, JEOL JSM-6500F). 2.3 Flow Cytometry (FCM) The particle numbers in samples from equine and bovine milk were estimated by flow cytometry, using a MACSQuant Analyzer flow cytometer (Miltenyi Biotec, Bergisch Glad-bach, Germany) and the related software. Before measurement, the milk was diluted by saline to obtain a measurable dilution (200x bovine and equine whole and skimmed milk). The following instrument settings were employed: FSC: 458 V; SSC: 467 V with a trigger set to 1.48; B3: 300 V; and R1: 360 V. Particles were detected from the forward (FSC) and side scatter parameters (SSC). 2.4 Interferometric Light Microscopy (ILM) The average hydrodynamic diameter (Dh) and the number density of small particles in milk were determined by interferometric light microscopy using Videodrop (Myriade, Paris, France). Before measurement the milk was diluted by saline for injections (Braun, Melsungen, Germany) to obtain a measurable dilution. The range of dilution was 8x - 500x for bovine whole milk, 32x - 500x bovine skimmed milk and 16 x - 50x for equine whole and skimmed milk. Signals of the saline were under the detection limit. The threshold value 4.2 was used. 7 µL of sample was placed between cover glasses and illuminated by 2W blue LED light. The light scattered on the particle was imaged by a bright-field microscope objective and allowed to interfere with the incoming light. The image was recorded by a complementary metal–oxide–semiconductor high resolution high speed camera. The Proceedings of 10th Socratic Lectures 2024 76 of 234 obtained pattern that includes contrasting black and white spots was recognized as a particle and its position in the sample was assessed. Number density of the particles is the number of the detected particles within the detected volume (e.g. 15 pL). Dh was determined by tracking the position of the imaged particle within the recorded movie. It was assumed that particles undergo Brownian motion due to collisions with surrounding particles. The diffusion coefficient D of the motion of the particle is taken to be proportional to the mean square displacement d of the particle between two consecutive frames taken in the time interval ∆t, = <4D ∆t> while Dh was estimated by assuming that the particles were spherical and using the Stokes-Einstein relation Dh = kT/3πηD. Each particle that was included in the analysis was tracked and processed individually and the respective incident light signal was subtracted from each image. Processing of the images and of the movies was performed by using the associated software QVIR 2.6.0 (Myriade, Paris, France). 2.5. Statistical analysis All measurements were performed in triplicates and presented by the average values and standard deviations. The differences were evaluated by the t-test using the Excel software. The value p = 0.05 was taken as a threshold for statistical significance. 3. Results Concentration and size of the particles in the equine milk was obtained by FCM and ILM (Table 1). SEM micrographs revealed presence of globular particles’ morphology (Figure 1). Table 1. Concentration and size of particles in the equine whole and skimmed milk determined by FCM and ILM. The measurement was done on the 91st day of lactation. Average ± Standard Deviation assessed from 3 measurements are given. Sample ILM (size range 80 nm - 400 nm) ILM FCM Number density (109 /mL) Dh (nm) Number density (109 /mL) 91st day whole milk 28.89 ± 2.14 308 ± 12 0.23 ± 0.06 91st day skimmed milk 36.80 ± 2.00 292 ± 7 2.14 ± 0.41 p (t-test) 0.009 0.12 <0.00001 4. Discussion According to Walstra (1969), in the bovine milk there is a subpopulation of milk fat globules with a diameter smaller than 1 µm. They seem to represent 80% of all milk fat globules but only approximately 5 % of the volume of the milk fat (Walstra, 1969).We found significantly higher concentration of EPs sized 80 – 500 nm in the skimmed milk than in the whole milk indicating that fat globules occupy larger space but are not very numerous. Equine milk fat globules were reported to be relatively small, their average volume-surface diameter being about 2.75 µm (Welsch et al., 1988). In the cow’s milk, a population of medium globules (ranging from 1 to 8 µm) was measured; it was estimated that these globules constitute approximately 94% of the fat volume (Walstra, 1969). However, when the average size of EPs in the equine milk was assessed by ILM, there was no significant difference between the whole and skimmed milk, which might be explained by the low fat content in the equine milk. Proceedings of 10th Socratic Lectures 2024 77 of 234 Figure 1. SEM micrographs of equine milk: A – Equine milk on the 1st day after parturition, B – Equine milk on the 1st day after parturition, C – Equine milk on the 2nd day after parturition, D – Equine milk on the 3rd day after parturition, E – Equine cream. The content of fat in the equine milk was found lower (1,21%) than in the bovine milk (3,61%) and in the human milk (3,64%) (Malacarne M, et al., 2002 ). Similarly, the concentration of particles measured by FCM was significantly higher in the skimmed milk and lower in the whole milk. The average size of the EPs measured by ILM was significantly higher in the whole milk which might be due to milk fat globules (present in the whole milk) and casein micelles (present in the whole and skimmed milk). Funding: Authors acknowledge support from ARIS, grants J3-3066, J2-4447, P2-0232 and P3-0388. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki. Conflicts of Interest: The authors declare no conflict of interest. References 1. Musaev A, Sadykova S, Anambayeva A, et al. Mare's Milk: Composition, Properties, and Application in Medicine. Arch Razi Inst. 2021; 76: 1125-1135. DOI: 10.22092/ari.2021.355834.1725. 2. Malacarne M, Martuzzi F, Summer A, Mariani P. Protein and fat composition of mare's milk: some nutritional remarks with reference to human and cow's milk. Int Dairy J. 2002; 12: 869-77. DOI: https://doi.org/10.1016/S0958-6946(02)00120-6 3. Ong SL, Blenkiron C, Haines S, et al. Ruminant Milk-Derived Extracellular Vesicles: A Nutritional and Therapeutic Opportunity? Nutrients. 2021; 13: 2505. DOI: 10.3390/nu13082505. 4. Palmeira P, Carneiro-Sampaio M. Immunology of breast milk. Rev Assoc Med Bras (1992). 2016; 62: 584-593. DOI: 10.1590/1806-9282.62.06.584. 5. Welsch U, Buchheim W, Schumacher U, et al. Structural, histochemical and biochemical observations on horse milk-fat-globule membranes and casein micelles. Histochemistry. 1988; 88: 357-65. DOI: 10.1007/BF00570295. 6. Walstra P. Studies on milk fat dispersion. The globule size distribution of cow’s milk. Neth. Milk Dairy J. 1969; 28: 3–9. Proceedings of 10th Socratic Lectures 2024 79 of 234 Review An Insight into the Use of Cannabis in Medical and Veterinary Dermatological Applications and its Legal Regulation Mišič Jančar Jakob1,#, Schofs Laureano2,3,#, Pečan Luka Irenej 4,5, Oblak Tine6, Sánchez Bruni Sergio2,3, Kuhar Aleš4, Ponikvar-Svet Maja6, Tavčar Gašper 6, Hupli Aleksi7, Jeran Marko6,* 1. University of Ljubljana, Faculty of Law, Ljubljana, Slovenia 2. Laboratory of Pharmacology, Faculty of Veterinary Medicine, Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, Argentina 3. Veterinary Research Center of Tandil (CIVETAN), CONICET-CICPBA-UNCPBA, Tandil, Argentina 4. University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Chair for Agrarian Economics, Policy and Law, Ljubljana, Slovenia 5. University of Trieste, Department of Life Sciences, Trieste, Italy 6. “Jožef Stefan” Institute, Department of Inorganic Chemistry and Technology, Ljubljana, Slovenia 7. Emerging Technologies Lab, Faculty of Social Sciences, Tampere University, Tampere, Finland # JMJ and LS contributed equally to this work * Correspondence: Marko Jeran, marko.jeran@ijs.si Abstract: Cannabis contains more than 140 cannabinoid compounds, of which cannabidiol and Citation: Mišič Jančar J, Schofs L, tetrahydrocannabinol are the most exposed ones, as they have the potential for numerous Pečan LI, Oblak T, Sánchez Bruni S, applications in various fields of health and medicine. Many scientific findings and Kuhar A, Ponikvar-Svet M, Tavčar numerous clinical studies over the last decade report results related to pain relief, G, Hupli A, Jeran M. An insight into treatment of chronic diseases and certain neurological disorders in human medicine. In the use of cannabis in medical and veterinary dermatological line with the growing trend of research and application in human medicine, the research applications and its legal regulation. and application potential in veterinary medicine is also increasing. Of the biologically Proceedings of Socratic Lectures . active molecules contained in the plant, cannabidiol has attracted the most interest. The 2024, 10, 79-91. https://doi.org/10.55295/PSL.2024.I13 cultivation of cannabis for medical and pharmaceutical purposes requires, among other things, compliance with legal regulations. An analysis of the legal acts regulating the use for medical and pharmaceutical purposes shows that the current regulation at state level Publisher’s Note: UL ZF stays neutral with regard to jurisdictional is complex and should be amended. claims in published maps and institutional affiliations. Keywords: Cannabis, Cannabinoids, Human medicine, Veterinary medicine, Inflammation, Legal Regulation 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/). Proceedings of 10th Socratic Lectures 2024 80 of 234 1. Introduction Cannabis sativa stands as a botanical enigma deeply ingrained in human history, weaving a rich tapestry across ancient cultures. From being a crucial agricultural crop and industrial raw material to its multifaceted roles in contemporary society – medical, nutritional, and industrial cannabis has evolved as a subject of intense scientific inquiry. Its origins date back to more than a millennium BC and cannabis played a central role in agriculture until the late 19th century (Zuardi, 2006). The year 2019 marked an important turning point with the European Parliament's resolution supporting the medicinal use of cannabis (Pečan et al., 2021). This has driven efforts towards decriminalisation in the European Union, the promotion of research and the integration of medicinal cannabis in EU countries. At the same time, the Commission on Narcotic drugs recognised cannabis, particularly ∆-9-tetrahydrocannabinol (∆9-THC, THC) (Figure 1 (b)), as a substance that primarily causes no harm, which shaped the international monitoring framework (Pečan et al., 2021; Štukelj et al., 2019). Cannabis, which botanically belongs to the Cannabaceae family, is a dioecious, flowering plant characterised by remarkable adaptability. The leaves are palmately compound or toothed with toothed leaflets. The phenotypic expression of the plant is strongly influenced by environmental factors, which makes it an interesting subject for detailed botanical studies (Amaducci et al., 2008). Modern scientific research has identified over 420 compounds in cannabis (Atakan, 2012). Trichomes, specialised secretory glands concentrated in the female inflorescences, contain cannabinoids, terpenes and flavonoids, fundamental components of the secondary metabolism of cannabis (Tanney et al., 2021). Cannabinoids, more than 90 different substances, have a binding affinity to cannabinoid receptors and thus trigger a spectrum of physiological effects (Andre et al., 2016). As the primary psychoactive component, THC plays a central role in the pharmacological profile of cannabis (Bridgeman and Abazia, 2017). Classified as a narcotic, THC acts as a partial agonist at the cannabinoid receptors CB1 and CB2, particularly in the central nervous system and the immune system, and thus unfolds its psychotropic effect (Pertwee 2008). THC is approved for medical applications and plays an important role in drugs such as Marinol®, CesametTM and Sativex® (Tavčar Benković et al., 2019). Cannabidiol (CBD) (Figure 1 (a)) was reported in 1940 (Burstein, 2015) and has developed into an important phytocannabinoid with a wide range of applications. CBD has analgesic, anti-inflammatory, antineoplastic and chemopreventive effects and differs from THC in that it is not psychoactive (Martínez et al., 2020). Its complex mechanisms include stimulation of endoplasmic reticulum stress, inhibition of AKT/ m TOR signalling and activation of autophagy, which offers diverse therapeutic potential (Pertwee, 2006). Figure 1. Structures of (a) cannabidiol (CBD) and (b) ∆-9-tetrahydrocannabinol (THC). Cannabis and its derivatives have made a name for themselves in the pharmaceutical industry, particularly for dermatological problems (Martins et al., 2022). Cannabinoids such as CBD have anti-inflammatory and analgesic properties, making them promising candidates for the treatment of conditions such as psoriasis and eczema (Baswan et al., 2020). Pharmaceutical formulations containing cannabis extracts are being investigated for their potential for topical treatments that provide targeted relief (Baswan et al., 2020). In veterinary medicine, cannabis and its derivatives are currently being carefully investigated for their potential therapeutic benefits. Studies suggest that cannabinoids may Proceedings of 10th Socratic Lectures 2024 81 of 234 be useful in the treatment of certain conditions such as osteoarthritis and epilepsy in animals (Di Salvo et al., 2023). This emerging field offers the possibility of tailored veterinary treatment, taking into account species-specific responses and dosing considerations (Gupta et al., 2019). Cannabis, deeply rooted in antiquity, is evolving into a complex botanical entity with profound implications for historical understanding and contemporary applications. Research into the complex botanical and pharmacological dimensions of cannabinoids such as THC and CBD forms the basis for careful scientific investigation. The dynamic legal landscape and increasing applications underscore the need for rigorous research that places cannabis at the forefront of scientific exploration, offering potential advances in human and veterinary medicine and opening new dimensions for specialized pharmaceutical formulations (Pečan et al., 2023). 2. Cannabis extracts and cannabinoids for the treatment of skin diseases in human medicine The long history of the use of cannabis for medicinal and recreational purposes has also led to its use for various skin applications. Cannabis extracts have been investigated as a potential remedy for skin problems and disorders (Shao et al., 2023). The therapeutic potential of the cannabinoid CBD has been particularly researched and studied as it is abundant in the cannabis plant and has a relatively favourable legal status compared to THC. Recently, extensive review articles have been published on the therapeutic potential of phytocannabinoids, particularly cannabidiol, focusing on its effect on skin health, disease (Baswan et al., 2020; Martins et al., 2022) and delivery mechanisms (Filipiuc et al., 2023). Natural cannabinoids, including CBD, have been shown to have a positive effect in the treatment of many skin diseases (Baswan et al., 2020). Their effect is mainly due to the fact that the endocannabinoid system (ECS) has an important regulatory function in the skin (Baswan et al., 2020) and therefore effective preparations can be used when applied topically. Cannabinoids are an interesting field of research in dermatology due to their anti-inflammatory, antipruritic and antinociceptive properties (Shalaby et al., 2018). 2.1. Atopic dermatitis and eczema Atopic dermatitis (AD) and eczema are chronic conditions that lead to inflammation, redness and irritated skin. Many factors can lead to atopic dermatitis, such as a damaged skin barrier, an unwanted immune response and an imbalance in the microbiome (Dimitriu et al., 2019). The anti-inflammatory effect of CBD was investigated in an experimental model (Petrosino et al., 2018). This study has shown that CBD inhibits the production of the chemokine MCP-2 and several other proinflammatory cytokines in poly- (I:C)-stimulated keratinocytes more effectively than other non-psychotropic phytocannabinoids tested. Clinical studies have yet to be conducted. Several studies have suggested using the skin ECS as a potential treatment for AD (Mounessa et al., 2017). Studies in mice showed that activation of cannabinoid 1 receptor (CB1R) in skin cells has a positive effect on epidermal barrier function and reduces a Th2-type inflammatory response (Gaffal et al., 2014; Kim et al., 2015). In an earlier study, the consumption of cannabis seed oil was compared with olive oil (Callaway et al., 2005); in a 20-week randomized, single-blind crossover study with AD patients, the treatment was carried out with a daily intake of 30 mL cannabis seed oil or olive oil (Callaway et al., 2005). The intake of cannabis seed oil led to significant changes in plasma fatty acid profiles and a reduction in skin dryness, skin irritation and itching compared to olive oil. The authors assume that this effect is due to the high proportion of polyunsaturated fatty acids (PUFAs) in cannabis seed oil. An unbalanced skin microbiome could also have an important influence on the severity of dermatitis; the studies suggest that the main antimicrobial effects of cannabis come from its essential oil (Mediavilla et al., 1997), i. e. the volatile component of cannabis. Its main components are terpenes such as myrcene, α-pinene, β-caryophyllene and others. The antimicrobial effect has been proven against S. aureus (Zengin et al., 2018). Proceedings of 10th Socratic Lectures 2024 82 of 234 2.2. Acne and seborrhea Acne and seborrhea are well-known skin diseases characterized by irregularly increased sebum production by the sebaceous glands (SC). Acne as a skin disease occurs when the hair follicles become clogged with oil and dead skin cells. Sebum overproduction, inflammation and altered sebocyte proliferation are the most important factors in the appearance of acne on the skin. The endogenous endocannabinoids (ECBs) are involved in the maintenance of homeostatic sebum production in the human sebaceous glands (Dobrosi et al., 2019). Several studies confirm that CBD could play an important role in the treatment of acne due to its effect on acne-related factors (Oláh et al., 2014). An important study was conducted to test the anti-inflammatory effect of CBD on human sebocytes (Oláh A et al., 2014). The study suggests that CBD is not limited to a direct ECS interaction, but has a more generalized effect. More importantly, CBD not only acts as a lipid-lowering agent but also stabilizes lipid imbalance by normalizing lipogenesis (Jin and Lee, 2018). In addition, CBD has been shown to reduce overall cell proliferation at some relevant doses (Oláh et al., 2014). The same research group has tested the anti-inflammatory potential of CBD and demonstrated its effect (Oláh et al., 2014). The anti-inflammatory effect was attributed to the A2a adenosine receptor-dependent upregulation of Tribbles homolog 3 (TRIB3) and inhibition of the NF- κ B signaling pathway. The study concludes that CBD could be a promising therapeutic agent for the treatment of acne due to its combined lipostatic, antiproliferative and anti-inflammatory effects (Oláh et al., 2014). An unbalanced skin microbiome can contribute to the occurrence of acne, especially the overgrowth of C. acne (Platsidaki and Dessinioti, 2018). The study (Jin and Lee, 2018) using cannabis seed hexane extract showed that it has an antimicrobial effect on C. acnes. It also induced inflammation and lipogenesis in sebocytes. Cannabis seeds have a minimal CBD content (Callaway, 2004), so we cannot draw parallels regarding their effect on the acne skin microbiome. More promising results could be expected from the use of cannabis essential oil, which mainly contains terpenes (Vuerich et al., 2019). These have been shown to have an antimicrobial effect against C. acnes (Raman et al., 1995). 2.3. Psoriasis Psoriasis is a chronic, inflammatory autoimmune disease of the skin in which the immune system becomes overactive and causes hyperproliferation of skin cells, followed by skin changes known as psoriatic plaques and inflammation (Michalek et al., 2019). Recent research (Sangiovanni et al., 2019) has shown that CBD and cannabis extract ( C. sativa) standardized to 5% CBD (CSE) inhibit TNF-α-induced NF- κ B inhibitory effects in HaCaT cells. NF- κ b is a family of transcription factor protein complexes that regulate DNA transcription, cytokine production and cell survival (Wu et al., 2003). It plays an important role in inflammatory skin diseases, usually associated with psoriasis (Sangiovanni et al., 2019). CSE treatment reduced all 26 genes associated with inflammation, while CBD alone reduced only 15 genes (Sangiovanni et al., 2019). This suggests that a broad range of cannabinoids could achieve better results than CBD alone. The use of phytocannabinoids for the treatment of psoriasis should be used with caution. A study on the treatment of psoriasis with CBD (Casares et al., 2020) has also shown that it has proliferative effects on keratins 16 and 17. This could lead to additional complications for psoriasis patients. 3. Cannabis in veterinary dermatology Dermatologic diseases are the main reason for consultations in general veterinary small animal clinics after preventive health care (Hill et al., 2006). Skin diseases encompass a variety of different conditions, but in most cases an inflammatory response triggered by an infectious, parasitic or immunologic stimulus is the main mechanism behind the pathologic process (Scott & Paradis, 1990). Therefore, pruritus is the most commonly observed sign, especially in dogs (Khoshnegah et al., 2013). In cats, swelling of the skin appears to be the predominant clinical presentation (Hill et al., 2006). Bacterial skin infections are also common in dogs, with S. pseudintermedius being the main cause in most cases (Bourguignon et al., 2013). Consequently, the most commonly prescribed drugs for the treatment of skin diseases in small animals are systemic antibiotics and glucocorticoids Proceedings of 10th Socratic Lectures 2024 83 of 234 (Hill et al., 2006). Topical treatments may also be prescribed for localized skin lesions (Mueller et al., 2012). The commercially available formulations used in veterinary medicine are very similar to those used in humans. They are mainly topical creams and ointments with antibacterial or anti-inflammatory agents to heal skin lesions. The inclusion of novel molecules with anti-inflammatory, immunomodulatory and antimicrobial effects such as cannabinoids (Izzo et al., 2009) could expand the therapeutic arsenal for dermatological diseases. The endocannabinoid receptors (CB1 and CB2) are expressed in different cell types of the epidermis and dermis of healthy dogs (Campora et al., 2012), cats (Miragliotta et al., 2018) and horses (Kupczyk et al., 2022). CB1 receptors have also been found in the inner root sheath cells of the primary and secondary hair follicles of dogs (Mercati et al., 2012). The ECS appears to play a protective role in inflammatory dermatological conditions, as ECS expression has been reported to be increased in canine atopic dermatitis (Campora et al., 2012) and in cats with hypersensitivity dermatitis (Miragliotta et al., 2018). Cannabidiol (CBD) and ∆-9-tetrahydrocannabinol (THC) are the best-studied phytocannabinoids in veterinary medicine (De Mendonça Lima et al., 2022). The use of CBD and THC is considered safe for dogs (Vaughn et al., 2020) and cats (Kulpa et al., 2021). CBD is better tolerated than THC, as escalating doses of CBD resulted in mild adverse effects in both dogs and cats (Vaughn et al., 2020; Kulpa et al., 2021). High doses of THC can cause moderate to severe adverse effects in pets, such as lethargy, hypothermia and ataxia (Vaughn et al., 2020; Kulpa et al., 2021). However, THC has a high therapeutic index, as maximum doses of this compound do not cause death in dogs (Beaulieu, 2005). Pharmacokinetic studies in dogs have shown that the oral bioavailability of CBD is low, ranging from 13 to 19% (Samara et al., 1988). Although other administration methods have also been tested, oral administration is still the preferred route of administration in most pharmacological studies on cannabinoids in veterinary medicine (Corsato Alvarenga et al., 2023). For focal dermatological conditions, cannabinoids can be administered via the transdermal route to maximize skin concentration while allowing absorption into the bloodstream (Bartner et al., 2018; Hannon et al., 2020). To this end, cannabinoids could be formulated as ointments, preferably as native acid derivatives, as acidic forms of CBD and THC have been shown to be better absorbed than their decarboxylated counterparts (Hannon et al., 2020). In veterinary medicine, the clinical efficacy of CBD has been tested mainly in osteoarthritic pain, epilepsy and behavioural disorders (De Mendonça Lima et al., 2022; Di Salvo et al., 2023). For skin diseases, two studies focused on testing the potential benefits of cannabinoids in atopic dermatitis in dogs (Loewinger et al., 2022; Mogi et al., 2022). In both studies, cannabinoids were able to reduce itching, although no clinical differences in skin lesions were observed compared to the control groups (placebo) (Loewinger et al., 2022). Infectious diseases of the skin could be another potential clinical target for cannabinoids. Infections on the skin surface of animals can not only prolong the patient's recovery time, but also spread to other parts of the body, invade the bloodstream and migrate to the internal organs, which can prove fatal if left untreated for too long. Amoxicillin and cefalexin are the most commonly prescribed systemic drugs for skin infections in veterinary dermatology (Hill et al., 2006). Cannabis derivatives exert antimicrobial properties on a broad spectrum of pathogenic bacteria (Schofs et al., 2021). Cannabinoids could act against S. pseudintermedius, the main cause of skin infections in dogs (Bourguignon et al., 2013; Aiemsaard et al., 2022), but also against skin infections caused by methicillin-resistant Staphylococcus aureus (MRSA), a pathogen important for both public health and animal health (Haag et al., 2019; Pečan et al., 2023). These antimicrobial effects of cannabinoids could be further explored in clinical trials in veterinary medicine to test in vivo efficacy against skin infections. Laboratory animal models are not always of translational use, as in many cases they have not been able to predict clinical efficacy and toxicity in humans (Ritter et al., 2020; Marshall et al., 2023). As part of a “One Health” approach and utilising naturally occurring diseases in dogs and cats, cannabinoids can therefore be used as a potentially useful tool in both veterinary and human medicine. Proceedings of 10th Socratic Lectures 2024 84 of 234 4. Legal regulation of medical cannabis in Slovenia The history of cannabis regulation is marked by an evolving understanding and shifting legal frameworks. Initially used for medicinal and recreational purposes in various cultures, cannabis' perception underwent a significant transformation in the 20th century. This period saw the international community, through agreements like the 1961 Single Convention on Narcotic Drugs, categorize cannabis as a controlled substance, significantly restricting its use globally (United Nations, 1961). However, recent decades have witnessed a re-evaluation, especially in the context of medical cannabis. This shift has been propelled by a growing body of scientific research that underscores the therapeutic potential of cannabis, particularly in managing chronic pain and other medical conditions (Whiting et al., 2015). Consequently, many countries have begun to adapt their legal frameworks, creating distinct regulations for medicinal and recreational cannabis use. Today's regulatory landscape of cannabis is a complex amalgamation of medical research, legal policies, and evolving societal attitudes (Hall & Lynskey, 2016). This article focuses specifically on cannabis as a plant and does not address cannabinoids that are synthesized independently of the cultivation of the plant. 4.1. Cannabis as an illegal drug Zakon o proizvodnji in prometu s prepovedanimi drogami (Production of and trade in illicit drugs act: Law 1) specifies the conditions under which the production and trafficking of illegal drugs, as well as the possession of illegal drugs, are permitted. The law requires the Government of the Republic of Slovenia, upon the proposal of the minister responsible for health, to adopt the classification of illegal drugs into the following three groups: • Group I: Plants and substances that are extremely dangerous for human health due to the severe consequences that can result from their abuse and are not used in medicine; • Group II: Plants and substances that are extremely dangerous due to the severe consequences that can result from their abuse but can be used in medicine; • Group III: Plants and substances that are moderately dangerous due to the consequences that can result from their abuse and can be used in medicine. According to the currently valid Uredba o razvrstitvi prepovedanih drog (Decree on the classification of illicit drugs: Decree 1), cannabis, or scientifically Cannabis sativa L., as a plant, or its resin and extracts are classified in Group II. Cannabis is thus an illegal drug according to Slovenian law. 4.2. Under the gaze of Article 186: The regulation of cannabis in Slovenia In the context of illegal drugs, the Slovenian Kazenski zakonik (Criminal code: Law 2), specifically Article 186, addresses the unauthorized production, processing, sale, offer for sale, or acquisition for the purpose of sale or distribution of illegal drugs. This legal framework is crucial for understanding the criminal implications of cannabis-related activities in Slovenia. Article 186 states that anyone who unjustifiably produces, processes, sells, offers for sale, purchases, stores, or transports drugs, or facilitates their sale or purchase, is subject to legal penalties. This article is applied in situations involving cannabis when these activities are conducted without proper authorization or in violation of existing regulations. The application of Article 186 to cannabis-related activities underscores the seriousness with which the Slovenian legal system treats the control of illegal drugs. This includes cannabis, which, despite its varying legal status in different jurisdictions (including Slovenian), remains under strict regulation in Slovenia. The law aims to prevent unregulated and potentially dangerous drug trafficking, ensuring public health and safety by controlling the production and distribution of such substances. 4.3. Exclusion of illegality Although in the case of the specific criminal act under Article 186, we could even talk about the non-fulfillment of the statutory characteristics, as it emphasizes unauthorized production, sale, etc., due to systematic correctness and the possibility of future Proceedings of 10th Socratic Lectures 2024 85 of 234 amendments to the text, the author adopts a position that will always exist regardless of the text of the criminal code, as it originates from the theory of criminal law. To understand how exactly legal acts then permit its use for medical purposes, it is necessary to briefly explain the theory of a criminal offense. For an act to be considered criminal, an individual's conduct must meet specific conditions, which are described in the Kazenski zakonik ( Law 2). When an individual does this, it is necessary to examine whether they acted in circumstances that, despite meeting all the characteristics, make their conduct completely in accordance with the law. This is called the exclusion of illegality. One way to exclude illegality is through exclusion by another law, meaning another law, not the criminal code, permits a certain behaviour that would otherwise be criminal according to the criminal code. 4.4. Regulation of registered cannabinoid medicines in Slovenia Under the Slovenian Law on the Zakon o proizvodnji in prometu s prepovedanimi drogami (Production of and trade in illicit drugs act: Law 1), registered medicines that contain cannabis and its naturally derived active substances are not considered illegal drugs, provided they are obtained and handled legally. Article 6 exempts such substances from being categorized as illegal drugs when they are used in accordance with specific regulations ( Zakon o lekarniški dejavnosti (Pharmacy practice act: Law 3) in Zakon o zdravilih (Medicinal products act: Law 4)). Consequently, these substances are not implicated in the criminal offense described in Article 186 of the Criminal Code, which deals with the unauthorized production and trafficking of illegal drugs. As of the time of research, there are 24 such medicines registered in the central database of drugs, including those that are only authorized for import and not for distribution within Slovenia. 4.5. Supply of cannabis for pharmaceutical production In the context of importing active substances, the exclusion of illegality operates in a somewhat different manner which will be described in the following paragraphs. This approach is essential in jurisdictions where the use of certain substances is permitted for specific purposes, but production is not and therefore all active substances in Slovenia are imported. The import of active substances for the manufacture of (magistral) drugs with cannabinoids in Slovenia is governed by a complex legal framework, ensuring that only high-quality and safe ingredients are used. The primary legal documents regulating this area are the Zakon o zdravilih ( Law 3) specifically Articles 119 to 122, and the Government rules ( Rules on the manufacture, wholesale distribution and import of active substances and on registers of manufacturers, wholesalers and importers of active substances : Rules 1). These laws stipulate that only registered importers, who are either holders of a medicinal product manufacturing license, active substance manufacturers, or wholesalers of active substances, are authorized to import such substances. In terms of magistral drugs with cannabinoids, Slovenian regulations permit the prescription of isolated or synthetically obtained cannabinoids, standardized cannabis flower extract, and phytocannabinoids in the form of floral and fruit tops of medical cannabis. However, the import of standardized cannabis flower extract and its components requires a demonstrated medical need (JAZMP, 2023). Furthermore, the import of drugs without marketing authorization in Slovenia is permissible only with a specific import permit. The Agency for Medicinal Products and Medical Devices of the Republic of Slovenia (JAZMP) issues these permits within 30 days upon receipt of a complete application for drugs necessary for individual emergency treatments, essential or urgently needed medicines, and in extraordinary cases or for reasons of public health protection (JAZMP, 2023). The applicant for these permits must be a holder of a wholesale drug trading license or a manufacturer authorized to import drugs. In summary, the Slovenian legal framework ensures that the import of active substances for (magistral) drugs with cannabinoids is strictly regulated. Importers must be registered and comply with EU or equivalent production standards. Prescribing and importing Proceedings of 10th Socratic Lectures 2024 86 of 234 cannabinoid-based drugs are subject to stringent controls, emphasizing the necessity of medical justification and adherence to public health interests. 4.6. Industrial hemp cultivation regulations in Slovenia In Slovenia, the cultivation of industrial hemp is strictly regulated under the Pravilnik o pogojih za pridobitev dovoljenja za gojenje konoplje in maka ( Rules on conditions for obtaining a permit for hemp and poppy cultivation: Rules 2). This legislation mandates that cultivation is permissible solely for certain varieties that are included in the national "Sortna lista" for 2023. These varieties must be listed within the EU and contain a THC content of less than 0.3%. To engage in hemp cultivation, farmers are required to satisfy various criteria. These include possessing a minimum land area of 0.1 hectares, which is reduced to 0.01 hectares for organic farmers. Additionally, they must register their cultivation activities, providing comprehensive details about the area of cultivation and its intended use. These stringent measures ensure that Slovenian hemp cultivation adheres to both national and European Union standards. It's crucial to note that the cultivation of medical cannabis, or cannabis suitable for the production of medicines, is currently not permitted in Slovenia. This distinction is important as the term "medical cannabis" specifically refers to varieties used for medicinal purposes, different from those allowed for industrial cultivation. 5. International Resonance and Comparative Perspective The significance of medical cannabis in the context of human rights has also already been acknowledged. The European Court of Human Rights (ECHR) in the case of Thörn v. Sweden ( Case 1) examined whether domestic authorities violated the appellant's right to respect for private life by not exempting him from general criminal liability, which applies to such actions. The case emphasizes the cultivation of cannabis for personal medical use and it underlines the necessity for domestic authorities to consider individual circumstances in criminal prosecutions involving medical cannabis cultivation, balancing public drug regulation interests against individual health needs, considering THC content, distribution risk, medical necessity, and legal available alternatives. In contrast to Slovenia, some EU countries like Germany and the Czech Republic have more liberal approaches towards medical cannabis. Germany has a regulated medical cannabis program with strict quality control measures and a tightly regulated supply chain to ensure patient safety and product consistency. The regulation also covers the import and manufacturing of medicinal cannabis, with strict licensing requirements (Düwel et al., 2023). The Czech Republic permits medical cannabis use with a prescription and allows individuals to cultivate up to five cannabis plants for personal use. As of 2022, decrees regulate the cultivation and processing of cannabis plants for medical use, setting the rules for proper cultivation practice and licensing for Czech growers. There have been efforts to increase the number of suppliers, enable export, and reduce patient costs. The Czech legal framework involves reimbursement of medical cannabis costs and ongoing developments in the regulation of cultivation and supply (KOPAC, 2023) which can sometimes prove ineffective (Medical cannabis network, 2019). The benefits of liberal medical cannabis regulation in the EU have been recognized. More EU Member States are viewing the clinical benefits of cannabis and its derivatives favorably. There is a movement towards ensuring fair and equal access to medical cannabis products and establishing a harmonized policy framework at the EU level (Lipnik-Štangelj and Razinger, 2020). 6. Conclusion Cannabis has been very popular throughout human history due to its versatility, as it can be used in various fields such as the textile, paper, food, furniture and energy industries. It contains more than 140 cannabinoid compounds, of which cannabidiol and tetrahydrocannabinol are the most exposed as they have the potential to treat diseases (Čulić et al., 2021). Numerous scientific findings and clinical studies over the last decade agree that the use of cannabis in medicine has undoubted clinical benefits for many Proceedings of 10th Socratic Lectures 2024 87 of 234 conditions. The best-known effects of cannabis use are pain relief and treatment of chronic diseases and certain neurological disorders in human medicine (Čulić et al., 2021). The use of cannabis products by humans is increasing worldwide. In veterinary medicine trend is related to the growing interest of clients and veterinarians in the treatment of animal diseases with these molecules. In general, CBD is of primary interest in veterinary medicine (Temmerman, 2023). In summarizing Slovenia's approach to cannabis regulation, it is essential to recognize the nuanced interplay between legal rigidity and emerging recognition of cannabis's medicinal value. The journey through Slovenia's legal frameworks, particularly the Zakon o proizvodnji in prometu s prepovedanimi drogami ( Law 1) and Article 186 of the Kazenski zakonik ( Law 2), highlights a cautious yet evolving stance towards cannabis. A critical aspect yet to be fully explored is the potential for domestic production of medical cannabis. The current legislative framework, while stringent on illicit drug activities, has not fully embraced the cultivation of cannabis for medicinal purposes (Štukelj et al., 2018). Developing a regulatory system that permits controlled cultivation for medical production under strict quality standards could significantly enhance Slovenia’s healthcare landscape and contribute to global medicinal cannabis research. This review of Slovenia's cannabis regulation reveals a nation balancing control with emerging medicinal perspectives. The potential for regulatory reform, accommodating both public safety and medical advancement, looms as a promising horizon. Such a future, where regulation aligns with the scientific and compassionate use of medicinal cannabis, could position Slovenia as a leader in this field, reflecting a commitment to both legal integrity and medical innovation. Funding: This research was supported by the Slovenian Research and Innovation Agency (ARIS) through the Core Funding no. P1-0045 and by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) from Argentina, PICT 2016 nº 608 ANPCyT. Conflicts of Interest: The authors declare no conflict of interest. List of regulation documents 1. Law 1: Zakon o proizvodnji in prometu s prepovedanimi drogami (Uradni list RS, št. 108/99, 44/00, 2/04 – ZZdrI-A in 47/04 – ZdZPZ) 2. Law 2: Kazenski zakonik (Uradni list RS, št. 50/12 – uradno prečiščeno besedilo, 6/16 – popr., 54/15, 38/16, 27/17, 23/20, 91/20, 95/21, 186/21, 105/22 – ZZNŠPP in 16/23) 3. Law 3: Zakon o lekarniški dejavnosti (Uradni list RS, št. 85/16, 77/17, 73/19 in 186/21) 4. Law 4: Zakon o zdravilih (Uradni list RS, št. 17/14 in 66/19) 5. Decree 1: Uredba o razvrstitvi prepovedanih drog (Uradni list RS, št. 69/19, 157/20, 162/21, 8/23 in 113/23) 6. 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DOI: https://doi.org/10.1590/S1516-44462006000200015 Proceedings of 10th Socratic Lectures 2024 93 of 234 Invited lecture/Research A Multiplex GC-MS/MS Analysis for the Quantitative Moni- toring of Bilobalide, Ginkgolides and Ginkgotoxin in Ginkgo biloba-Derived Products and Biomaterials Árva Zsolt 1,2, Barbulova Ani1, Fiume Immacolata1, Moubarak Maneea1,3, Pocsfalvi Gabriella1 1. Laboratory of Extracellular Vesicles and Mass Spectrometry, Institute of Biosciences and BioResources, Italian National Research Council, Naples, Italy 2. Faculty of Science and Technology, University of Debrecen, Hungary 3. Faculty of Agriculture, Damanhour University, Damanhour, Egypt * Correspondence: Gabriella Pocsfalvi; gabriella.pocsfalvi@ibbr.cnr.it Abstract: The bioactivity Ginkgo biloba-derived extracts and other preparations is attributed to the presence of secondary metabolites, especially terpene trilactones (ginkgolides and bilob- alide), flavonoids but also toxic constituents, like ginkgotoxin. In this study, we set up a Citation: Árva Z, Barbulova A, multiplex method using a gas chromatography coupled with tandem mass spectrometry Fiume I, Moubarak M, Pocsfalvi G. (GC-MS/MS) for the simultaneous quantitative analysis of six of these characteristic me-A Multiplex GC-MS/MS Analysis tabolites, namely ginkgolide A, B, C, J, bilobalide and ginkgotoxin. Parameters were set for the Quantitative Moni-toring of up and optimized for multiple reaction monitoring (MRM) to allow the sensitive and se-Bilobalide, Ginkgolides and lective monitoring of specific collision induced dissociation transitions for each analyte. Ginkgotoxin in Ginkgo biloba-Derived Products and Biomaterials. The method was applied to quantify and compare the above ingredients after derivatiza-Proceedings of Socratic Lectures . tion in the methanol extracts of a commercial supplement, ginkgo nuts, ginkgo cells from 2024, 10, 93-108. suspension and callus cultures, and microvesicles (MVs) and nanovesicles (NVs) isolated https://doi.org/10.55295/PSL.2024.I14 from ginkgo seeds. As a result of the MRM analysis, the commercial supplement contained 7,77% terpene lactones per tablet. The seed samples contained 50 parts per million Publisher’s Note: UL ZF stays neu- (ppm) ginkgolide A and 14 ppm ginkgolide B, but ginkgolide C and J were below the limit tral with regard to jurisdictional of quantitation. Ginkgo biloba cells from in vitro cultures, MVs and NVs contained con-claims in published maps and insti- siderably less bilobalide and ginkgolides than the seed and the commercial supplement. tutional affiliations. Keywords: Ginkgo biloba; extracellular vesicles; GC-MS/MS; derivatization; quantitative analysis, multiple reaction monitoring, ginkgolides, bilobalides, ginkgotoxin Copyright: © 2024 by the authors. Submitted for possible open access publi- cation under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecom- mons.org/licenses/by/4.0/). Proceedings of 10th Socratic Lectures 2024 94 of 234 1. Introduction Ginkgo biloba L., the only survivor of genius ginkgo is a living fossils revered for its longev-ity and elegance of its leaves (Chi et al., 2020). Different parts of ginkgo tree such as leaves, seeds and roots have been used in traditional Chinese medicine for thousands of years. A standardized extract of ginkgo biloba (GB) dried leaf, Egb 761 containing 24% Ginkgo Fla-vone Glycosides and 6% Terpene lactones is among the most studied and most popular herbal supplements today (Nor-E-Tabassum, 2022). Egb 761 represents the only herbal alternative to synthetic antidementia drugs in the therapy of Alzheimer's disease and cognitive decline (Nor-E-Tabassum, 2022). Sora et al developed and validated a liquid chromatography tandem mass spectrometry (LC–MS/MS) assay of terpene trilactones in (GB) extracts and pharmaceutical formulations through standard addition method and found bilobalide content 2.8-3.2 mg and ginkgolides (A, B, C) content 2.9-3.2 mg (Sora et al., 2009). Gas chromatography - mass spectrometry (GC-MS) has also been successfully applied to quantify ginkgolides in ginkgo dietary supplements (Deng and Zito, 2003). The edible seeds of GB are also used in traditional Chinese medicine for different purposes, including the treatment of senility, asthma, bronchitis, and kidney and bladder disorders. GB seeds known to contain toxic compounds like ginkgotoxin (N-methyl pyridoxine), an analogue of Vitamin B6, that raised concern about its use (Boateng and Yang, 2022). LC-MS and LC-MS/MS methods have been developed for the identification and quantitation of ginkgotoxin (Scott et al., 2000) and determined 174 ppm ginkotoxin in seed extracts. In this study, 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). Figure 1. Chemical structures of ginkgolides A, B, C, J, bilobalide and ginkgotoxin. The developed method was used to investigate the quantities of these primary ginkgo specific 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). While there have been LC-MS/MS based studies on terpene lactones that investigates commercial pharmaceutical products containing standardized extracts (Sora et al., 2009; Scott et al., 2000), to the best of our knowledge there is no GC-MS/MS method available today for the simultaneous analysis of terpene lactones and ginkgotoxin. Proceedings of 10th Socratic Lectures 2024 95 of 234 Figure 2. Samples analysed; A) Gloryfeel ginkgo biloba supplement prepared from ginkgo leaves, B) ginkgo seeds C) embryo, callus and cell suspension cultures. 2. Materials and methods 2.1. Sample preparation and standards GB dietary supplement was from Gloryfeel BmbH (Hamburg, Germany). Tablet (274.05 mg) was pulverized in a mortar and sample (235.6 mg) was extracted in 5 mL methanol (Romil MS grade) for 30 minutes under orbital shaking at room temperature. Samples were centrifuged at 14000 x g at 26 °C for 20 minutes two times. GB seeds were collected in Naples Piscinola in November, 2023. Seed shell and inner layer were removed and 5 samples containing 5 seeds were dried for 72 h at 40 °C. Samples were pulverized in a mortar and 50 mg were extracted in 600 µL methanol. Callus and cell suspension cultures were established using GB embryos as starting material. The seeds were surface sterilized with sodium hypochlorite and subsequently, viable GB embryos were isolated and put for callus induction on Gamborg B5 growth medium (Duchefa Biochemie), supplemented with 2 mg/L of 2,4-dichlorophenoxyacetic acid (2,4D) and 2 mg/L of naphthyl acetic acid (NAA). White and friable callus cultures were obtained after 8 weeks of cultivation in dark conditions and were maintained by subculturing every 4 weeks. To initiate cell suspension cultures, 5 g of callus were resuspended in 50 mL of the same growth medium and incubated at 26 °C in dark conditions under constant orbital stirring (110 rpm). The cultures were maintained by subculturing every 7 days. To prepare the samples, 40-45 days old callus cultures and 7 days old suspension cultures were used. Samples were homogenized in methanol using a Tissuelyser (Qiagen) at 30 Hz, 5 x 1 minutes. Microvesicles (MVs) and nanovesicles (NVs) were isolated from the homogenate of GB seeds using the differential ultracentrifugation method (Bokka et al., 2020). Protein concentration was measured by Qubit assay (Invitrogen). 300 µg MVs and NVs expressed in protein quantity were extracted in 300 µL methanol. The samples were homogenized in methanol using a Tissuelyser (Qiagen) at 30 Hz, 5 x 1 minutes. Five replicates were prepared of the suspension and callus cultures, seeds and tablet, and triplicates for MVs and NVs samples. All methanol extracts were centrifuged at 15000 x g for 20 min. 600 ng D-Mannitol-13C6 (Cambridge Isotope Laboratories, Canada) was added as internal standard (IS). Samples were vacuum dried and then further dried under nitrogen flow. Derivatization was performed using 50 µL silylation derivatization reagent, (N,O-bis[trimethylsilyltrifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) (Sigma Aldrich) and 35 µL pyridine (Merck) at 70 °C for 1 hour at 500 rpm agita-tion in a thermomixer. Proceedings of 10th Socratic Lectures 2024 96 of 234 The calibration solutions were prepared in the following ranges: ginkgolide A, B, C and J from 0.05 µg to 50 µg; bilobalide from 0.05 to 30 µg and ginkgotoxin from 0.01 to 20 µg in 85 µL. The quantity of the IS was maintained at 600 ng in 85 µL in each standard solution. 2.2. GC-MS/MS measurement parameters GC-MS/MS analyses were performed using a Thermo Scientific Trace 1300 GC coupled to a TSQ 8000 Duo mass spectrometer equipped with an electron ionization (EI) ion source and a triple quadrupole (QqQ) ion analyzer. Separation was performed using a TG-SQC 30 m, 0.25 mm x 0.25 µm capillary column. GC parameters were as follows: 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. Dionex Chromeleon 7 Chromatographic Data System version 7.2.10 (Thermo Scientific Fisher, USA) was used for data acquisition and processing. The tentative identification of the compounds was based on computer matching with the National Institute of Standards and Technology (NIST) library. Collision induced dissociation (CID) was performed using Argon as collision gas. Two ion transmissions of the single analytes were selected by using the Thermo Scientific AutoSRM application run under Chromeleon 7 software package. Concentrations of the analytes in the samples were calculated using calibration curves values corrected against the internal standard. 3. Results and Discussion Full scan MS-analysis using chemical derivatization to make the compounds volatile provides an unbiased identification of metabolites in plant extracts (Fiehn, 2016). In this study, after silylation derivatization we have performed full scan GC-MS analyses of the methanol extracts of the following ginkgo biloba-derived samples: i) seed, ii) callus iii) cells from suspension culture, iv) microvesicles isolated from GB seeds, and v) nanovesicles isolated form GB seeds. Compounds that were identified in at least 3 samples were considered. Appendix A shows the compounds tentatively identified in each sample. Mostly primer metabolites, i.e. aminoacids, lipids, organic acids, aldehydes, ketones and alcohols and sugars could be identified in this analysis. 100 compounds were identified in the GB seed extract, 60 in callus culture, 68 in cell suspension derived cells, 35 in microvesicles and 39 in nanovesicles derived from GB seeds. Venn diagrams compare the presence of different metabolites in seed and seed-derived micro- and nanovesicles samples (Figure 3A) and in seed and seed explant-derived callus and suspension cultures (Figure 3B). Only 10 compounds were commonly present in MVs, NVs and seed extracts, 7 compounds in NVs and seed, 8 compounds in MVs and seed, and 13 compounds in MVs and NVs (Figure 3A). 30 compounds were commonly identified in callus, suspension cultures and seed, 3 compounds in callus and seed, 6 compounds in suspension cultures and seed, and 19 compounds were commonly identified in callus and suspension cultures (Figure 3B). Interestingly, only two secondary metabolites, the flavonoid monomeric unit containing catechin (2R-trans) and epigallocatechin, were detected in callus and suspension cultures. Characteristic GB metabolites, like bilobalide, ginkgolides or ginkgotoxin could not be identified through the full scan GC-MS analysis in seed and seed derived cells cultured in vitro or seed-derived micro and nanovesicles. Proceedings of 10th Socratic Lectures 2024 97 of 234 A B Figure 3. Venn diagram of A: the metabolites identified in MVs, NVs and ginkgo biloba seeds and B: callus, suspension cultures and ginkgo biloba seeds (lists of compounds are reported in Appendix A). To setup the GC-MS/MS targeted analysis method, we have determined and optimized two characteristic precursor-to-product ion transitions for the internal standard mannitol 13C6 and six ginkgo biloba secondary metabolites, i.e. ginkgotoxin, bilobalide and ginkgolides A, B, C and J. These are reported in Table 1. Collision energy (CE) for each transition was optimized using the Chromeleon AutoSRM software and the optimal CE values are reported in Table 1. Table 1. Precursor / product ion’s m/z as well as collision energy values. GGvvv Compound m/z Precursor/Product Ion Collision Energy (eV) 280.1 / 206.1 10 ginkgotoxin 295.1 / 280.1 10 426.1 / 264.1 10 D-mannitol 13C6 426,1 / 336.14 10 299.1 / 271.1 10 bilobalide 398.3 / 223.1 10 537.2 / 187.0 25 ginkgolide A 537.2 / 391.2 10 625.3 / 479.2 10 ginkgolide B 625.3 / 597.2 5 713.3 / 567.1 10 ginkgolide C 713.3 / 595.1 10 478.3 / 264.2 10 ginkgolide J 478.3 / 422.2 10 Based on these results we setup a quantitation method suitable for the monitoring of these analytes in a single data acquisition. Figure 4 shows a representative MRM spectrum of ginkgotoxin, bilobalide, ginkgolide A, B, C, J and internal standard D-mannitol-13C6 in MS quantitation mode. GC conditions were set to achieve baseline separation for all the four ginkgolides studied. The data of calibration curves, i.e. retention times, ranges, R2 values, slopes are reported for each standard in Table 2. The results of the 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 limit of quantitation (LOQ), methanol extracts of Ginkgo biloba cells from callus and cell suspension, MVs and NVs contained considerably less bilobalides and ginkgolides than the seed and supplement extracts. We Proceedings of 10th Socratic Lectures 2024 98 of 234 found that MVs sample contained 269 ppm, NVs 61 ppm, suspension culture (dry) 3 ppm, seed 266 ppm and tablet 37 ppm of ginkgotoxin. Figure 4. Representative MRM spectra of ginkgotoxin, bilobalide, ginkgolide A, B, C, J and internal standard the D-mannitol-13C6 (MS quantitation). Table 2. Calibration data (Retention Times (RT), Ranges, R2, Slopes). Compound RT (min) Calibration points Range (g) R2 Slope ginkgotoxin 15.40 7 0.01-20 0.9944 13502.73 bilobalide 21.41 6 0.05-30 0.9917 343.02 ginkgolide A 27.53 5 0.50-50 0.9939 16.15 ginkgolide B 28.20 5 0.50-50 0.9965 14.44 ginkgolide C 28.35 5 0.50-50 0.9982 14.55 ginkgolide J 27.60 5 0.50-50 0.9804 14.76 Table 3. Results of the quantitative GC-MS/MS analysis of nanovesicles, microvesicles, cells from suspension culture wet and dry, callus wet and dry, seed and ginkgo supplement tablet – weight: mass used for the methanol extraction in milligrams (mg, average values); ginkgotoxin, bilobalide, ginkgolide A, B, C and J analytes expressed in part per millions (ppm) (average values). LOQ is for limit of quantitation. Sample Weight Ginkgotoxin Bilobalide Ginkgolide Ginkgolide Ginkgolide Ginkgolide (mg) (ppm) (ppm) A (ppm) B (ppm) C (ppm) J (ppm) Nanovesicles 0.3 61